key: cord-1006267-ahxwa34k
authors: Makino, Shinji; Taguchi, Fumihiro; Hirano, Norio; Fujiwara, Kosaku
title: Analysis of genomic and intracellular viral RNAs of small plaque mutants of mouse hepatitis virus, JHM strain
date: 1984-11-30
journal: Virology
DOI: 10.1016/0042-6822(84)90335-0
sha: ddd687875254747862c30e116e2fe7beea13e9e7
doc_id: 1006267
cord_uid: ahxwa34k

Abstract The genomic RNA and intracellular RNA of mouse hepatitis virus, strain JHM (MHV-JHM) and two plaque mutants (la and 2c), which have been isolated from a persistently infected culture (JHM-CC), have been analyzed by T1-resistant oligonucleotide finger-printing. The genomic RNA of the virus population (JHM-CC virus) released from different passage levels of the same persistent infection has also been analyzed. The analysis shows the locations within the genomic and intracellular RNAs of more than 45 T1-resistant oligonucleotides and confirm earlier studies (J. L. Leibowitz, K. C. Wilhelmsen, and C. W. Bond (1981), Virology 114, 39–51), showing that the six subgenomic RNAs of MHV-JHM form a 3′ coterminal nested set which extends for different lengths in a 5′ direction. The analysis also identifies in each subgenomic RNA those large T1 oligonucleotides derived from noncontiguous regions of the genome during mRNA synthesis. Two important conclusions can be reached from analysis of the mutant viruses. First, the virus population released from the persistent infection represents a fairly constant mixture of viruses, and the fluctuating emergence of variants as predominant species in the culture does not occur. Second, the data indicate that for particular intracellular RNAs of mutant viruses the sequence rearrangements occurring during subgenomic mRNA synthesis are different from those in the corresponding intracellular RNA of wild-type virus. The result may indicate a potential flexibility in the leader/body fusion process that has not been previously recognized.

Coronaviruses infect a variety of vertebrates, including man, and are responsible for disease of clinical and economic importance . The most studied member of the group is mouse hepatitis virus (MHV) which has a single, positive-stranded genomic RNA of 5.4-6.7 X lo6 molecular weight (Lai and Stohlman, 1978; Leibowitz et al, 1981; Spaan et &, i 1981; Wege et al, 1978 Wege et al, , 1981 Yogo et aL, 1977) . In cells infected with MHV six species of subgenomic mRNA, together with a genomic-size mRNA, are produced (and are numbered in order of decreasing size). These mRNAs form a 3' coterminal "nested" set but each has at its 5' terminus a common leader, derived from sequences at the 5' end of the genome (Baric et al, 1983; Cheley et ak, 1981; Lai et ah, 198313; Spaan et ah, 1982 Spaan et ah, , 1983 . As MHV replicates in the cytoplasm Wilhelmsen et aL, 1981) and mRNA synthesis does not involve the processing of larger precursor RNAs (Jacobs et d, 1981) , coronaviruses appear to have evolved a novel mechanism of mRNA synthesis involving the cytoplasmic fusion of noncontiguous transcripts (Spaan et al, 1983) . In the infected cell the synthesis of viral polypeptides is initiated independently (Siddell, 1982 (Siddell, , 1983 and in tivo and in vitro translation studies have shown that mRNA7, mRNA6, and mRNA3 encode the virion nucleocapsid protein (NP), the matrix protein (El), and the precursor of the peplomer protein (E2), respectively (Leibowitz et c& 1982; Rottier et aL, 1981b; Siddell et d, 1980 , Siddell, 1983 .

One MHV strain, MHV-JHM, is of particular interest in that it has the ability to induce central nervous system disorders in mice and rats and can be used as a model for virus-induced demyelination (Herndon et aL, 1975; Knobler et ah, 1981; Nagashima et u& 1978) . Previously, we have established an in vitro persistent infection with MHV-JHM in a DBT cell line (JHM-CC), and found that the virus population released from this culture (JHM-CC virus) formed small plaques on DBT cells and possessed lower virulence to ICR mice when compared with original MHV-JHM (Hirano et aL, 1981; Makino et uL, 1982) . Histopathological examination revealed that after infection with JHM-CC virus, the acute encephalitis characteristic of infection with wild-type virus was absent and instead sharply delineated demyelinating lesions were produced in the brain and spinal cord. The JHM-CC virus will therefore be very useful for studying the pathogenesis of demyelination. In our initial studies (Makino et cd, 1983) we have characterized the virological features of two small plaque mutant viruses, la and 2c, which were isolated from the JHM-CC virus population. In contrast to wild-type MHV-JHM these mutants grew more slowly, produced no prominent cell fusion during infection, and possessed different amounts and molecular-weight forms of the virion peplomer protein E2.

In this report we have analyzed the genomic and intracellular subgenomic RNAs of MHV-JHM, la and 2c, as well as the genomic RNA of the JHM-CC virus population at different passage levels of the persistent culture. These studies indicated that the population of JHM-CC viruses represents a fairly constant mixture of viruses and that different variants do not emerge to predominate during the persistent infection. In some of the subgenomic RNAs of mutant viruses noncontiguous transcripts appeared to have been combined which contain different sequences compared to wild-type MHV-JHM.

Cell culture and viruses. MHV-JHM, la, and 2c viruses were grown on DBT cells as described previously (Makino et cd, 1983) . Viruses from the persistent culture JHM-CC were harvested at different passage levels and propagated once on DBT cells before use.

Prepurutim of vi&n RNA. 32P-labeled RNA was extracted from purified viruses by procedures described previously (Makino et r& 1983 (Makino et r& , 1984 . In some experiments, the 60 S virion RNA was obtained by separation on a 15-30s sucrose gradient in a SW50.1 rotor at 45,000 rpm for 1.5 hr.

Prepurutim of intracellular viral RNA. DBT cells were inoculated with MHV-JHM at a multiplicity of 1.0. After adsorption for 1 hr at 37", cultures were incubated for 2.5 hr with Eagle's minimal essential medium (MEM, Nissui, Tokyo) containing 5% dialyzed calf serum. Then, cultures were incubated with MEM containing 5% dialyzed calf serum, l/10 normal concentration of phosphate, and 2.5 pg of actinomycin D (AMD)/ml (Sigma, St. Louis, MO.) (l/lOP-medium). At 6 hr postinfection (p.i.) the culture medium was replaced with phosphate-free MEM containing 5% dialyzed calf serum, 2.5 pg of AMD, and 250 &i/ml of "P (Japan Radioisotope Association) (32P-medium). Intracellular viral RNA was extracted at 9 to 10 hr p.i. For the labeling of the intracellular RNA of the mutant viruses, la and 2c, the culture medium was replaced at 9 hr p.i. with l/lOP-medium. After 12 hr of infection, cultures were incubated with =P-medium and viral RNA was extracted at 24 hr p.i. For the extraction of intracellular viral RNA, monolayers of infected cells were chilled on ice, and washed three times with chilled phosphate-buffered saline, pH 7.2. Then the cells were scraped by a rubber policeman and centrifuged at 3000 rpm for 10 min at 4". The cells were lysed in NTE buffer (0.1 MNaCl, 0.01 MTris-HCl, pH '7.2, and 1 m&f EDTA) containing 0.5% NP-40. After centrifugation at 3000 rpm for 15 min at 4" to remove nuclei, the RNA was isolated from the supernatant following phenol/chloroform extractions as described previously (Makino et al, 1934) .

Oligo(dT)-cellulose column chrwnuztography. Ethanol-precipitated RNA was dissolved in binding buffer (0.01 1K Tris-HCl, pH 7.5, 0.5 MLiCl, 0.5% SDS, and 1 mM EDTA) and applied to an oligo(dT)cellulose (type 3, Collaborative Research) column. After washing with binding buffer, poly(A)-containing RNA was eluted with elution buffer (0.01 M Tris-HCI, pH 7.5, 0.05% SDS, and 1 mM EDTA), and RNA was precipitated with ethanol in the presence of 0.1 M NaCl.

Agarose gel electrophoresk Analytical gel electrophoresis was conducted following denaturation of RNA with glyoxal treatment, as described previously (McMaster and Carmicheal, 1977) . Preparative gel electrophoresis in 1% ureaagarose gels was performed as described previously (Makino et aL, 1934) . Since individual intracellular viral RNA species excised from the gels were occasionally contaminated with smaller-size RNAs, the gel slices were melted at 75" and applied to a second urea-agarose gel. After the second electrophoresis, contaminating RNAs were no longer detected. The RNA was eluted from gel slices by the methods of Langridge et al. (1980) . O~igonucEeotide jingerprinting. =P-labeled and purified viral RNA was digested with ribonuclease T1 and the products were analyzed by two-dimensional polyacrylamide gel electrophoresis as described previously (Makino et a& 1934) .

Fragmentation of virion RNA for oligonwleotide mapping. The =P-labeled purified virion RNA was dissolved in a buffer containing 0.01 M Tris-HCI (pH 7.5) and 1 mM EDTA, and divided into four aliquots. Sodium carbonate was added to each at a final concentration of 0.05 M and then each solution was incubated at 25" for 1 min or 3 min or at 50" for 1 min or 3 min. Then the solutions were neutralized with acetic acid and the RNA was precipitated with ethanol in the presence of 0.1 M NaCl. These alkali-degraded RNA fragments were pooled and applied to a urea-agarose gel. After electrophoresis, the gel areas containing RNA fragments of different sizes were excised and RNA was eluted, precipitated by ethanol, selected by oligo(dT)-cellulose column chromatography, and each sized poly(A)containing RNA was analyzed by oligonucleotide fingerprinting.

of getwmk RNA of MHV-JHM, la, and 2~. The genomic RNAs of MHV-JHM, la, and 2c, were analyzed by oligonucleotide fingerprinting and the results are shown in Figs. lA, B, and C. Prior to RNase T1 digestion the RNAs were analyzed on glyoxal agarose gels (data not shown) and found to have identical electrophoretic mobilities, corresponding to an estimated molecular weight of 5.4 X lo6 (Lai and Stohlman, 1978; Makino et aL, 1934) . In order to positively identify each oligonucleotide, mixtures of the genomic RNA of MHV-JHM and la and also MHV-JHM and 2c were digested together and the resulting oligonucleotide maps are shown in Figs. 1D and E, respectively. Careful examination of these fingerprints shows that more than 45 characteristic oligonucleotides can be identified in the genomic RNA of MHV-JHM. A single oligonucleotide (indicated by an arrow in Fig.  1A ) was not reproducibly found. An oligonucleotide fingerprint of this complexity corresponds well to a single-stranded RNA of this size which does not contain extensive sequence reiteration. The oligonucleotide fingerprint of MHV-JHM shown here is also very similar to that described by Stohlman et al. (1982) . We have therefore adapted the numbering system used by Stohlman et al, additionally numbering four oligonucleotides which are unique to our MHV-JHM isolate as 2a, 2b, 26b, and 37a. When the oligonucleotide fingerprint of the wild-type MHV-JHM is compared to those of the mutant viruses la and 2c, a number of significant differences can be found and these differences are summarized schematically in Fig. 2 . Essentially both mutants lacked the oligonucleotides 2b, 4, 6, 17, and 26b (indicated by arrow heads in Fig. 1A ) of the wild-type MHV-JHM, and contained one new oligonucleotide, 26~ (arrows in Figs. 1B and C). The mutant 2c also contained three additional oligonucleotides 6c, 48, and 49 (arrows in Fig. 1C) .

Location of the RNase Tl-resistant oligonuckotides on the g-of MHV-JHM. The genomic RNA of MHV-JHM was degraded by mild alkali treatment and frag-ments were then fractionated into five size classes by electrophoresis in ureaagarose gels. The 3' coterminal fragments were selected by oligo(dT)-cellulose column chromatography and oligonucleotide fingerprints of each class were prepared. The results are shown in Fig. 3 . If the genome of MHV-JHM is assumed to be 18 kilobases (kb) in length (Lai et al, 1981; Siddell, 1933) it can be deduced that the oligonucleotides 4, 17, and 6 and the oligonucleotides 2b and 26b (i.e., those absent from the mutant viruses) were located respectively, between 5.5 to 8 kb, 3 to 10.6 kb, 10.6 to 14.3 kb, and 16.5 to 18 kb from the 3' end of the genome. It should also be noted that the localization of oligonucleotides presented here does FIG. 2 . Diagrammatic representation of the relationships between the oligonucleotide fingerprints of genomic RNA of MHV-JHM and mutant viruses. The numbering of the oligonucleotides is according to Stohlman et al (1982) . Closed spots denote the oligonucleotide spots detected only in MHV-JHM.

Cross-hatched spots denote the oligonucleotide spots found in mutant viruses.

not differ significantly from that previously reported with the exception of oligonucleotide 8 (see also below), which in our study is located near the 5' end of the genome, in contrast to Stohlman et al. (1982) who located this oligonucleotide 5 to 7 kb from the 3' terminus.

Oligomcleotide jingerprinting of intracellular viral RNAs of MHV-JHM, la, and 2~. In agreement with previous reports (Lai et c& 1981 , 1982 Spaan et d, 1981; Wege et aL, 1981) seven major poly(A)-containing viral RNA species (numbered RNA1 to RNA7 in order of decreasing size) were found in MHV-JHM infected cells (Fig. 4a) . These RNAs had estimated molecular weights of 5.4, 3.3, 2.6, 1.25, 1.12, 0.86, and 0.6 X 106. No discrete RNA bands were identified in mock-infected extracts (data not shown). A number of minor virus-specific RNA species were also found in cells infected with both wild-type and mutant viruses (labeled a, b, c, d, and e in Fig. 4 ) and the relative proportions of different RNA species was also variable. For example, RNA2 was missing in mutants la and 2c infected cells, RNAs 5 and c were particularly prominent in mutant 2c infected cells and RNAa was only irreproducibly found. Oligonucleotide maps of the intracellular RNA species of all these viruses were prepared. In some cases it was difficult to separate major from minor RNA species (e.g., RNA5/c) but possible mixtures are clearly indicated in the figures.

The oligonucleotide fingerprints of intracellular viral RNAs of JHM are presented in Fig. 5 . As reported by Lai et al. (1981) the pattern of RNA1 was identical to genomic RNA and the fingerprinting pattern was more complex with increased sizes of RNAs. Most oligonucleotides of the small RNAs were included in neighboring larger RNAs and the localization of oligonucleotides of the intracellular viral RNAs was well matched with the order of oligonucleotides determined in genomic RNA. However, major exceptions were found for oligonucleotide 8, which has been located close to the 5' end of the genomic RNA but appeared in every intracellular viral RNA, and several oligonucleotides (indicated by arrow heads in Fig. 5 ) which are present in subgenomic RNAs (with the exception of RNAZ) but were not found in genomic RNA (see Discussion). The oligonucleotide 34 which was found only in RNAb was numbered 34*.

As the intracellular RNA species of MHV-JHM form a 3' coterminal nested set , careful analysis of these fingerprints allows for a more detailed localization of the oligonucleotides on the genome, as is shown in Fig.  6 . Thus oligonucleotides 1'7 and 6 could be more accurately positioned between 8 and 8.7 kb and 11 and 14.8 kb, respectively from the 3' end of the genome.

The oligonucleotide fingerprints of the intracellular viral RNA of la and 2c are shown in Figs. 7 and 8 . The structures of the RNAs were in general agreement with that of MHV-JHM. Oligonucleotide 8 is again present in each subgenomic RNA ET AL. and subgenomic RNAs contain oligonucleotides which are not found in genomic RNA. Most surprisingly oligonucleotide 2, which in MHV-JHM infected cells was found only in RNA6 and larger RNAs, was conspicuously present in RNA7 of mutant 2c (see Discussion).

Using these fingerprints it was also possible to locate the position of the oligonucleotides which was specific to the mutant viruses. Oligonucleotide 26c (indicated in both mutants but not in MHV-JHM) was located 5.5 to 8.7 kb from the 3' end of the genome. In the mutant 2c, the oligonucleotides 6c, 48, and 49 are located 8.7 to 18 kb, 3.8 to 4.1 kb, and 8.7 to 18 kb from the 3' end of the genome, respectively. A number of oligonucleotides appeared to give locations in the mutant intracellular RNAs which were inconsistent with their locations in MHV-JHM intracellular RNA. These oligonucleotides are designated 43*, ll*, and 34* (for example, oligonucleotide 43 is found in RNAs 1 to 5 of MHV-JHM, but only in RNA1 of the mutant viruses). Oligonucleotide 35 was found in RNA1 to RNA5 of MHV-JHM, but only in RNA1 to 4 of mutant viruses. Although we are sure these anomalies are significant we cannot readily explain their occurrence.

Oligxmucleoti5!f3&gerprinting of genmnic RNA of viruses released frm d~srent vsage levels of JHMCC. The mutant viruses la and 2c were isolated from the JHM-CC culture at the 133rd passage. In order to search for any genotypic changes in the RNA of viruses released from the JHM-CC culture over a period of time, we infected DBT cells with JHM-CC virus from different passage levels, labeled with [32P]orthophosphate, purified the progeny virus, and extracted and analyzed the viral RNA by oligonucleotide fingerprinting. At no time did we detect any alteration in the size of the genomic RNA (data not shown) and the fingerprints of passage 93 virus (93V), passage 110 virus (llOV), passage 133 virus (133V), and passage 185 virus (185V) are shown in Fig. 9 . These fingerprints show that the genomic RNA of the virus populations 93V, llOV, and 133V were identical to that of the isolated FIG. 5 . Oligonucleotide fingerprints of the intracellular MHV-JHM-specific RNAs. The virusspecific RNAs were eluted from urea-agarose gels, digested with RNase T1, and oligonucleotides were separated by two-dimensional polyacrylamide gel electrophoresis. mutant la. The genomic RNA of 185V has JHM, and the related MHV strain A59 one additional oligonucleotide (48) which (Lai et d, 1981 is also found in the isolated mutant 2~.

1981; Spaan et al, 1981) . The detailed DISCUSSION location of a large number of RNase TIresistant oligonucleotides on the genomic The results presented here confirm and RNA and oligonucleotide mapping of the extend previous studies on MHV, strain seven intracellular RNA species provide FIG. 6. Map order of T1 oligonucleotide spots on the RNA genome of MHV-JHM, la, and 3~. The order of the oligonucleotides within every region separated by vertical lines is arbitrary. The proposed seven genetic regions are based on the data described in the present study and from others (Lai et al, 1981; Siddell, 1983) . Each region corresponds to the position of each viral intracellular RNA species which does not overlap with the neighboring-intracellular RNA species. a firm basis for further studies on the genetic structure and expression of a virus which is becoming increasingly useful for the study of pathogenetic aspects of virusinduced demyelination (Herndon et aL, 1975; Knobler et a& 1981; Nagashima et aL, 1978) . The presumptive location of every large oligonucleotide on the genome of MHV-JHM is shown in Fig. 6 . These experiments also identify for the first time oligonucleotide 8 as being derived from sequences which are encompassed within the leader of MHV-JHM. Also in each subgenomic RNA of MHV-JHM (with the exception of RNAZ) oligonucleotides can be identified which are not found in genomic RNA and it can be assumed that these "unique" oligonucleotides have arisen from the combination of sequences during the, synthesis of mRNAs involving the joining of leader and body transcripts (Lai et aL, 1983b; Spaan et aL, 1933) . It is interesting to note that at least for some of the subgenomic RNAs (e.g., RNA7 and RNAG) these "unique" oligonucleotide are apparently very similar in their electrophoretic behavior, suggesting that the sequences combined during RNA synthesis may be very similar. In other cases, however, e.g., RNA5, the "unique" oligonucleotide is clearly different, suggesting the combination of different sequences (the differences most probably being derived from the mRNA body). In one case, RNAb, two such oligonucleotides are identified. The explanation of this result requires further study but it is perhaps noteworthy that in the case of mutant virus RNAs (see below) such a result is found when a mixture of two RNAs are analyzed. Oligonucleotide analysis of the mutant virus genomic RNA and subgenomic RNAs leads to two conclusions. First, regarding the synthesis of subgenomic RNAs; in RNA7 of mutant 2c one oligonucleotide, 2, is clearly found which has been mapped FIG. 7. Oligonucleotide fingerprints of the intracellular la-specific RNAs.

in wild-type MHV-JHM to the sequences may be joined. It is also important to note unique to RNA6 and large RNAs. The that in mutant 2c the "unique" oligonusimplest explanation of this result would cleotide in RNA'7 appears to be the same be that in the mutant virus the body of as in wild-type MHV-JHM subgenomic RNA7 extends further in a 5' direction RNA, but is clearly different in the case and includes sequences not found in the of RNAG. These conclusions should howbody of wild-type MHV-JHM RNA7. One ever be considered tentative, because alpossible interpretation would be therefore ternative more complex explanations inthat during mRNA synthesis alternative volving rearrangements of sequences in combinations of leader and body sequences the genomic RNA are conceivable. Even- MAKINO ET AL. tually sequence analysis of these RNA species and the corresponding regions of the genomic RNA will be necessary to provide a full explanation. The second conclusion that can be reached by comparison of the wild-type MHV-JHM and mutant viruses concerns the locations of detectable sequence changes in the viruses which replicate in the persistent culture JHM-CC. In Fig. 6 it can be seen that with the exception of the sequences surrounding oligonucleotide 48, all of the sequence changes in the mutant viruses are located (or in the case of oligonucleotides 6c, 49, and 43*, may be located) in one of two regions of the genome, namely in the unique sequences found in genomic RNA (or RNAl) and FIG. 9 . Oligonucleotide fingerprints of genomic RNA of viruses released from JHM-CC at different passage levels. The viruses isolated from JHM-CC at passage levels 93, 110, 133, and 185, respectively are indicated as 93V, llOV, 133V and 135V. the sequences between the 5' termini of into cells (Holmes et uL, 1981 ; Niemann RNAs b and 3. These regions of the geand Klenk, 1981; Rottier et aL, 1981a) . The nome are believed to encode the RNA fact that in tissue culture both mutants polymerase functions (RNAl) (Brayton et grew more slowly than wild-type MHV-aL, 1982; Mahy et a& 1983) and the E2 JHM, and possess altered E2 proteins peplomer protein of the virion (RNA3) (Makino et aL, 1983) may, but need not which is responsible for virus adsorption necessarily, be related to these changes.

In the infected animal it has been suggested that MHV-JHM induced demyelination is a consequence of the lytic infection of oligodendrocytes (Knobler et o& 1981) and the observation that both mutants produced reduced neural degeneration and delayed demyelination (unpublished observation) might be related to the inability of the viruses to infect neural cells, together with reduced rates of replication in oligodendrocytes.

It is now recognized that most, if not all, RNA viruses have mutation frequencies, in part because of the lack of proofreading enzymes that assure fidelity of DNA replication. The high rates of replication of RNA viruses also contribute to this high mutation frequency (Holland et al, 1982) . Oligonucleotide fingerprinting of the virus populations released from different passages of the JHM-CC culture and isolates from the culture provides evidence that mutations are readily produced and accumulated in the MHV-JHM genome. This conclusion would also support previous descriptions of MHV gene diversification reported by Lai et al. (1983a) . The oligonucleotide fingerprintings also show that the virus population released from the JHM-CC culture is relatively constant, although the differences shown for the mutants la and 2c, which were both isolated from the 133V population, demonstrate that individual viruses within the population can have different genetic structures. In the JHM-CC culture there does not appear to be any fluctuating emergence of variants which predominate the culture at any one time. This is in contrast to the situation reported in several other virus systems (Holland et al, 1979 (Holland et al, , 1982 Meinkoth and Kennedy, 1980) , although in these cases defective interfering (DI) particles appeared to play a role, while in the JHM-CC culture there is no evidence for the presence of DI particles (Hirano et aL, 1981) .

Characterization of replicative intermediate RNA of mouse hepatitis virus: Presence of leader RNA sequences on nascent chains

Host cell nuclear function and murine hepatitis virus replication

Characterization of two RNA polymerase activities induced by mouse hepatitis virus

Intracellular murine hepatitis virus-specific RNAs contain common sequences

Mouse hepatitis virus-induced recurrent demyelination

Persistent infection with mouse hepatitis virus JHM strain in DBT cell culture

Evolution of multiple genome mutations during long-term persistent infection of vesicular stomatitis virus

Rapid evolution of RNA genomes

Tunicamycin resistant glycosylation of a coronavirus glycoprotein: Demonstration of a novel type of viral glycoprotein

The synthesis of the subgenomic mRNAs of mouse hepatitis virus is initiated independently: Evidence from UV transcription mapping

Selective localization of wild type and mutant mouse hepatitis virus (JHM strain) antigens in CNS tissue by fluorescence, light and electron microscopy

Mouse hepatitis virus A59 mRNA structure and genetic localization of the sequence divergence from hepatotropic strain MHV-3

Genetic heterogeneity of murine coronaviruses

Presence of leader sequences in the mRNA of mouse hepatitis virus

Further characterization of mRNA's of mouse hepatitis virus: Presence of common Y-end nucleotides

RNA of mouse hepatitis virus

Extraction of nucleic acids from agarose gels

The virus-specific intracellular RNA species of two murine coronavirus: MHV-A59 and MHV-JHM

Cell-free translation of murine coronavirus RNA. .I. Viral

RNA-dependent RNA polymerase activity in murine coronavirus-infected cells

Defective interfering particles of mouse hepatitis virus

Heterologous response of antiserumtreated cell clones from a persistently infected DBT cell line to mouse hepatitis virus

Characterization of small plaque mutants of mouse hepatitis virus

Analysis of single-and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange

Semliki Forest virus persistence in mouse L929 cells

Coronavirus induced subacute demyelinating encephalitis in rats: A morphological analysis

Coronavirus glycoprotein El, a new type of viral glycoprotein

Viral protein synthesis in mouse hepatitis virus strain A59-infected cells: Effect of tunicamycin

Translation of three mouse hepatitis virus strain A59 subgenomic RNAs in Xenqpus la& Oocytes

Coronavirus JHM: Tryptic peptide fingerprinting of virion proteins and intracellular polypeptides

Coronavirus JHM: Coding assignments of subgenomic mRNAs

Coronavirus JHM: Cell-free synthesis of structural protein P60

The biology of coronaviruses

Coronavirus mRNA synthesis involves fusion of noncontiguous sequences

Isolation and identification of virus-specific mRNAs in cells infected with mouse hepatitis virus (MHV-A59)

Sequence relationships between the genome and the intracellular RNA species 1, 3, 6 and 7 of mouse hepatitis virus strain A59

Murine coronaviruses: Isolation and characterization of two plaque morphology variants of the JHM neurotropic strain

Genomic RNA of the murine coronavirus

Coronavirus JHM: Characterization of intracellular viral RNA. .I. Gen ViroL 54

The replication of murine coronaviruses in enucleated cells

Polyadenylate in the virion RNA of mouse hepatitis virus

We express our gratitude to Dr. S. G. Siddell for valuable discussion and correction of the manuscript, and Dr. Y. Yoshikawa for his suggestions and comments.