key: cord-023120-jcgf2401
authors: nan
title: Animal virus genetics
date: 2004-06-18
journal: J Supramol Struct
DOI: 10.1002/jss.400140505
sha: 
doc_id: 23120
cord_uid: jcgf2401

nan

We have cloned the viral DNA together with its flanking host sequences from several lines of cells transformed by adenovirus 2 and SV40. From analyses of these clones we draw the following conclusions: those of the viral DNA. Deletion of viral sequences is common and at least in two cases inversions have been found within the integrated viral DNA. t h i s configuration, a complete copy of v i r a l RNA i s positioned "downstream i n a t r a n s c r i pt i o n a l sense" from sequences present a t t h e 3' end of v i r a l RNA, suggesting t h e p o s s i b i l i t y t h a t the "3 'I sequences might promote and r e g u l a t e t r a n s c r i p t i o n of v i r a l genes. nearly wild type levels at the permissive temperature. The level of phosphotyrosine in mutant infected cells rises rapidly upon shift from the restrictive to the permissive temperature, being substantially increased within 1 hour. Elevated levels of phosphotyrosine are not found inall p p 6 0 C itself has two phosphorylation sites. The level of phosphorylation at the site in the C-terminal half is affected by mutations in the SIC gene and it has been suggested that this site undergoes autophosphorylation. Consistent with these notions we have now found that this site is a phosphotyrosine. This is the first demonstration of the presence of phosphotyrosine in a protein labeled in vivo. Phosphot rosine is also present in a 50,000 dalton phosphoprotein which co-precipitates with p p 6 d from transformed chick cells. this protein is an intracellular substrate of ~~60%. It should clearly be possible to identify further substrates of p p 6 e on the basis of their containing phosphotyrosine. p p 6 e , the closely related cellular homoloque of viral p p 6 e , is present in all vertebrate cells. We have found that this normal cellular protein, obtained from both chicken and human cells, also phosphorylates tyrosine. This is additional evidence of the functional similarity of these structurally related proteins. It also demonstrates that all uninfected cells contain at least one protein kinase that phosphorylates tyrosine.

The immunoglobulin' heavy chain is phosphorylated by the protein kinase activity Despite the fact that a protein kinase types of bansformed cells,however,for example being low in polyoma transformed cells.

The r i g h t o p e r a t o r ( 0 ) i n t h e chromosome o f bacteriophage A c o n t a i n s t h r e e contiguous sites t h a t R a r e recognized by t h e A phage r e p r e s s o r .

overlaps, and c o n t r o l s divergent t r a n s c r i p t i o n from, t h e promoters P and P . 

Cro i s a negative r e g u l a t o r required f o r l y t i c phage growth. Reaction mixtures c o n t a i n a d i a l y z e d and c o n c e n t r a t e d HeLa c e l l e x t r a c t , small molecules and c o f a c t o r s r e q u i r e d f o r t r a n s c r i p t i o n , and exogenously added DNA. When a cloned 2 . 2 kb r es t r i c t i o n fragment o f adenovirus 2 (Adz) DNA which c o n t a i n s t h e major l a t e promoter i s added t o t h e system, w e d e t e c t l a r g e amounts of a 1 . 7 kb t r a n s c r i p t . I f a n o t h e r cloned 2.2 kb

Ad2 r e s t r i c t i o n fragment, which does n o t contain a promoter, i s added i n s t e a d , no t r a n s c r i pt i o n occurs. W e have shown t h a t t h e 1 . 7 kb t r a n s c r i p t i n i t i a t e s a t t h e major l a t e promoter by s e v e r a l methods, i n c l u d i n g f i n g e r p r i n t a n a l y s i s of t h e 5' end. T o t a l Ad2 DNA a l s o funct i o n s e f f i c i e n t l y a s a template and i n v i t r o t r a n s c r i p t i o n i n i t i a t e d a t t h e major l a t e promoter continues f o r a d i s t a n c e of a t l e a s t 4.5 kb. W e have a l s o t e n t a t i v e l y i d e n t i f i e d t r a n s c r i p t s o r i g i n a t i n g from e a r l y Ad2 promoters, u s i n g cloned DNA r e s t r i c t i o n fragments, a s well a s t o t a l Ad2 DNA, a s templates. Avian erythroblastosis (AEV) and avian myelocytomatosis virus strain 29 (MC29) have previously been shown to transform hematopoietic cells as well as fibroblasts in vitro even after clone purification of the respective viruses (1, 2) . Hematopoietic cells transformed by AEV have the properties of erythroblasts whereas MC29-transformed hematopoietic cells resemble macrophages (3) . Cloned fibroblasts transformed by these strains can bk distinguished by t h e pattern of transformation parameters they express (4) . The different biological properties of these two virus strains have been attributed to two new transformation specific sequences designated erb for AEV and mac for MC29 (5, 3) . W e have now been able to i s o l a x a mutant of A m ( t d 3 5 9 AEV) (5) and 3 mutants of MC29 (td4A MC29, tdlOC MC29 and tdlOH MC29) which have lost t h e x i l i t y to transform hematopoetic cells6vt which E v e retained t h e 3 i l i t y to transform fibroblasts. These mutants show a n increase in the mobility on SDS-PACE of their presumptive transforming a -erb fusion protein p75 AEV (7) and in the gag-mac fusion protein p l l 0 MC29 (8), respectively. I n s n , tryptic peptide analysis of these proteins revealed that they lack some of the transformation specific sequences and that they acquired in some cases additional =-specific sequences. Nearly all mice of the inbred AKR strain develop lymphoma before one year of age, but F1 mice from crosses of AKR with mice of various low-lymphoma strains develop the disease only at low incidences and/or much later in life in the large majority of such crosses that have been studied. It appears that one or more dominant gene transmitted from the low-lymphoma parental strain can suppress the development of the disease.

AKR mice express high levels of ecotropic murine leukemia virus (MuLV) from birth or soon thereafter, and MuLV with xenotropic and polytropic host ranges are usually detectable in their thymuses at six to eight months of age. (Gross MuLV Passage A is a strain of leukemogenic virus originally isolated from lymphomatous AKR tissues, but the relation between the relatively well characterized MuLVs present in AKR tissues and the lymphomagenic agent in Gross MuLV preparations remains to be clarified.)

This laboratory has previously reported that genetic analysis of two such crosses, AKR x BALB/c and AKR x RF, indicated that alleles at the F v -1 locus transmitted from the lowlymphoma parent were major factors for lymphoma suppression. In each case, the F v -1 resistance allele concomitantly suppressed some aspect of MuLV expression. We now report results from three more such crosses: AKR x DBA/1, AKR x C57L and C58 x DBA/2. Mice of segregating generations of these crosses were examined for their phenotypes at the Gpd-1 locus (closely linked to F p 1 ) and observed for occurrence of lymphoma. In none of the three crosses was there an association between Gpd-1 type and the disease, indicating that genes other than F v -1 suppress lymphoma in these cases. Moreover, no significant association between lymphoma and endogenous MuLV expression was detected in two of the three crosses, suggesting that the lymphoma-suppressing genes operate by affecting some lymphomaassociated factor other than endogenous MuLV. In only one of the three crosses was an association noted between lymphoma and H-2 type, although the H-2 complex has often proved to be a significant determinant of lymphoma susceptibility in other crosses. DNA extracted from several mouse clones transformed by chemical carcinogens has been applied to mouse fibroblast NIH3T3 cultures, using the transfection technique of Graham and Van der Eb. Foci of transformed cells have been derived from such transfections. The transformants are able to grow in soft agar, and are tumorigenic in newborn mice. These results show that the phenotype of transformation can be transferred by naked DNA. They also indicate that in those cases the transforming allele is dominant.

Treatment of one of the transforming DNAS (extracted from a C3HlOT1/2 cell line transformed by 3-methylcholanthrene) with a series of restriction enzymes showed that BamHI does not inactivate its biological activity. Using a cloning strategy developed in yeast (1). we have cloned a DNA fragment having sequence homology with the gene carrying the transforming allele. A homologous gene can be demonstrated to exist as a single copy in normal mouse cells, using the Southern blotting technique. In cells which have been transformed via transfection. an additional copy of a homologous sequence is shown to be acquired. The cloned sequence has no apparent homology with the sarc regions of Moloney murine sarcoma virus and Avian sarcoma virus.

The potential use of the cloned DNA fragment will be described. whether cells transformed by independent carcinogenic events, always transfer the same transforming gene during DNA transfections. The results would indicate whether a single or multiple normal cellular genes are potential targets for the carcinogenic events. In order to study the effect of the carcinogen on the gene, differences between the transforming allele and its counterpart sequence in normal cells will be looked at. Finally the transcription of the gene will be monitored in normal and transformed cells, to ask whether the carcinogen is affecting the promoter of the gene. Rxcmbinants derived fran rewiruses type 1, 2 a d 3 have been used to s t d y virus-bst i n a t i o n s . We have foold that the S1 dsmA s-t, the segment that enccdes the 01 p l m e , ~ZE-Z&S the viral henagglutinin. The h n q g l u t i n h i s responsible for specificity in hunural a d cellular innunity. detetmines cell tropian i n i?e nervous systen, binairq to cellular microtuhles, an3 shtoff of h t cel1.m synthsls. h t thus deterrmne s several facets of viral host interactions and virulence.

Thus the 01 polypeptide is the henagglutinin.

The specific interaction of the hanagglutinin w i t h the

Although a great deal of information has accumulated about viral penetration and replication in many types of host cells, comparatively little is known about the initial binding of virus to cells or &out specific receptors for viruses. The product of the major histocompatibility gene complex (MHC) is of biological interest because of its fundamental role in certain cell-cell membrane recognition systems, its control of important gene products, and i t s association with susceptibility to some diseases. Despite t h e hosts humoral and c e l l mediated immune response, the v i r u s p e r s i s t s i n t h e animal. Viruses i s o l a t e d from a p e r s i s t e n t l y i n f e c t e d sheep e a r l y a f t e r i n f e c t i o n were n e u t r a l i z e d by e a r l y immune sera from t h i s animal t o t h e same extent as t h e p a r e n t a l v i r u s (1). p a r e n t a l type as w e l l as a n t i g e n i c a l l y d i s t i n c t viruses. n e u t r a l i z i n 8 antibody t o these v a r i a n t viruses. The sheep subsequently developed These a n t i g e n i c v a r i a n t s are g e n e t i c a l l y Antigenic v a r i a n t s o f visna v i r u s have been compared u s i n g t h e genomic RNA and analyzing Mutants i s o l a t e d from a p e r s i s t e n t l y i n f e c t e d t h e l a r g e RNase T i -r e s i s t a n t oligonucleotides.

sheep contained a small number of changes i n t h e i r o l i g o n u c l e o t i d e p a t t e r n s when compared w i t h p a r e n t a l v i r u s . d i f f e r e n c e between t h e p a r e n t a l s t r a i n and t h e a n t i g e n i c mutant used f o r mapping were located w i t h i n 2 kilobases from t h e 3' terminus. nucleotides which d i f f e r e d from t h e p a r e n t a l t o t h e mutant suggest t h a t they might be derived by simple mutation. n u c l e o t i d e sequence of several oligonucleotides were determined. a number o f t h e oligonucleotides which were d i f f e r e n t between t h e p a r e n t a l and mutant could be accounted f o r by s i n g l e base changes.

Thus, based on these s t u d i e s we propose t h a t m u l t i p l e m u t a t i o n a l events i n t h e r e g i o n of t h e genome which codes f o r t h e v i r a l antigen t h a t e l i c i t s n e u t r a l i z i n g antibody permit t h e v i r u s t o escape t h e h u n e response and may, i n addition, be responsible f o r t h e slow progressive n a t u r e of t h e disease. The biological roles of paramyxovirus envelope proteins (HN, F, and M) have been studied. F is involved in virus penetration, cell fusion and hemolysis, and is activated by cleavage by a host protease into 2 disulfide-bonded subunits (F1 and Fz). undergo multiple cycle replication, spread, and cause disease is dependent on the presence of an activating protease in the host; thus cleavage of F is a major determinant of pathogenesis. The N-terminus generated on F1 by cleavage is involved in its biological activity. The amim acid sequence in this region is hydrophobic and similar in Sendai, SV5 and NDV. Inan attempt to design specific inhibitors of virus replication, cell fusion and hemolysis, oligopeptides analogous to this region of F1 were synthesized and found to be specific and highly-active. Structure-activity studies showed that amino acid sequence, peptide length, and N-terminal carbobenzoxy group, C-terminal esterification, and steric configuration of amino acids affect inhibitory activity. Specific inhibition of influenza virus has also been obtained with peptides which resemble the N-terminal region of HAz. These results have provided information on the.mechanism of action of viral proteins in penetration and membrane fusion, and have also suggested a new approach to specific inhibition of viral replication.

Studies with mono-specific antibodies have shown that antibodies to HN can inhibit dissemination of infection by released virus, ,but not spread from cell to cell via membrane fusion, whereas antibodies to F completely inhibit spread of infection. Thus to be effective a paramyxovirus vaccine must stimulate antibodies to F. These results, coupled with findings by Norrby and co-workers that formalin-inactivated paramyxovirus vaccines did not elicit antihemolyzing antibodies, provide an explanation for vaccine failure, and for atypical and severe diseases, e.g., atypical measles, that occurred in individuals who received inactivated vaccines and were later infected. In the absence of F antibodies, infection could spread at the same time that there was a secondary immune response to HN and other viral proteins, setting the stage for a pathological immune reaction.

virus infection, lack antibodies to M protein despite high serum and cerebrospinal fluid (CSF) titers of antibodies to other measles proteins, suggesting a host-dependent lack of M synthesis in the brain. Evidence supporting this has been obtained in cultures of SSPE brain cells which synthesized other measles proteins but not M. We also found that a patient with severe mental retardation and epilepsy 24 years after measles encephalitis had high titers of serum and CSF antibodies to all measles proteins except M, suggesting that: 1) virus persisted in the brain; 2 ) chronic neurological disease caused by measles virus is not limited to SSPE; 3) persistent measles infection may cause some cases of mental retardation and epilepsy. 

the group which the temperate coliphage lambda and the temperate Salmonella phage P22) suggest that such families evolve as a group of interbreeding interchangeable modular elements. Natural selection, in this view, acts upon the efficiency with which each module does its job and upon retention of easy interchangeability without loss of function and proper regulation. Thus the product of evolution is not a given bacteriophage, but the group of interbreeding interchangeable modular parts. Each virus found in nature is seen as a sample of a large variety of possible modular assemblies which work well in a given ecological niche.

possible applicability of this idea to animal virues will be discussed.

The evidence for this view of bacteriophage evolution will be summarized, and the ORIGINS AND DISTRIBUTION OF GENETICALLY TRANSMITTED RETROVIRUSES OF PRIMATES, George J. Todaro, Laboratory of V i r a l C a r c i n o g e n e s i s , National I n s t i t u t e s of H e a l t h , Bethesda , Maryland 20205

Six d i f f e r e n t endogenous r e t r o v i r u s e s o f primates have been i s o l a t e d d u r i n g t h e p a s t few years.

Two a r e f r a n New World primatesa type C v i r u s f r a n owl monkeys and a t y p e D v i r u s from s q u i r r e l monkeys.

Four a r e from Old World p r i m a t e st y p e C ' s from baboons, macaques and colobus monkeys and t y p e D ' s f r a n langur monkeys. The colobus t y p e C v i r u s came f r u n a kidney c e l l c u l t u r e , and was i s o l a t e d on an u n u s u a l l y permissive human carcinoma l i n e , A549. 50-100 c o p i e s of v i r a l r e l a t e d information a r e found i n colobus c e l l u l a r DNA with r e l a t e d sequences i n r e l a t e d primate DNAs. The colobus v i r a l RNA and p r o t e i n s a r e r e l a t e d t o macaque t y p e C v i r u s e s and n o t to baboon v i r u s e s . The i s o l a t e s a r e sumnarized a s follows:

A n c e s t r a l Primates We are using endogenous viruses and recombinant3 of endogenous and exogenous viruses to test for the role of envelope antigens and c regions in disease. Four cloned envE cx viruses (RAV-60s) have been compared for their oncogenic activity wit= eiivA cx virus (RAV-1). These experiments demonstrated that envelope antigens are not critical for disease and that cloned isolates of ALVs cause more than one disease3. being tested foroncogenesis.

3' portion of its c region has oligonucleotides characteristic of sx 4. results to date of these tests suggest that cx is the oncogenic agent in nonacute disease. events? We suggest that, (1) c regions contain promotors for viral RNA synthesis, (2) cx contains a more efficient promotor than c" and (3) non-acute disease foliows the formation of critical viral-cell recombinants in which ( 2 ) The r e s u l t i n g CDNA i s f r a c t i o n a t e d and a l l fragments having a length g r e a t e r than 300 nucleotides are i s o l a t e d and c u t with a r e s t r i c t i o n endonuclease capable of d i g e s t i n g single-stranded DNA. polymerase I, the ends were f l a t t e n e d using S1 nuclease, and poly dC tails were added with terminal deoxynucleotide t r a n s f e r a s e . The product was i n s e r t e d i n t o pBR322 by annealing t o poly dG t a i l s added a t the plasmid's P a t I r e s t r i c t i o n s i t e and propagated i n E . X1776 . A 905 210 base p a i r fragment including the simian v i r u s "strong stop" sequence, p l u s a d j a c e n t sequences, was obtained. Happing and sequence d a t a w i l l be presented. These include full genome length ds DNA, circular DNA, a deleted linear DNA missing the 5 ' end 600 bp repeat and a 600 bp repeat unit. All these forms have been cloned in pBR 322. Moreover, subclones containing the "src" region of the genme have been obtained. Sequencing of both the repeat unit and the sarcoma specific region is in progress.

These cloned vDNA molecules have been used to analyze the formation of viral mRNA in vivo. We have shown that spliced mRNA-like molecules are present in virions and cellular cytoplasms. The src mRNA, about 205 in size has been purified by hybridization to cloned DNA and characterized by electron microscopy and S l mapping. Preliminary data indicate that the purified cellular vRNAs can be translated into sarcoma-specific polypeptides which are also made by virion RSA.

Moloney sarcoma virus (MSV) contains a sequence (src) preswbly acquired from the host during passage of the parent Moloney murine leukemia virus through Balb/c mice. This sequence is apparently a prerequisite for the tumorigenic potential of the virus and its ability to mrphologically transform cells in vitro. Using recombinant DNA techniques we have cloned in phage lambda a sequence (sars fromlb/c muse DNA homologous to the MSV src sequence. The cloned DNA fragment was comparal to two cloned integrated proviruses of MSV (the ml and HT1 isolates) by heteroduplex mapping and restriction endonuclease analyses. Only one region of homology was observed corresponding to the src region of the proviruses and measuring 1.17 kb for m l and 1.3 kb for HT1 MSV. Nine identical restriction sites were mapped in Balb/c sarc and HT1 MSV src. The cloned fragment also contains 14 kb of muse cell sequences flanking sarc, which showed no homology to other regions of MSV. The sarc fragment was tested in a direct DNA transfection assay for its ability to induce foci of transformation on NIH-3T3 cells. In this assay the cloned provirus of m1 MSV produces 4.7 x 104 focus forming units per pmole DNA. Subgenomic fragments of K 3 also transform NIH-3T3 cells. However, in repeated transfections even at high DNA concentrations the sarc fragment or subcloned fragments containing sarc were inactive in the assay. t h e i n t e g r a t e d provirus of two HSV isolates. together w i t h flanking cellular sequences, from transformed mink c e l l s i n t o bacteriophage A ( 1 ) . and have subsequently cloned subgenmic MSV fragnents into the plasmid pBR322. Utilizing these cloned D N A ' s i n transfection assays, we have attempted to define the role of specific viral sequences i n transformation.

Cloned whole p r o v i r a l DNA t r a n s f e c t s and transforms w i t h high e f f i c i e n c i e s (5~10'-8xlO' ffu/pnole). and i n the majority of cases these c e l l s contain rescuable HSV. Cloned %-containing subgenanic fragments, f r a which either the terminal 5' 0' 3' leukemia and mink-derived sequences were deleted, also transform w i t h o n l y a &fold reduced efficiency. S i m i l a r c l o n e s which lack terminal leuk sequences (pHT10) t r a n s f o n w i t h efficiencies %OOO-fold lower than cloned whole MSV genome.

Activity can be restored by the i n v i t r o addition of non-transforming proviral sequences which contain the 600 b p terminal rerundancy (TRS). Furthermore, co-transfection w i t h the poorly transforming pHT10 fragment together w i t h a non-transforming clone containing o n l y TRS and associated m i n k c e l l sequences transforms 50-100 f o l d more efficiently than pHT10 alone. Collectively, the data indicate t h a t sequences i n addition t o are necessary for e f f i c i e n t c e l l transformation and t h a t TRS may function to enhance the insertion and/or expression of transforming sarcoma viral sequences. Unintegrated supercoiled spleen focus-forming virus (SFFV) DNA was extracted from r a t c e l l s newly infected with a woolly monkey virus pseudotype of the Lilly-Steeves s t r a i n of SFFV.

The DNA was linearized by digestion a t the unique Hind I11 endonuclease s i t e , inserted into the plasmid pBR322, and cloned in an approved EK2 host. Six independent clones containing SFFV DNA inserts were isolated and analyzed by restriction endonuclease digestion. One clone had an insert of 5.7 kbp with the same restriction enzyme s i t e s as the unintegrated linear SFFV DNA except that it lacked a copy of the terminally redundant sequence. Recom 

is acute myeloblastic leukemia. mic myeloblast DNA show: 1) Specific proviral fragments are present in a l l leukemic myeloblasts regardless of the chicken genetic background, 2) These AMV specific fragments are not found in cells infected by helper virus alone, and 3 ) The same AMv specific fraqments are present regardless of the AMV pseudotype used. Analysis of DNA from colonies arising from single leukemic myeloblasts (containing or not containing the helper genome) permitted the identification of restriction endonuclease fragments which belong to either the helper or the presumptive AMV genome. Furthermore, the juncture fragments of the clonal DNAs show that both AMV and its helper(s) can integrate at different cellular DNA sites. n o h Charon 4A clones containing proviral DNA from leukemic myeloblast have been characterized by restriction endonuclease digestion. Clone X 10A2-1 contains 85% of the helper genome as well as host sequences flanking the 5' end of the genome with respect to the viral RNA. Clone h 11Al-1 contains an entire presumptive AMV genome with flanking chicken sequences. putative AMV genome is approximately 4.8 md compared to approximately 5.25 md for its natural helper(s). The difference in size between AMV and its helpercs) is relatively small compared to other defective leukosis viruses relative to their helper(s), i.e., MC29 is approximately 3 . 4 md while its helper is approximately 5.2 md. The nature of the defectcs) in the AMV genome rendering it both apparently defective and leukemogenic is under investigation. We have analyzed the FNA genome of RadLV/VL , a highly oncogenic murine leukemia virus which is produced by a permanent cell line derived f ! o m a RadLV-induced thymic lymphoma of C57BL/Ka mice. Two distiict FNA components were found in the virions: a 705 dimer containing two 8 kb RNA subunits and a 545 dimer containing two 5.6 kb WAS. A non-oncogenic retrovirus, BL/Ka(B), endogenous in the same strain of mice, contains only 8 kb viral RNA subunits. We made long strands of RadLV/VL CDNA in the absence of actinomycin D. Two discrete DNA molecules, 9.0 kb and 5 . 6 kb in l&th were synthesized. fied 5.6 kb RadLV/VL RNA, followed by treatment with S1, we showed the t w o RNA subunits share 2.3 kb of thei! sequences.

BY hybridizing these DNA products with an excess of puri-Lily Sun and Thomas G. Kawakami. Comparative Oncology Laboratory, University of California, Davis, California, 95616. A type4 v i r u s isolated from a gibbon with myelogenous leukemia, GaLV-3M, is oncogenic for host animals since the same disease has been induced in several gibbons. A similar virus isolated from a clinically normal gibbon, GaLV-5, is not oncogenic for host animals since gibbons infected by this virus remained persistently viremic for several years without leukemia. between GaLV-3M and GaLV-5, we analyzed the genomes of these viruses by molecular hybridization. adsorption procedure using cellular DNA carrying proviral sequences of GaLV associated with various forms of leukemia. with myelogenous leukemia and designated as "WE". disease-specific "WE" sequences are present in genome of GaLV-5, the genome of GaLV-3M was adsorbed by cellular DNA carrying proviral sequences of GaLV-5 and separated into sequences homologous t o and sequences non-homologous to GaLV-5. can readily detect high levels (46-529) of complementary DNA sequences only in myelogenous leukemic tissues. non-homologous fraction of GaLV-3M and thus not in genome of GaLV-5. specific sequences in genome of GaLV-5 may account for its apparent non-oncogenicity so that animals infected by GaLV-5 remained persistently viremic without development of leukemia.

To define genetic factors that could be responsible for the biological differences

The genome of GaLV-3M was separated into four subgenomic fractions by a sequential One fraction was found to contain RNA sequences identifiable only

In an attempt to determine if these

The RNA of the non-homologous fraction This indicates that the major portion of the "MYE" RNA is contained in the The lack of the disease- yet, undetermined nature. Antisera specific for the FeLV and FeSV-specific domains of each of these polyproteins have been obtained from goats immunized with purified polyproteins or autologous FeSV-transformed cells as well as from tumor bearing animals. These antisera, along with tryptic peptide fingerprinting analysis techniques, have been utilized to characterize these FeSV-coded polyproteins. The SM-FeSV primary translational product is a 180,000 dalton polyprotein which is processed into an unstable 60,000 dalton molecule containing the p15-plZ-p30 fragment of the FeLV = gene-coded precursor protein and a 120,000 dalton FeSV-specific polypeptide. The GA-and ST-FeSV genomes code for relatively stable polyproteins (half-lives around 16 hours) of 95,000 and 85,000 daltons, respectively, which in addition to the amino terminal moiety, also contain FeSV specific polypeptides. latter appeared to be antigenically cross-reactive and exhibited common methionine-containing peptides. of the FeSV-coded polyproteins will be presented.

Finally, evidence indicating a cellular origin for the sarcoma-specific moiety 637 biology, University of Illinois, Urbana, Illinois 61801.

Studies of the synthesis and processing of viral proteins in simian sarcoma associated virus (SSAV)infected and SSV(SSAV)transformed marmoset and human cell lines has demonstrated polyprotein precursors precipitable by anti-SSAV p30 and anti-SSAV gp70. 60,000 dalton precursor polypeptide (pr60) and lesser amounts of 52,000 and 37,000 dalton polypeptides are precipitable from 35s-methionine-labeled cell extracts by anti-p30 serum Immunoprecipation by anti-gp70 revealed large amounts of intracellular gp70 precursor of 80.000 daltons. Precursorproduct relationships and post-translational modification of gag and env precursors will be discussed. infected with SSV in the absence of SSAV were evaluated for expression of virus-specific proteins precipitable by antisera prepared against SSAV p30, gp70 and tween-ether disrupted virus. No p30 and no polypeptides precipitable by anti-SSAV gp70 were detected in any of the cell lines. Polyprotein precursors precipitable by anti-SSAV p30 were identified in some HFISSV-NP clonal cell lines but absent in others. Results of studies comparing proteins precipitated from extracts of normal HF, untransformed HFlSSAV and HF/SSV-NP transformed cell lines by antisera from marmosets bearing SSV-induced tumors w i l l be discussed. Avian erythroblastosis virus (AEV) is an avian acute leukemia virus which can transform both bone marrow cells and fibroblasts in tissue culture. It has a 28s RNA genome. Previous data obtained from DNA.RNA hybridization, oligonucleotide fingerprinting and heteroduplex mapping showed that more than half of the 28s RNA represented AEV-specific transforminq gene sequences which occupied a 3.25 kb antiquous segment in the middle of the 6 kb AEV qenome. I n vitro translation of AEV RNA showed that these AEV-specific transformation sequences code for two proteins p7S and p40. unique peptides not shared with other viral proteins. Which of these two proteins is directly responsible for leukerngenesis is not clear.

variety of manmalian cells. The chrormsomal location of these sequences in the chicken cells will be discussed. This protein has been found in the cytoplasm of RSV transformed cells, at gap junctions between tumor cells, and as a perinuclear spot. In addition to these patterns, we &?e maw found by indirect immunofluorescence using rabbit anti-tumor serum specific for pp60 , a speckled pattern of fluorescence on the ventral surface of RSV (strain SR-D) transformed NRK cells (SR-NRK). This pattern, as well as all others, was not seen when normal serum was substituted for anti-tumor serum. Likewise anti-tumor serum $!$ not stain uninfected NRK cells. Interference reflection microscopy has been used to analyze the pp60 This technique has been used to visualize the adhesion plaque or points at which cells attach to a substratum.

Simultaneous immyppuorescence and interference reflection microscopy has demonstrated that each speckle of pp60 specific fluorescence on the ventral surface of the SR-NRK cells corresponds to an adhesion plaque. In addition we have shown that SR-NRK cells could be removed from the glass surface leaving behind the adhesion &qws or "feet" still attached to the glass substrate. These isolateds;Jeet" stain specifically for pp60 by indirect immunofluorescence. The RSV src gene pmduct (pp60 ) also has been observed in adhesion plaque of SR-D transformed CEC and B&/c fibroblasts as well as tsLA91 infected rat cells grown at the permissive t e m g p t u r e . When the tsLA91 infected rat cells were maintained at the nonpermissive temperature, pp60

was not observed in the adhesion plaques. Adhesion plaques or cellul.a&feet" appear to act as focal points of microfilament attachment and our observations swgest that pp60

fluorescence on the SR-NRK ventral surface.

may affect cellular stress fiber integrity by acting at the adhesion plaques. properties has led to the isolation of nonproducer transformed cells in which the new cell derived transforming functions are stably expressed. produced by the transforming viruses i n the nonproducer cells will be described. Supernatants from cultures have been quantitatively analyzed for albumin, alpha-fetoprotein (AFP) and HBsAg during the phases of cell growth. thesized with the amount in the culture supernatant related to cell number and length of time in contact with the cells. AFP is continuously synthesized but its rate of production increases during late log phase, paralleling HBsAg synthesis. In contrast, HRsAg is n o t detect able during the lag or early log phases of growth, but first becomes detectable in the supernatant and cells during late log phase. during this phase and reaches a peak near the end of the culture's growth. synthesis will be discussed in terms of cell density, cell contact and regulation through the culture supernatant fluid. as to polypeptides present. of this tumor-derived line and somatic cell hybrids between murine cells and this line is under investigation. Results are compared to the PLC/PRF/5 line, which is the.only other known line that produces HBsAg. Digestion of intact Sindbis virions with a-chmotrynsin gives rise t o a particle of lichter density contninine: two protense-resj s t a n t frapnents of the glycoproteins embedded in the lipid bilayer as well ss the intnct nucleocnpsid. The npmrent moleculPr weights of these frnmments nre 10.000 m d 5.000 dsltons. TM-dimensional thin 1s.yer as well as high pressure ljquid chromntographic techniques for sepnration of tryptic and/or chymotrypic digests of these framents have shown that the IOK dalton frament is derived from E2 (IiE2). and the 5% i?alton frament from El (PF1). ln n two-phase, aqueous-orgnnic system at neutral p!i. RE1 is nuch more soluble (sit-fold) in the orpnj.c phase than is PE2. both fragments are rich in hydrophobic amino acids, but ?El has R larger proportion of hydrophobic residues then AE2. harvesting only the v i r u s r e l e a s e d w i t h i n t h e f i r s t two hours following t h e end of t h e l a t e n t period. An a t t e n u a t e d v a r i a n t (SB-RL) was cloned from t h e eleventh such passage. Gne hundred percent m o r t a l i t y was observed i n l i t t e r s of one day o l d mice i n j e c t e d S . C . with 25 o r more PFU of SB. In SB-RL i n f e c t e d l i t t e r s , t h e s u r v i v a l r a t e decreased from 60 t o 30% with doses ranging from 2 . 5 t o 2 . 5~1 0~ PFU/mouse. decreased w i t h i n c r e a s i n g dose, while t h i s parameter i n SB i n f e c t e d mice was independent of dose. SB-RL was found t o p r o t e c t mice from challenge with SB. In t i s s u e c u l t u r e , SB-RL w a s characterized by a s i g n i f i c a n t reduction i n l a t e n t period and an increased rate of penetration. Cell clones obtained fromtwo linesof cultured cells persistently infected with Japanese encephalitis virus (JEV) initially neither expressed viral antigen nor released infectious virus but retained viral RNA. able from parent persistent infections (PI): i.e. showed no viral CPE. expressed viral antigen in most cells and released low levels of infectious virus which suggested replication interference. Superinfection of clones with a temperature sensitive (ts) mutant of JEV at the permissive temperature resulted in production only of ts mutant JEV indicating no activation of latent virus. Continued passage of clones resulted in spontaneous release of infectious virus fran some non-superinfected clones. Superinfected non-releaser or spontaneous releaser clones examined by EM showed little evidence of viral replication even when all cells expressed viral antigen. clones indicated that only the two small subgenomic W A S (12 and 15s) were produced in large amounts while spontaneous releasers and superinfected clones produced genome size (405) RNA as well but in lesser amounts than w t JEV control infections. The RNA patterns observed are similar to the early events in wt JEV replication and suggest that interference with virus replication occurs at the level of genome-length RNA synthesis. 126 and 6811201s in an attempt to localize the genetic basis of the mutant's attenuated virulence. was shown t o differ from the parent virus with respect to virion structure-dependent, particularly surface structure-dependent, characteristics: temperature lability. plaque sizes in Vero cells, and btnding properties to hydroxylapatite. of all these phenotypic differences was demonstrated by the isolation of a number of independently-arising, stable genetic revertants of 126. These revertants exhibit rhararteristics identical in every respect to 6811201 s.

indicates that the genetic lesion responsible for the loss of virulence by g 126 is most

probably located in the structural gene(s) coding for one of the two virion surface glycoproteins.

We describe here further phenotypic differences between 126

The common genetic basis The virions were dissociated and iodinated with lZ5I.

Four structural proteins were obsenred with molecular weights of 44,000, Rubella virus has been purified using 2 5 4 5 % discontinuous and 30-45% continuous renografin gradients. labeled proteins were then subjected to polyacrylamide slab gel electrophoresis and autoradiography. 41,000, 24,000 and 19,000 daltons, and a total molecular weight of 128.000 daltons. These data are not in close agreement with previous reports which have employed different purification and labeling technics. itivity afforded by the technics described here provide a more accurate analysis of the structural proteins of Rubella virus than has been possible previously. Rubella virus has been propagated i n murine f i b r o b l a s t s (L c e l l s ) and purified using two renografin gradients. The virus was grown i n the presence of 2 pCi/ml 3H-uridine, pelleted from t i s s u e culture media 5 days post-infection and applied t o a 25-45% discontinuous gradient. A single, sharp band was observed a t the interface. This band was collected and applied t o a 30-45% continuous gradient which separated i n t a c t labeled virus from a large amount of 3H-labeled, l i g h t density material. degree of separation.

good yields of infectious virus from t i s s u e culture. The yield appeared t o depend on both the v i r a l stock and the type of cell culture employed. (greater than lo7 PFU/ml) were never obtained. consistent plaque formation with any cell c u l t u r e tested. Therefore, hemagglutination (HA) and enzyme-linked immunosorbent assays (ELISA) and d i r e t microscopic examination were employed t o d e t e c t the virus during purification. The H-labeled l i g h t density peak contained only ELISA a c t i v i t y and no i n t a c t virions were found upon microscopic examination. A second 3H-labeled peak with a density of 1.19 9m/cm3 demonstrated both HA and ELISA a c t i v i t i e s and upon microscopic examination was found t o consist primarily of Rubella virus. was digested w i t h T~ ~~a s e and the ribosanes w i t h protected oii-qiiGFGtides were isolated on a glycerol gradimt. After deproteinization, the olig~nucleotides were analyzed by two dimnsio~l polyacrylamide gel electrophoresis. to be protected by ribo-s &iereas other fragmnts were protected by mn-ribosanal prote-Secordary digests by T and pancreatic RNase follcved by two dimensional gel e l e c t r q h r e s i s d c m n s t r a w that eadilof these three r -protected oligonw~eotides was a distinct specie. digestion w i t h FWse I11 ard p l y A-Oontaining fragmnts selected by p l y U sepharose affinity chraratography. In vitru translation of different size classes of fra-ts s h c~e d that proteins ccu~Te7iZS fran +sm olipnwleotides ~c h were app&ely 0.55 and 0.92 m p units fran the 5' end, as ell as fmn the full length genom. Corroborative evidence for ribbinding a t three sites was obtained by electron mianscope studies. 

LDV replicates rapidly in all strains of mice so far tested producing a persistent infection. A relatively high level of infectivity is continually present in the blood o f infected mice, but no disease symptoms are observed. Recently,we have isolated a variant of LDV which was associated with an Ib leukemia cell line that had been passaged in C58 mice for about 2 0 years. This LDV strain (C58-LDV) is able to produce a polioencephalomyelitis in C58 mice. Unique characteristics of both the host and the virus strain are required for the production o f this paralysis. LDV's have been previously isolated in a number of laboratories. but no technical methods existed for distinguishing differences between these isolates. The assessment of the ability to induce paralysis i n C58 mice and the measurement of antigenic crossreactivity by R I A have provided initial means for characterization. Persistently infected mice circulate virus complexed with antiviral antibody. These complexes are infectious. We have found that challenge of persistently infected mice with homologous virus results in an increase in the level of antiviral antibody. The C58-LDV appears to be more immunogenic than other LDV's, since it elicits a 4D-fold increase in antibody as compared to a 5-fold increase elicited by other LDV's. We have recently observed that C58-LDV i s capable of replicating in neurons as assessed by EM analysis of spinal cord lesions in C58 mice and by infection of CNS cells in culture. Macrophage-like cells had been thought to be the only cells replicating LDV. Levels of infectivity and antiviral Ab in the CSF are being evaluated to determine the abilities of the various LDV strains to enter the CNS of C58 and control mice.

SCRAF'IE--AN EXPIANATION FOR ITS EXTREME RESISTANCE TO RADIATION, Robert G. Rohwer,

The infectious ,agent of scrapie disease is two times more resistant to inactivation hv ionizing radiation than are the most resistant viruses yet characterized in this wav. Computation of its rediation target size using conventional assumptions suggests a suhviral nucleic acid size of only 60,000 to 15I7,OOO daltons. Values at least this small are i n d lcated for the related human diseases, kuru and Creutzfeldt-Jakoh Disease.

The% small target sizes have led to the suggestton that these diseases are caused hy viroids or Pven novel mechanisms which do not involve nucleic acids. However, unlike the vfroid agents, scrapie infectivity is very sensitive to phenol extraction. Also, other physical methods such as filtration, sedimentation and bouyant density suggest virus-like properties for the infectious agent.

One solution to this paradox is a model in which the titratable infectious unit o f these diseases exists as an aggregate of several competent viruse particles, all of which must be inactivated before the infectious unit itself is inactivated. Depending upon the size distribution of these presumed aggregates, the inacttvatlon curve observed for the scrapie agent can be fit exactly by conventionally sized viruses.

Ample qualatative evidence for aggregation of the sort required in this model exists in the scrapie literature. In an attempt to quantitate this phenomenon. This suggested t h a t the cT-ag phenotype was dominant over the nT-ag phenotype. The dominance of t h e cT-ag phenotype was also manifest in ceUs co-infected with PARA(cT) and either SV40 or WT PARA, both of which induce nT-ag during single infection. Two lines of evidence indicate t h a t t h e absence of nuclear T-ag reactivity in co-infected cells is due to a failure of migration of WT T-ag into t h e nucleus, rather than an inhibition of its synthesis. First, co-infection of cells with SV40 and either helper adenovirus or PARA-ademvirus populations, at the multiplicities of infection employed in these studies, did not reduce the yields of infectious SV40. Second, cells co-infected with PARA(cT) and deletion mutants of SV40 which encode T-ag polypeptides of reduced molecular weight expressed t h e cT-ag phenotype, and t h e presence of the deleted forms of T-ag was confirmed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis.

The dominant e f f e c t of t h e c T mutation was specific for transport of SV40 7-ag, since normal migration of adenovirus tumor and virion antigens as well as SV40 virion antigen occurred.

The early region of polyoma virus encodes three tumor antigens of molecular weights 8 8 K , 51K, and 25K daltons. The coding regions for the three T antigens have been analyzed by comparing the tryptic peptides of the isolated proteins to the nucleotide sequence of polyoma DNA. A protein kinase activity, having the unusual specificity of phosphorylating tyrosine, is associated with polyoma T antigen immunoprecipitates. We are studying the effect of Sixty-five clones of rat cells (Fisher F-111 and NFX) obtained by mixed infections between hr-t (NG-18, 11-5, SD-15) and ts-a (ts-25D, ts-616, ts-48, ts-a) mutants have been studied in terms of the T-antigens expressed and the nature of the virus obtained by fusion with permissive cells or revealed by Southern blotting. Viral proteins are expressed both from the hr-t and ts-a alleles. All clones analyzed express middle and little t-antigens in both Fisher and NFZ clones. Thirty percent of NRK and about 10% of the Fisher clones have either no or a thermolabile expression of large T-antigen. Virus can be rescued from the majority of the Fisher clones and from a minority of the NRK clones. Both hr-t and ts-a alleles are rescued. In addition, wild type recombinants are observed in the majority of the Fisher rat clones. are testing the hypothesis that wild type genomes arise by packaging DNA from concatemers containing hr-t and ts-a alleles in tandem configuration. The SV40 tsA58 transformed m u s e clone A21 displays a temperature sens&tive transformed phenotype, but colonies can be obtained in soft agar at 40 C wlth a frequency of less than 1%. Clonal derivatives of A21, isolated by growth in soft agar at 4OoC, were analyzed by restriction endonuclease cleavage and Southern blot hybridization. In 10 of 12 clones examined, the conversion of A21 cells to anchorage independent growth at 4OoC is accompanied by the acquisition of new sites of integrated SV40. By using the Bgl I1 endonuclease which lacks specificity for SV40, it was shown that the parental A21 clone contains SV40 sequences in 9 kb and 15 kb fragments of cellular DNA. clonal derivatives generally retain the 9 kb and 15 kb integration sites but possess additional SV40 insertions within Bgl I1 fragments of 15 to 25 kb. The viral DNA monomer in both the 9 kb and 15 kb Bgl I1 inserts is a deletion fragment of 3 . 8 kb which is arranged in a tandem head to tail fashion in the cellular DNA. These results suqgest that the change in biological expression of the transformed phenotype may be accompanied by the rearrangement of integrated SV40 specific sequences.

Cell transformation with bovine papilloma virus (BPV) and BPV DNA is described. Primary hamster embryo cells were obtained by trypsinization of minced embryonic tissue from Golden Syrian hamsters (Graffi or LVG strains) and cell cultures established. Confluent cell cultures were treated with bromodeoxyuridine for about 24 hours prior to infection with BPV or treatment with calcium phosphate-precipitated BPV DNA. Other cells were mock-infected with plain Dulbecco's medium to serve as controls. Infected cells underwent striking mrphological changes during the next few weeks; long, spindle-shaped cells became predominant. These cells grew in patterns resembling foci, thus they were tested for anchorage-indendent growth in dilute agarose containing medium. Colonies of these cells became'evident in 5-6 weeks. Colonies were removed from the agarose and transferred to dishes of liquid medium. Using cotransformation with the HSV-1 thymidine kinase (tk) gene, we have constructed several cell lines that contain and express different segments of the genome of adenovirus type 2 (Adz). Five lines were derived from the human tk-143 cell line (gift of C. Croce) which are both tk+ and support the growth of an adenovirus type 5 deletion mutant (d1312) that lacks the DNA sequences located between coordinates 1 and 4.5. Although five lines maintain the Ad2 sequences, their expression is unstable. Two lines lost their ability to complement d1312 after several generations even while maintained under selective pressure for tk expression. three lines continue to support d1312's growth after more than 150 generations although some clones derived from them show different levels of complementation. its transcription will be discussed as will attempts to construct cells expressing other regions of the viral genome.

The viral DNA present in these cells and

A 1.5 kilobase DNA fragment carrying the rat insulin I gene, including its intervening and adjacent sequences, has been cloned in the late gene region of the monkey virus SV40. Two types of hybrid viruses have been constructed, with the rat insulin gene inserted either in the same orientation as the SV40 late protein coding sequence, or in the inverted orientation. Thus in the latter case the expression of the rat insulin gene should be under the control of its own transcription promoter whereas in the former case transcription could occur from both the viral late region promoter and the rat insulin promoter. by the analysis of rat insulin RNA and protein synthesized in monkey kidney cells infected with a helper phage and either type of hybrid virus. Our interest in developing SV40 derivatives as vectors for eukaryotic cells has led to the construction of the recombinant vector pSV2. segment containing the origin of replication and the amp region. Hindltt site (or between Hind111 and BgIII sites) about 70 base pairs from the SV40 origin/ early promoter. We have inserted several eukaryotic cDNA and prokaryotic coding segments into pSV2 to generate "secondary" recombinants (pSV2-X). After propagation i n E. coli tnese may be transferred into eukaryotic cells, where the SV40 early region provides promoter, splicing sequences, and termination/polyadenylation site(s) for the generation of insert-specific mKNA.

insert coding sequence in monkey, human, and mouse cellsi.e. cells in which the SV40 early promoter is constitutively transcribed.

pSV2 is a hybrid between PBR322 (a 2 . 3 Kb gene) and a modified SV40 early Foreign UNA sequences may be inserted into the SV40 portion of pSV2 at a unique When sucrose gradients used to purify the virion core are assayed for transcriptional activity, all of the template activity co-sediments with the virion core. plexes are formed in vitro and then RNA synthesis is allowed to proceed for increasing lengths of time, the-labeled RNA at first co-sediments with 110s virion core; as the length of transcription time Increases, a progressive increase in the sedimentation of the SV40 cores occur, due to the growth of RNA chains. as a fairly homogeneous species with a sedimentation value of 16-18s. analysis indicates that no loss of protein occurs during the process of transcription. rate of incorporation of ribonucleoside triphosphates into acid insoluble RNA with SV40 cores as the template is 7045% of that obtained with naked supercoiled SV40 DNA. SV40 minichromosomes, under identical transcription assay conditions, have an incorporation rate which is 20% of that obtained with naked SV40 DNA. These results provide the first suggestive evidence that the "late" viral proteins of SV40 may play a role in the regulation of expression of the SV40 genome.

Under optimal conditions 95-100% of the SV40 nucleoprotein cores are able to form RNA polymerase. We have characterized a small RNA (approximately 70 nucleotides) which is induced late in SV40 lytic Infection. This SV40-associated small RNA (SAS-RNA) is specific in size and sequence and is not selected by oligo(dT)-cellulose. The SAS-RNA is complementary to the viral early mRNAs (and the late DNA strand) at a point 260 nucleotides from the 3'end of the early mRNAs (0.21 map units) and i s homologous to at least 50 nucleotldes in this region. This area of the genome is interesting because it marks the start of a second open translational reading frame at the carboxy terminal end of T-antigen. It is also the region deleted by ts1499, a deletion mutant which is temperature sensitive for growth but cold sensitive for transformation (Pintel s. 1979, CSHSQB 44, in press). The function of the SAS-RNA in the viral cycle and its source (host or viral) are unknown at this point; however its temporal expression, unique sequence and its interesting region of homology on the early mRNAs (or the viral genome) suggest a potential role in SV40 gene expression. *Yale Univ. Sch. of FIed., New Haven, Ct. 06510 and **Univ. of I l l . Med. Ctr., Chicago, Ill. 60680.

Utilizing the transcription of S W O as a model system to study the formation and function of leader sequences in m a l i a n m W , we have examined the structure of late viral mRNAs produced in cells infected with each of 3 viable deletion mutants of W d O .

These mutants, dl 1659, dl 1626 and dl 1613, lack non-werlapping s e w n t s of the IXiA encoding the major leaders of late Sr40 mRNA. ing the reverse transcriptase catalyzed extension of s h o r t SV40 DNA fragments hybridized at specific points in t k FNA sequence. direct the of synthesis 1ES and 1% mJNAs, each type containing splices found in wildtype &NA. Cicantly from that of wild-type m A s . which are ercodcd by DYA sequences lying upstream from DNA encoding the major 5'-termini of wild-type mRNA. A relationship between the selection of 5'-termini and the lengths of the late leaders is suggested. wild-type 19s mlwAs, we have found striking differences i n the ratio bf mutant 19s mRNAs which conCain one of the three wild-type splices to those which lack a splice upstream from the b d y of the message. Cells infected by the mutants dl 165Q and dl 1626 produce an unusually high proportion of the "unspliced" forms of 10s RNA. these "unspliced" forms of lqS FXiA contain SOM internal or 3 s i s t e n t w i t h the hypothesis t h a t v a r i a n t 3049 has an a l t e r e d s p l i c i n g mechanism f o r l a t e messenger RNA.

The v a r i a n t phenotype was termed CyCt.

Unique

The DNA between coordinates 45.0 and W e i n t e r p r e t these and other data as being con-687 polis, Indiana 4 6 2 2 3 . While knowledge of the steps involved in synthesis of papovavirus DNA is quite sophisticated, relatively little is known of the factors determining whether newly synthesized DNA is used as a template for further DNA synthesis (re-enters replicatiodor whether it becomes encapsidated. geny polyoma DNA is greatest early in the infectious cycle. However, regardless of the time post-infection examined, progeny molecules appear to be removed from the replicating pool approximately three hours after their initial synthesis. lication whether encapaidation of pulse-labeled DNA is the direct cause of cessation of re-entry we are examining the kinetics of encapsidation. Preliminary data suggest that as the fraction of pulse-labeled SV40 DNA in previrions and virions increases the fraction of pulse-labeled DNA which re-enters replication decreases. Early in the infectious cycle a smaller percentage of pulse-labeled DNA becomes encapsidated during a six hour chase than late in the cycle. In addition, pulse-labeled DNA is more rapidly encapsidated late in infection. The data suggest that the factor(s) removing viral DNA from the replicating pool (causing cessation of re-entry) is involved in virus maturation.

We have shown previously that the rate and extent of re-entry of pulse-labeled pro-Pulse-labeled SV40 DNA re-enters repover the same time period and then similarly ceases to re-enter. To determine early in the infectious cycle; s a p y e relatioyhiw betieen the region 1 plypeptides translated in vitro has been detenlurd by l-dunens i d psptide mapiny. related and mrrespnd to proteins isolated fran infected cell extracta. proteins frun region 1E share M mthionine peptides. identical to virion polypeptide IX and shares no mthionine pepticks with 52K 01 15K.

2 early mRNA4 frun region 1A. the aryinine analcg -mine.

Prelimimry eqerimmts have rZZledhiyher mlec~lar .

weight polypzptide pmducts, suyyestimj that formation of these peptides may include a proteolytic processing event.

Filled -s represent mWA8 and pro& ?he all spies are made at late times.

All 5 polypeptides f r u n -G IA are highly The 12.% pmdmzt fran region 1B is 

In each case the identity of the mRNA was defined

The significance of this will be discussed. C57BL mice share P s t l r e s t r i c t i o n fragments a t 3.3, 1.1, 0.9 and 0.5 megadaltons i n common.

(C57BL x GR)F 3 backcross mice. i.e., 50 percent of t h e livers examined contain P s t l fragments a t 3.5 and 0.6 megadaltons. presence of these MMTV(GR) s p e c i f i c P s t l fragments with t h e occurrence of e a r l y mammary tumgrs i n t h e [C57BL x (C57BL x GR)F1l backcross mice. u t i l i t y of r e s t r i c t i o n mapping i n t h e diagnosis of a n a t u r a l l y occurring neoplasm.

incidence.

The GR and Fragments cont a i n i n g the v i r u s -c e l l " j o i n t s " a t t h e 3' end of t h e integrated genomes were prepared and subjected t o sequence analysis. These sequences were compared w i t h the 3' end o f unintegrated MMTV DNA as inferred from t h e MMTV "strong-stop" sequence determined by Lovinger and Schochetman (personal comnunication). Interferon (IF) affects the production of murine leukemia viruses in chronically infected cells. In some systems, IF treatment inhibited virus release while in others, the virions released were deficient in infectivity. To determine whether the observed differences in IF effect were dependent on the infecting virus or the host cell, a clone of Maloney murine leukemia virus (M-MuLV) was used to establish persistent infections in several mouse cell lines. All of these cell lines were capable of establishing an antiviral state, as shown by resistance to challenge by EMC virus in the standard CPE test after IF treatment. Our data indicated that IF treatment had no effect on the production of M-MuLV in NIH 3T3 cells, as determined by reverse transcriptase activity and infectivity of virus released into the culture fluid. cells resulted in significant decreases in the reverse transcriptase activity and infectivity of released virions. Additionally, preliminary data indicated that low levels of non-infectious particles were released from one of these clones after IF treatment. The We have developed a technique for the isolation and characterization of newly transcribed murine leuke ia viral RNA in chronically infected cells. Cellular RNA was pulse labeled with H-uridine and virus specific sequences were annealed with an excess of mercurated complementary DNA. Based on the affinity between mercurated cDNA and sulfhydryl-Sepharose. the hybrid was specifically selected by affinity column chromatography. The specificity of this method was dependent on the purity of the cDNA, and it was necessary to remove non-viral sequences from the cDNA in order to isolate virus-specific RNA. The kinetics of cytoplasmic viral DNA synthesis was followed by thymidine labeling of MLV-infected cells. An increased rate of synthesis is observed up to 3.5 hours after infection followed by a decrease. Infection of interferon-treated cells results in a reduced rate of viral DNA synthesis. Preliminary results show that a cellular factor, probably a protein, is involved in the synthesis of viral DNA. Its amount is increased in infected cells.

Size analysis on the polyribosomal viral RNA indicates that 38s temperature-sensitive and deletion mutants. been able to study the complementation patterns between various mutant and wild-type viruses. We have obtained genetic evidence for a virally coded, trans-acting factor involved in the proteolytic processing of prCigag. We have also found that protease treatment of intact cells can effect a bypass of the maturation and assembly block in a late temperature-sensitive mutant of MuLV. Finally, we are investigating various polymerase mutants of F L V , both those with thennolabile enzyme activities and those defective in prlda'-po processing.

clones. their nucleic acid inheritance. recombinant viruses has been investigated. Preliminary results indicate that these experiments will aid in mapping the viral functions that determine the target cells and organs of the MuLV-induced leukemias.

We have investigated the steps involved in murine retrovirus maturation using By analysis of doubly infected clones, we have 2 , 86-93) . We will present the results of cloning experiments using strong-stop DNA, designed for the large scale isolation of avian tumor virus specific mRNAs from infected and uninfected cells. We present an analysis of the structure of RSV genomic and RSV-specific 35s polysomal RNA with respect to the 5'-end, up to and including the initiation site for GAG protein synthesis. The methodology employed for sequence analysls and ribosome interaction in vitro with the 5'-end of genomic and 355 poly-soma1 RNA will be r e p o r t e m m p l i c a t i o n of the 5'-end sequence arrangement on the nature of in vitro translation products immunologically related to the GAG protein will also be discussed. 

The messenger activities Of 70s virion RNAs from a variety of avian leukosissarcoma viruses have been examined by in vitro translation. A polyadenylated 18s RNA which codes for a polypeptide of 34,000 daltons has been found in all non-defective sarcoma viruses analyzed. It is the only major messenger activity other than the 355 genomic RNA found in a transformation defective deletion mutant. has revealed that the 34K proteins synthesized in response to different virion RNAs are identical. RNA from uninfected SPAFAS gs-chf-chick embryo fibroblasts contains an abundant messenger activity for a polypeptide which comigrates in SDS polyacrylamide gels with the 34,000 dalton protein made in response to virion RNA. The tryptic peptide map of this 34K protein is identical with the virion RNA 34K protein. In addition, the uninfected cell RNA 34K shares 3 methionine containing tryptic peptides with Pr76, the precursor to the virion internal structural proteins. These observations suggest that the 34K messenger activity found in 705 virion RNA is the result of selective packaging of an abundant host cell mRNA. We are investigating the possibility that this RNA represents-a transcript of a deleted endogenous viral genome. Analysis by two-dimensional isoelectric focusing--SDS polyacrylamide gel electrophoresis of MuLV proteins revealed interstrain and intrastrain heterogeneity of the major core protein p30. All viruses analyzed had one mgjor and at least one minor p30 component, with regard to isoelectric point (PI Minor 6.7 6.3, 6.2, 6.0 6.5 6.3' 6.7 6.6, 6.3. 6.2 6.7 6.3 6.3 6.1 6.3 6.1 6.3 6.1 6.3 6.1 7.1 6.7, 6 . 4 , 6.3

The p30's of 8-tropic and xenotropic viruses have similar PI'S, and lack the more basic components present in N-tropic and NB-tropic viruses. Though only a limited number of viruses have been analyzed the pattern that has emerged supports recent evidence su gestin a relationship between Fv-1 [rapism and p30, and the possible recombinational origin of some #-tropic viruses from xenotropic viruses.

CROSS-REACTING NEUTRALIZING SITES BY THE USE OF MONOCLONAL ANTIBODY Lenore P. Pereira and J. Richard baringer, Veterans Administration Medical Center, San Francisco, CA Monoclonal antibody with neutralizing activity for HSV-1 and HSV-2 was used to identify the viral glycoproteins which bind neutralizing antibody. antibody of the IgG o r IgG subclass were cloned. In comparative reactions they were found to differ in their &eutraliffng activity for HSV-1 and HSV-2. Three clones were characterized by antibody which reacted with HSV-1 while two clones produced antibody which neutralized both type 1 and type 2. Fluorescent staining reactions showed that the binding of monoclonal antibody to antigens expressed on the surface of HSV-infected cells correlated with the pattern of neutralization. In immune precipitation reactions with soluble antigen preparations, two glycoproteins were precipited by monoclonal antibody ofdifferent site specificities. Glycoprotein 2 (137,000 MU) specifies neutralizing sites which are specific for type l. In contrast, 2glycoptotein D (55,000 MW) specifies neutralizing determinants which are shared by type 1 and type 2. bind neutralizing antibody specific for HSV-I.

Five hybrids which produced neutralizing

In aidifion this glycoprotein also appears to specify determinants which 1, 2 , and 3) have been subjected to both O'Farrell two-dimensional analysis of virion proteins and restriction enzyme analysis of the viral DNA in an attempt to determine the genetic relationship between the three herpesvirus types and the degree of variation within each type. proteins and/or restriction fragments and the tropism of the herpesvirus type, which may be useful in future epidemiological studies. Individual isolates were found to be quite stable, showing no detectible changes over several passages in either cows (following reactivation of the latent virus) or tissue cultures. certain loci may have a comparatively high proclivity for successful mutation. having the greatest apparent variation among the isolates are being analyzed as to their location and function i n the virion.

This approach allows a preliminary correlation between specific Differences between isotypic isolates suggest that

The proteins 725 several properties. Of special interest are those which are mnsense mutants as shmm by their suppressibility in vitro with yeast suppressor m. Hmun cykmegalovirus (HcWrr). one of the herpenvirusas. is medically signiffcurt both an a cause of birth defects ud as a source of problems in bmosuppressed individuals.

Becmse the virus is highly call-associated, it i s difficult to obtrin luge quantities of pure viral DNA. AclIv genome to aid in further studies of the molecular biology of HCW infaotions. Approximately 9,000 colonies were selected on the basis of tetracycline resistance pnd chloramphenicol sensitivity. plamids, the cloned fragments were analyzed for co-mieration on a g m s e gels w i t h authentic Eco R I di ested HcMl ENA. The viral origin of the fr.gmsnts was deterpined by hybridization of !b HCHV DNA to Southern blots of the inserts. To date, we have an& ly?.ed 80 clones md the viral inserts obtained represent at least 70$ of the genome. We are currently constructing a physical map of the genme for ordering the fragments.

For thls reaaon, we have sought to construct a cloned library of the The cloning was acconplished as follows:

After isolation and Ew RI cleavsge of the recmbinant

Cellular and viral antigens appearinq after human CMV infection were studied in the presence of phosphonoformate (PFA). Complete Inhibition of CMV replication was obtained at 500 uM, 50% plaque reduction at 100 pM PFA. Early nuclear CMV antigens (EA) \?ere nbtinhibited, but the formation of nuclear inclusion bodies, late cytoplasmic antigens (LA), Fc-receptors and cytopathic effects were inhibited by PFA.

By microimmunofluorometry, antigens of cells in different stages of CMV infection were quantified. In naturally aborted infection, the largest amount of EA was seen between days 3-5. In PFA treated cells, high EA levels persisted up to day 14, by 35 days it was hardly measurable. Late antigens and complete infectious virus appeared delayed after PFA had been deleted. It seems possible to induce an artificial latent CMV infection of all infected cells by the use of PFA. by electrophoresis.

EcoRI, 12 for Hind I l l , 15 for Bam HI, 4 for Pvu I and 10 for Sal I . The total molecular weight of the fragments ranges from 94 to 107 x lo6 daltons.

A exonuclease prior to endonuclease digestion. EcoRl A and J fragments and Hind I l l A and H fragments were found to be sensitive to 4 exonuciease. Furthermre, Southern hybridization showed that the EcoRI A fragment hybridized to the Hind I f 1 H fragment and EcoRI J to Hind Additional higher molecular weight RNAs have been detected and mapped by the above blotting technique as well as by el electrophoretic analysis of nuclease S1-resistant hybrids formed by annealing of RNA to 32P-labeled DNA strands. The cloned region of the genome appears to encode few mRNAs for specific late proteins. One of them has been mapped and appears to be quite heterogeneous in length. common property of many late mRNAs. Models for the generation of high molecular weight and variable length RNAs will be discussed.

Of interest is the apparent lack of transcription from the Additional data suggest that this heterogeneity may be a Virol. 29:1044-1055) and are currently constructing a genetic map by marker rescue of mutant genomes with wild-type restriction fragments. ts for occlusion, marker rescue involves distinguishing plaques with and without occluded viruses. I n addition to their basic virological interest, baculoviruses are currently the only viruses with potential as vectors for transducing invertebrate cells and a knowledge of gene organization will be valuable from this perspective. should be an excellent site for inserting passenger DNA since the Virus will be defective in transmission in nature. The partial denaturation and restriction enzyme maps of the different strains show great similarities and do not favor the idea, that different viralstrdhs are associated with different diseases. All strains showed variations in the number of internal repeats, the number of repeats being not even constant and stable within one viral strain. Adjacent to the internal repeats a sequence carrying a HindIII site is deleted in the nontransforming P3HR-1 strain. This region of the genome will be preferentially analyzed for transforming genes.

Additional variations between different strains were observed in the Hind111 4 and the HindIII E fragments at the right hand site of the EBV genome. Dinucleotide spacers (CA or GA) whose complements do not appear in the mRNAs, follow the polyadenylation signals and constitute the intergenic regions. Imediately after these dinucleotides are the sequences complementary to the 5'-tenninal sequences on each mRNA.

The amino acid sequence of the COOH-terminus of the VSV glycoprotein was determined from the sequence of a recombinant plasmid. flanked by basic residues.

Extensive homologies were found among the four junctions of the five VSV genes. 

Serial undiluted passage of Vesicular Stomatitis Virus (VSV) has been shown to generate defective interfering (DI) particles which contain the same proteins and RNA of the same polarity as the wild-type (wt) virus but are smaller and incapable of self-replication. Replication of DI particles requires co-infection with the wt VSV. The D I particles are capable of synthesizing in vitro only a 46 nucleotide long RNA which is different in sequence from the leader RNA Synthesized in vitro by wt VSV. obtained from a heat-resistant strain of VSV (DI-LT) that can transcribe the leader RNA and mRNA species coding for N,NS, M, and G. The former DI particles arise from the )'-terminal portion of the wt genome RNA and the latter from the 3'-terminal half.

In this report we show that when the purified cores prepared from three different DI Particles including DI-LT were pre-incubated with ATP and CTP, reisolated and subsequently incubated in the presence of the 6-y imido analogue of ATP and three other normal ribonucleoside triphosphates, the full-length complementary strands of the DI RNAs were synthesized. Using a similar approach we have also synthesized the full-length complement of the wt genome RNA in vitro. the precise length of deletion was measured by gel electrophoresis of the ribonuclease treated heteroduplex.

The exception is the DI particle 

The proteins in vesicular stomatitis virus particles are heterogeneous in electric charge when examined by isoelectric focusing, reflecting post-translational modifications. To assess the functional relevance of these changes, we compared free, nucleocapsid-associated, and membrane-associated viral proteins from infected cells with the proteins in virions. The major nucleocapsid protein, N, exhibited increasing numbers of electronegative variants as it moved through nucleocapsids into virions. In contrast, the transcriptase-associated protein, NS, was more heterogeneous in the free pool than in virions or intracellular nucleocapsids, due to loss of an electronegative species. Lastly, phosphorylated forms of the G , N, and M proteins were detected in virions, but not in infected cells. Thus, a complex series of post-translational modifications accompanies (and may regulate) stages in maturation of the virus. 

virus when the animals were less than one day old. Disease appeared from two months to one year after infection and was characterized pathologically by mononuclear cell infiltration, gliosis, and demyelination. Usually the disease was slowly progressive with prominent myoclonus of the hind limbs. In some animals the disease occurred as episodic paresis of the hind limbs with near total recovery between periods of paralysis. Virus could not be demonstrated in lesions by immunofluorescence or isolated by cocultivatlon of spinal cord with monkey kidney cells. The strain of virus used was distinctive in that it contained high levels of a naturally occurring viral variant, differing from typlcal measles in several ways. In cultures of monkey kidney cells the variant induces cell rounding and swelling rather than the usual measles syncytia formation. In acutely infected neonate hamster brain the variant grows less readily than does the syncytiagenic form of the virus and is less encephalitogenic. The variant has a decreased buoyant density in potassium tartrate gradients compared to typical measles, yet it expresses the same major structural polypeptides. However, the relative amounts of the two viral envelope glycoproteins are altered in the variant. The variant can be cloned and titered independently of the syncytiagenic type of virus, and is therefore unlikely to be a defective form of typical measles virus. This experimental system will be of value in the study of the :munological and pathogenetic aspects of chronic neurologic diseases associated with unusual or aberrant viral infections. We have produced stable mouse myeloma antibodies to individual measles virus polypeptides. Immune spleen cells were produced by immunization with either purified measles virus or persistently infected human lymphoblastoid cells. Cell fusion and hybrid isolation was performed by the protocol of Yelton g &. (1) .

Detection and characterization of antibody producing hybrids was performed in the following ways: i) Radioimmune assay using 1125 labeled anti-mouse light chain and virally infected cell extract; ii) neutralization of plaque forming ability of infectious measles virus: and iii) immunoprecipitation followed by SDS acrylamide gel electrophoresis of the disrupted immune complexes.

. different measles polypeptides:

Hybridomas have been isolated which are reactive against determinants of at least two (a) Nucleocapsid protein (NC) (b) Hemaglutinin (PI or G). Data will be presented concerning the use and specificity of these hybridomas including demonstration that the P4 polypeptide of measles virus is a breakdown product of NC. Rhv-Zr was recessive. Crosses designed t o discover s t r a i n s with t h e genotype Rhv-1s. Rhv-Zr indicate t h a t Balb/c c a r r i e s such a genotype since (SJL X Balb/cl F1 a r e r e s i s t a n t while all other F l s tested have been susceptible. A s i n g l e gene has been described which controls resistance t o MHV induced h e p a t i t i s . C3H/Bang s t r a i n i s r e s i s t a n t and the PRI is susceptible. A congenic l i n e , C3H.SS, c a r r i e s t h e susceptible a l l e l e . To t e s t whether this gene might be i d e n t i c a l t o e i t h e r Rhv-2 these mice were challenged i n t r a c r a n i a l l y with JHM virus. while C3ii/Bang is susceptible. This indicates t h a t e i t h e r resistance t o MHV induced h e p a t i t i s . The structure of several coronaviruses has recently been described but very little is known of their replication. Cells infected with the murine coronavirus strain JHM shut-off host cell protein synthesis and synthesize a number of virus-specific proteins including two structural proteins, p60 and p23. The poly A RNAs encoding p60 and p23 can be isolated from the cytoplasm of infected cells and translated in both reticulocyte and L-cell cell-free systems to give authentic products which can be identified by specific immunoprecipitation and tryptic peptide fingerprinting. The RNA encoding p60 sediments in sucrose-formamide gradients at 17s and that encoding p23 is clearly seperable and sediments at 19s. Both RNAs can be specifically released from infected cell polysomes and are therefore physiological mRNAs and they both constitute a large proportion of the viral RNA synthesized late in infection. Our interpretation of this data is that these RNAs represent two subgenomic coronavirus JHM mRNAs each of which encodes a different structural protein of the virus and if correct shows that the replication strategy of coronaviruses contrasts with other well-characterized non-transforming positive-stranded RNA animal viruses.

Stephen B. Cheley and Robert Anderson, Dept. of Microbiology d Immunology, University of Western Ontario, London, Canada Studies in our laboratory provide biochemical evidence f o r the derivation of a highly neurotropic (JHM) strain from the more prevalent and primarily hepatotropic (MHV3) strain of mouse hepatitis virus. Both strains code for three major primary gene products: a large glycoprotein, p'lBO', a basic, phosphorylated protein, p'56' and a low molecular weight protein, ~'22'. Proteins p'56' and p'22' give rise t o product polypeptides p'50' and p'24' respectively through post-translational modification mechanisms. All polypeptides of JHM have only slightly lower apparent molecular weights in SDS PAGE than the corresponding ones of MHV3. Comparison of polypeptides from MHV3 and JHM by proteolytic peptide mapping shows a high degree of structural relatedness between the two strains. Discrete peptide differences are. however, detected. Since virus-specified tissue tropism is connoonly determined by structural characteristics of viral polypeptides, it is suspected that the observed peptide differences between MHV3 and JHM may contribute to the biological differences exhibited by the two strains. Tunicamycin (TM) was used to inhibit the addition of sugars to the glycoproteins of 1.0 pg/ml of TM, administered 2 hrs post infection to cell cultures measles virus i n order to study the role of carbohydrates in the strucutre and function of these proteins. infected with virus, completely stopped the production of infectious progeny virus. This inhibition occurred at 33OC and 37,OC and affected both the Edmonston and Joy wild type measles strains. Synthesia of the nonglycosylatsd viral proteins (L, P, NP, M) appeared to be normal in the presence of the drug, while changes were observed in the synthesis o f H, the large viral glycoprotein. Particles released from TM-treated cultures lacked the normal H protein, but did contain a polypeptide, termed p90, which migrated more slowly than H on SDS-PAGE. p90 was also found in particles released from cultures infected in the presence of 2-deoxy-D-glucosc. Partial protease digests indicated that the p90 polypeptide might be related to H. The anomalously slow migration of p90 suggests that there may be proteolytic processing of the H protein. Antigenic v a r i a n t s of visna v i r u s have been compared u s i n g t h e genomic RNA and analyzing Mutants isolated from a p e r s i s t e n t l y i n f e c t & oligonucleotide p a t t e r n s when compared To detarmine whether t h e changes i n t h e nucleotide s t r u c t u r e were t h e l a r g e RNase T1-reaiatant oligonucleotides.

sheep contained a mall number of changes i n t h e i r with p a r e n t a l v i r u s . c l u s t e r e d i n one region of t h e genome, t h e oligonucleotides of t h e parental and a mutant RNA were ordered along the genome with respect t o t h e 3' polyadenylated end. A l l but one d i f f e r e n c e between t h e p a r e n t a l s t r a i n and t h e a n t i g e n i c mutant used f o r mapping were located within 2 kilobases from t h e 3' terminus. Gene 4 , the haemagglutinin gene, of both human (A/Victoria/3/75) and chicken influenza viruses (FPV) and gene 8 of FPV (coding for NS1 and NS2) have been reversed transcribed into double stranded DNA copies after in vitro polyadenylation of the viral RNA. transcripts were combined with pBR322, cloned in E. coli and complete sequence analyses obtained. mature HA protein and its precursor'and, in the case of gene 8, indicate the mechanism by which NS1 and NS2 are produced from this RNA segment.

The HA genes have been transferred to plasmids derived from the 9 promoter region of the E. coli genome and designed to achieve controlled expression of sequences inserted at a specific site. specifically with antiserum to FPV-HA.

The production, characterisation and relationship of this protein to authentic HA will be discussed.

The nucleotide sequences predict the complete amino acid sequence of the Such plasmids direct the synthesis of a protein that reacts PFU per ml at thirty hours post-infection. plaque forming units can be detected in the medium. The intracellular viral RNA and viral RNA isolated from extracellular virus pelleted from the medium from the persistently infected cell lines has been compared with RNA of virus isolated from infected BHK cells as well as from twenty-four post-infection Aedes albo ictus cells. ferential loss of the M and-ably ! h e S genome segments with the concomient appearance of a small (less than 4s) class of molecules. The profile obtained with rival RNA from both Uukuneimi and Inkoo persistently infected cells is nearly identical. The coinfection of wild type virus with virus from persistently infected Aedes albo ictus cells interfers with the pattern of wild type growth in BHK cells. I n t e r f e r e a s mos! striking at 2 8 O and is essentially undetectable at 37O. This suggests a temperature sensitive phenomina superimposed over a defective interfering activity. We propose that this system be used as a model for both the generation of the persistently infected cell state as well as for the horizontal transmission of this and similar viruses from invertibrate to vertihrate hosts. 

A persistent reovirus infection was established by infecting L cells with a serially passaged stock of temperature-sensitive (ts) mutant C(447) containing greater than 90% defective interfering (DI) particles. The nature of the virus produced in this carrier culture was examined over a period of 1 1/2 years. virus changes from ts to ts+ (wild type) within a few weeks of the initial infection ( 2 ) some of the ts+ clones contain extragenically suppressed ts lesions ( 3 ) & mutants in recombination groups B, G and a new group, designated H, were rescued from the suppressed clones (4) deletion mutants (01 particles) lacking genomic dsRNA segments L1, L3 and M1 are present in the carrier culture. during persistent infection.

Since persistently infected cells contain both infectious virus and 01 particles, there should be ample opportunity for genetic interaction between them. virus 01 particles can reassort genes with infectious virus and it is possible to rescue ts lesions from the 01s. ments also contain mutations in other genes. It is possible that 01 particles provide a source of mutant genes and that the genetic variability of virus isolated frwn the persistently infected cells is due to recombination between DI particles and infectious virus. The transcription of Cytoplasmic Polyhedrosis Virus (CPV) of Bombyx mori, a ten-segmented dsRNA virus, was investigated. Efficient in vitro synthesis of CPV mRNA was facilitated by the presence of sodium acetate, high concentrations of ribonucleoside triphosphates and proteinase K. Under optimal conditions, transcription was maintained for more than 24 hr, resulting in large quantities of full-size mRNAs which were active in cell-free translation. mixture of CPV mRNAs consisting of ten RNA species was resolved into nine discrete bands by agarose gel electrophoresis with 7 M urea. identified by hybridizing the separated, 32P-labeled mRNAs to a mixture of genome WAS. mRNA hybridized uniquely with its corresponding genome RNA segment in order of decreasing size. quantities by weight but different molar amounts of each of the separated mRNA species. These results imply that each of the genome segments is transcribed repeatedly by an equal number of template-associated RNA polymerases. The genome RNAs of CPV and reovirus, labeled at the 3'termini with 32pCp by RNA ligase, were separated by agarose gel electrophoresis into the plus and minus strands. than those of minus polarity for all CPV dsRNA genome segments, whereas the opposite was seen for most of the reovirus genome segments. preparation of purified single-stranded RNAs of plus or minus polarity from each segmented dsRNA genome for sequence analysis. Wertheimerl. Yih Shiong Wu2 and RonaldoBorchardt2, lRoche Institute of Molecular Biology, Nutley, NJ Cytoplasmic polyhedrosis virus (CPV) of the silkworm contains a ten-segment dsRNA genome, a virus-associated RNA polymerase and enzymes required for the formation of a 5'-cap in the mRNAs. Messenger RNA synthesis in vitro by CPV is stimulated by the presence of the methyl donor S-adenosylmethionine (AdoMet) (Furuichi, Nuc. Acids Res. 1:801, 1974) . effect by AdoMet is probably at the initiation step of RNA synthesis. mine the important structural features of the AdoMet molecule required for the stimulation, analogs of AdoMet have been tested for their ability to promote RNA synthesis and to inhibit AdoMet-stimulated methylation. Several potent inhibitors of cellular and viral methyltransferases, e.g., S-adenosylhomocysteine (AdoHcy), 3-deaza AdoMet, 7-deaza AdoMet, 7-deaza AdoHcy, and the antibiotic Sinefungin, inhibit methylation of CPV mR", but stimulate mRNA synthesis as effectively as AdoMet. In contrast, most of the analogs which fail to inhibit CPV methyltransferase also fail to stimulate CPV mRNA synthesis. Structural features of AdoMet needed for efficient mRNA synthesis apparently coincide with those recognized by CPV methyltransferase. recognizing protein) in CPV transcriptional complexes activates the RNA polymerase through an AdoMet-mediated allosteric conformational change resulting in the mRNA synthesis and the formation of the methylated cap structure. 07110 and 2University of Kansas, Lawrence, KS 66045

The stimulatory I n an effort to deter-These results suggest that binding of AdoMet to methyltransferase (or some other AdoMet-

POSSIBLE MECHANISM OF ROTAVIRUS PERSISTANCE, Vikram Misra and Lorne A. Babiuk, Dept. Vet. Microbiology, WCVM, Univ. of Saskatchewan, Saskatoon, Sask., Canada. Rotaviruses have been implicated as one of the etiological agents responsible for neonatal diarrhea in man and other animals. The disease in calves is seasonal although the mechanism by which the virus persists from season to season is not known. Most isolates of bovine rotavirus are cytolytic for BSC-1 cells. Infection causes breakdown of host DNA and cessation of host macromolecular synthesis followed by death of the cell and release of infectious virus. One isolate (2352) however, appears to be deficient in its ability to shut-down host synthesis and causes a persistant infection in BSC-1 cells (RP-BSC-1).

Viral persistance does not adversely affect these cells which grow at a rate comparable to that of uninfected cells. In addition, RP-BSC-1 cells continuously produce Rotavirus and are immune to super-infection with related viruses. To further characterize virus-cell interactions during persistant infections, viral expression was examined 'in synchronized RP-BSC-1 cells. Viral expression was limited to mid and late G1 when 80% of cells exhibited viral antigens detectable by imunofluorescence and PAGE. Entry into S-phase caused a degradation of viral proteins and a sharp decline in viral expression, nentially growing BSC-1 cells yielded only 'complete' virus particles. that some strains of rotavirus are unable to shut-down host macromolecular synthesis and therefore can only replicate in certain permissive phases of the cell cycle. As the infected cell traverses from 'G1' into 'S' viral replication and expression ceases, only to resume when the cells re-enter a permissive phase. Experiments to determine the implications of these observations to persistance in vivo are in progress.

Degradation was probably brought about by factors synthesized in S-phase as expo-

The results suggest Gardner's syndrome (GS) variant were reported. W e have confinned these findings and show, in addition, that these c e l l s are also susceptible to transformation by KiMSV w i t h a baboon retrovirus helper (KiMSV/BaEV) o r by feline sarcoma virus (FeSV). Both KiMSV/MuLV and KiMSV/ BaEV transformed the ACR and GS c e l l s w i t h 300-1000-fold greater efficiency than normal, agematched, control c e l l s . I t i s estimated that >lo' focus forming particles a r e required to induce few foci i n normal c e l l s whereas <lo0 particles are sufficient t o transform ACR and GS c e l l s . The relative focus inducing t i t e r s of KiMSV (BaEV) were higher than Ki MSV (MuLV) i n the c e l l s tested. However, the differential transformation efficiency o f these viruses i n ACR and GS as compared to control c e l l s was not due to differences i n the extent of virus infection o r replication. The increased transformability of ACR and GS c e l l s t h u s presumably operates a t the host genome level although permanent lines of completely transformed c e l l s have not y e t been established. T h i s assay i s highly reproducible and shows promise for detection of young ACR or GS gene-bearing "normal" individuals before development of colon polyps and other tumors. Hamburg 20. Federal Republic of Germany BALB/Mo mice carryin8 the Moloney leukemia virus ( = M-MuLV) as a n endoqenous virus become viremic soon after birth and develop leukemia a t a l a t e r age. hl-MuLV-specific gene expression a n d a n increase of virus-specific DNA copies i n lymphatic target organs a r e characteristic parameters of the preleukemic phase.

Passive immunotherapy of newborn BALB/Mo mice with anti-gp70 or anti-M-MuLV serum prevented viremia and subsequent development of leukemia.

Molecular hybridization experiments showed that both virus-specific genome transcription a n d virus-specific DNA amplification could be completely suppressed by antiserum treatment. Thus virus-specific R N A concentrations in target organs of immunized BALB/Mo mice of SIX months or older were as low a s i n normal BALB/c mice. This is a n age a t which untreated BALB/Mo mice have already developed malignant lymphoma. Our experiments demonstrate that treatment with antiserum interferes w i t h the e a r l y events of virus expression a n d prevents the subsequent steps leading to leukemia.

J.W.Streilein, M.Nelles, J.T.Phillips, W.Duncan, U.Tex.H.Sci.Ctr., Dallas,Texas 75235 Primary responsibility for anti-viral immunity is ascribed to genes of the major histocompatibility complex (MHC): while Class I1 genes (H-2I) of mice are important in immune regulation, recent attention has focused on Class I genes (H-ZK,D) which promote and restrict effector T lymphocytes that kill virus-infected target cells in vitro and protect against lethal virus infection. A relationship between extensive polymorphism for Class I antigens and ability of a species to survive viral epizootics has been postulated.

Syrian hamsters, captured from the wild over a 40 year span and many kilometers apart,display marked polymorphism for Class I1 (Ia-like) antigens, but "0 polymorphism for Class I antigens, although putative Class I molecules are expressed on hamster cells. To study the possible relationship between Class I antigenic variation and virus immunity, hamsters were infected with vaccinia virus and their lymphoid cells examined for cytotoxicity against virus infected target cells in vitro. Cytotoxic activity that was found among lymphoid cells of vaccinia-infected hamsters was not genetically restricted as expected from the lack of Class I polymorphism. More importantly, the cytotoxicity was not even T cell in nature! Instead, a fraction of cytotoxic activity was virus-specific, owing to an antibody-dependent cell-mediated process; the remaining cytotoxicity lacked viral specificity, probably effected by NK cells. Thus, hamsters fail to utilize cytotoxic T cell effectors during acute virus infection; instead, they rely upon an antibody dependent system, aided by activated NK cells, implying that imunity against virus infections need n o t require Class I MHC genes, and, perhaps, that Class I polymorphism co-evolves with a species' virus load. 

Certain inbred strains of mice contain highly oncogenic exogenous milk-transmitted mammary tumor virus (MMTV) and genetically transmitted endogenous MMTV. To study the ongenicity of the endogenous W , we established a cell.line producing MHTV from a mammary tumor of a C3H muse which had been foster-nursed on NIH Swiss mice to remove the highly oncogenic exogenous MMTV. lhis tissue culture-derived C3Hf HMTV has a low oncogenic potential since inoculation of Balblc and C3Hf mice with C3Hf virus have given no mammary tumors at the end of one year. Greater than 501: of the mice inoculated with equivalent amounts of C3H MHTV developed mammary tumors in less than one year. mice as revealed by analysis of large oligonucleotides generated by ribonuclease Ti digestion of their 70s R N h . inmunoassays for the envelope glycoproteins, gp52 and gp36, clearly demonstrate antigenic polymorphism for all four MMTVs. not been detected in radioimmnoassays for the major =-coded protein, p27. mammary glands by exogenous MMTV is a prerequisite for oncogenicity, cell surface receptors for MMTV were investigated. Cell surface receptor recognition resides with the gp52 molecule because only anti gp52 serum and monoclonal antibody to gp52 neutralize transforming capacity of a KiSV (C3H WTV) paeudotype containing gp52 as the surface antigen. Highly oncogenic C3H and GR MMTV bind common cellular receptors since they inhibit transformation by the pseudotype, whereas C3Hf MMTV does not inhibit. 'Ihe possibility that the greater oncogenicity of the exogenous MMTV m y reside with enhanced infectivity will be discussed. Tne C3Hf MMTV is genetically different from exogenous HMTV of C3H, RIII, and GR Some of the genetic differences apparently reside in the env region since radio- 

Antigenic polymorphism in the major envelope glycoprotein, gp52, of different strains of mouse mammary tunor virus (MMTV) was studied with monoclonal antibodies and sera from mammary tmor-bearing mice. With these imnologic probes. the functional domains of the gp52 molecule were studied b in vitro neutralization of infectivity, complement-dependent cytolysis, precipitation of lz3-F-7and a solid-phase radioimmunoassay. "be antigenic determinants could be grouped into three categories. Group I: Group-specific antigenic determinants found on C3H. GR, RIII MMTVs, and C3Hf MMTV, an endogenous virus of C3H mice. 'Ihese determinants function in precipitation and radioimmunoassays. Group 11: Class-specific antigenic determinants shared by some, but not all, HHTvs and detected with monoclonal antibodies and natural sera. Tnese include Subgroup A determinants found on C3H and GR WrITV, and Subgroup B determfnants found on RIII and C3Hf MMTV. These determinants are targets for neutralizing, cytotoxlc. and precipitating antibodies. Group 111: Type-specific determinants. unique to a HHN strain. have only been identified with monoclonal antibodies. Ileterminanta which are targets for neutralizing antibodies may be recognized by cell surface receptors. Based on cell hinding and competition assays, C3H and GR MMTV also share a common cell receptor which does not bind RIII and C3Hf H M N and correlates with Subgroup A identified antigenically. imrmnological properties, is analogous to groupings based on oncogenicities of the viruses in BALBIc mice. the viral genome. Some of these susceptibility loci were first identified because of their involvement in the regulation of different aspects of normal hemopoiesis (W, Steel). We have examined the effect of these host genes on the regulation of normal hemopoiesis as well as on the erythroleukemia induced by both the anemia-and polycythemia-inducing spleen focus-forming virus (SFFVA and SFFVp respectively) variants of Friend virus. indicated that (i) Friend virus specific sequences are expressed i n normal tissue and that their expression is under tissue control; (ii) the expression of these specific sequences are also under host genetic control (Fv-2, H-2); (iii) SFFVA and SFFVp induce dramatically different effects on erythroid progenitor cells; (iv) SFFVA Is regulated by the same host susceptibility genes (W, S1, Fv-2) that affect SFFVp. The mechanism of resistance of Friend erythroleukemia by these host genes will be discussed in the context of the following mechanisms: a) Inhibition of the replication of the transforming virus directly; b) reduction of the number of target cells available for transformation; c) suppression of the proliferation of infected or transformed target cells; and d) suppression of the transformed cells by the imune response. Several lines of mouse and rat cells infected with MSV have been isolated and characterized. All lines contained an integrated MSV proviral genome and expressed MSV specific information at various levels. A rat line, termed clone 60%-was isolated after injection and tumor formation in a nude mouse and shown to grow in suspension. Selection of revertants that could not grow in spinner cultures was performed Py growing the cells in suspension in the presence of 3H-thymidine or FdUr. and independently subcloned. An analysis of the mechanism of reversion of all these cell clones is in progress. tion of the virus genome with a loss of viral gene expression. A third class revertant representing a continuation of viral gene expression may be detected by determining viral ~R N A formation employing the techniques of RNA-RNA hybridization and R loop formation electron microscopy.

A large number of anchorage-dependent cell$ was isolated It is expected that reversion may be due to loss of the integrated viral genane or reten-

Proc. Natl

Proc. Natl. Acad. Sci

Replication-and transformation-competent viruses were recovered. Characterization of the RNA of these rescued viruses indicates that they originate by unequal recombination between the two defective parents, generating a nondefective virus.