key: cord-0745350-lfx2a3qi
authors: Cheley, Steve; Anderson, Robert; Cupples, Margaret J.; Lee Chan, Edwin C.M.; Morris, Vincent L.
title: Intracellular murine hepatitis virus-specific RNAs contain common sequences
date: 1981-07-30
journal: Virology
DOI: 10.1016/0042-6822(81)90305-6
sha: 17eb21c8881c783fb85d87b1750895b05f20e671
doc_id: 745350
cord_uid: lfx2a3qi

Abstract A major polyadenylated viral RNA of approximately 0.8 × 106 daltons was isolated from murine hepatitis virus (A59)-infected cells by preparative polyacrylamide gel electrophoresis in formamide. This RNA was shown to encode the viral nucleocapsid protein by direct in vitro translation in a cell-free, reticulocyte-derived system. Single stranded 32P-labeled complementary DNA was prepared from this RNA and was demonstrated to be virus specific. Using this complementary DNA in a Northern blotting procedure, we were able to identify six major virus-specific intracellular RNA species with estimated molecular weights of 0.8, 1.1, 1.4, 1.6, 3, and 4 × 106 daltons. All of these RNA species were polyadenylated. Our results support the idea that coronavirus-infected cells contain multiple intracellular polyadenylated RNAs which share common sequences.

Coronaviruses have a single-stranded, polyadenylated, nonsegmented RNA genome which is infectious (Lai and Stohlman, 1978; Lomniczi, 19'77; Lomniczi and Kennedy, 19'77; Schochetman et aZ., 1977; Wege et al., 1978) . Molecular weight estimates of this RNA range from 3 to 9 X lo6 daltons (Lai and Stohlman, 1978; Lomniczi and Kennedy, 1977; MacNaughton and Madge, 1977; Tannock, 1973; Watkins et al., 1975; Wege et al., 1978) . Although little is known about the RNA replication strategy of coronaviruses, Stern and Kennedy (1980a, b) have recently identified a set of five polyadenylated subgenomic RNAs in cells infected with the avian coronavirus, infectious bronchitis virus (IBV). Oligonucleotide fingerprint analysis of these RNAs suggested common sequences among them (Stern and Kennedy, 1980a, b) . Evidence for multiple intracellular messenger RNAs (mRNAs) has also been presented in the case of another coronavirus, i To whom reprint requests should be addressed. murine hepatitis virus (MHV). Siddell et al. (1980) reported the separation of polyadenylated RNAs from MHV (JHM strain)-infected cells by means of sucrose gradients; fractions from these gradients were able to direct the in vitro translation of two viral proteins. However, the limited degree of resolution obtainable on sucrose gradients makes a more definitive analysis of MHV subgenomic RNAs highly desirable.

In the present report we provide firm evidence for the presence, in MHV-infected cells, of a set of six overlapping, polyadenylated viral RNAs. Our results provide independent support for the "nested set" concept of coronavirus mRNAs, as advanced by Stern and Kennedy (1980a, b) .

CeuS and virus. The A59 strain of MHV was obtained from the American Type Culture Collection. Virus was propagated and plaqued on the L-2 strain (Rothfels Measurement of [3HJuridine incorporation into irlfected cells. Confluent cultures of L-Z cells in 35-mm petri dishes were inoculated at a multiplicity of infection (m.o.i.) of 5 and incubated at 37" in minimum essential medium (MEM) supplemented with 5% fetal calf serum (FCS) and containing actinomycin D (1 pg/ml). At intervals, medium was replaced with 0.5 ml of MEM containing actinomycin D and [5-3H]uridine (15 &i/ml).

After 30 min incubation at 37" the cell monolayers were washed with phosphate-buffered saline (PBS) and the cells lysed with 0.5% sodium dodecyl sulfate (SDS). After the addition of 10% trichloroacetic acid to cell lysates, RNA was trapped by filtration through Whatman 3MM filter disks and the radioactivity determined by scintillation spectrometry.

Isolation of 32P-labeled iqfected cell RNA for fomzamide gel electrophoresis. For large-scale preparation of infected cell RNA, seventy-five lOO-mm petri dishes of confluent L-2 cells were inoculated at a m.o.i. of approximately 2 and subsequently incubated at 37" in MEM supplemented with 5% FCS and containing actinomycin D (1 pg/ml). At 3.5 hr postinoculation (PI) the medium in five petri dishes was replaced with phosphate-free medium containing actinomycin D and [32P]phosphate (800 &i/ml). At 8.5 hr PI, at which time the infected cell monolayers were completely fused, medium was removed from all '75 dishes and the cells washed once with PBS. Cells were scraped with a rubber policeman in a total of 36 ml RSB (10 mM Tris, 10 mM NaCl, 1.5 mM MgC12, pH 7.4) and extracted with chloroform/ phenol/isoamyl alcohol according to the procedure of Rose and Knipe (1975) . RNA was recovered from the aqueous phase by the addition of 2 vol of ethanol and centrifugation for 20 min at 10,000 g. Prior to formamide gel electrophoresis RNA was poly(A) selected by passage through oligo(dT)-cellulose (see below). Isolation of RNA for hybridization and trader procedures. Cellular RNA was prepared according to the procedure of Strohman et al. (1977) as modified by Dr. G. Mackie (Department of Biochemistry, University of Western Ontario). Briefly, cell monolayers were washed with PBS, scraped into a solution of 7.6 Mguanidine-HCl, 0.1 M potassium acetate, pH 7.5, and homogenized with a glass dounce in a bath of salt-ice. After addition of 0.6 vol of 95% ethanol and cooling for several hours at -2O", the mixture was spun at 3000 g for 10 min. The pellet was taken up in buffer containing 7.6 M guanidine-HCl, 0.02 M EDTA, and gently vortexed at room temperature. After addition of 2 M potassium acetate, pH 5, to a final concentration of 0.1 M, ethanol (0.5 vol) was added and the mixture centrifuged at 3000 g for 10 min. The pellet was dissolved in 20 mM EDTA, pH 7, and extracted at room temperature with 3 vol of chloroform/butanol (4:1, v/ v). The aqueous phase was combined with two additional aqueous washings of the organic phase and made 3 M in sodium acetate, pH 6. After cooling at -2O", the mixture was centrifuged at 8700 g for 60 min to yield an RNA pellet which was subsequently precipitated twice from ethanol.

Oligo(dT)-cellulose chromatography. Ethanol-precipitated RNA was dissolved in application buffer (10 mM Tris, 0.5 M KCl, pH 7.5) and applied to a column of oligo(dT)-cellulose (0.5 g, Collaborative Research, Inc.). Stepwise elution with decreasing concentration of KC1 was performed according to Aviv and Leder (1972) . Fractions containing "unbound" (eluted with 0.5 M KC1 buffer) and "bound" (eluted with KCl-free buffer) RNA were diluted with 2 vol of ethanol. Precipitated RNA was recovered by centrifugation for 20 min at 10,000 g.

Formamide-polyacrylamide gel electrophoresis. RNA samples dissolved in formamide-glycerol sample buffer (Duesberg and Vogt, 1973) were carefully applied to formamide-filled wells of a slab gel containing 3.5% acrylamide in formamide (Duesberg and Vogt, 1973) . Electrophoresis was performed for 20 hr at 100 V. Gels were enclosed in Saran Wrap, frozen to facilitate subsequent manipulation, and autoradiographed for several hours at -70".

Recovery of %P-labeled RNA from fmamide gels. Using an autoradiogram as template, 32P-containing RNA bands were excised from the gel, macerated through an l&gauge syringe needle, and eluted with extraction buffer (10 mM Tris, 0.4 ikf sodium acetate, 0.02% SDS, pH 7.4; Rose and Knipe, 1975) . Eluted RNA was freed from minute gel debris by centrifugation at 10,000 Q for 10 min, precipitated by addition of 2 vol of ethanol, and recovered by centrifugation for 3 hr at 100,000 g. Recovered RNA was re-poly(A) selected by oligo(dT)-cellulose chromatography as described above.

Preparation of complementa?-y DNA. Complementary DNA (cDNA) was synthesized, using the procedure of Shank et a2. (1978) , in the presence of actinomycin D, calf thymus DNA primers, purified AMV polymerase (supplied by Dr. J. Beard and the Office of Program Resources and Logistics, National Cancer Institute) and [32P]dCTP (Amersham, 400 Ci/mmol). The cDNA product was isolated according to the procedure of Cohen et a2. (1979) . Before use the cDNA was annealed (0.6 M NaCl, 68') with 500 pg of uninfected L cell RNA to a C,t of 230. The annealing mixture was then made 0.12 M in phosphate buffer and the single-stranded sequences were eluted from a hydroxyapatite column at 60" with 0.12 M phosphate buffer; the doublestranded sequences were then eluted from the column at 60" with 0.4 M phosphate buffer (Morris et al., 1977) . The fractions containing the single-stranded [32P]cDNA (approximately 80% of the total radioactivity applied) were collected, pooled, and used for the subsequent experiments. Complementary DNA made with this technique has been shown to be representative of most, if not all, of a viral RNA by its ability to detect all the restriction endonuclease fragments of viral DNA with an efficiency equal to that of iodinated viral RNA (Ringold et aZ.,'1978) .

Hybridixaticm kinetic experiments. Hybridization was performed using the procedure of Varmus et al., (1973) . Hybridization mixtures contained 1150 cpm [32P]cDNA per 15-~1 aliquot, excess unlabeled RNA, and 0.6 M NaCl, 2 mM EDTA, 40 miW Tris-HCl, pH 7.4. Reactions were incubated at 68" under mineral oil in plas-tic tubes. Aliquots were removed and the percentage of single-stranded cDNA was determined using Sl nuclease (Calbiochem), which specifically digests singlestranded DNA, using the conditions of Leong et al. (1972) .

Analysis using gel electrophoresis and RNA trader procedures. RNA was denatured with glyoxal, subjected to electrophoresis in 1.5% agarose gels, transferred to diazobenzyloxymethyl (DBM) paper, and hybridized with [32P]cDNA using a modification of the technique of McMaster and Carmichael (1977) and Alwine et al. (1977) as described in Coulter-Mackie et al. (1980) . The [%P]cDNA used in these experiments was preannealed (0.6 M NaCl, 68') with 500 pg of uninfected L cell RNA to a CJ of 150. The A59 virus-specific RNA species were visualized by autoradiography (Swanstrom and Shank, 1978) .

DNA extraction and hybridization. Uninfected L cells were resuspended in 0.02 M Tris-HCl (pH 7.2), 0.01 M EDTA (pH 7). After incubating this suspension with Pronase (1 mg/ml, self-digested for 2 hr at 37") and 1% SDS overnight, the aqueous phase was extracted three times with phenol-chloroform (1:l). The DNA was then dialyzed extensively against 0.05 M Tris-HCL (pH 7.2), 0.1 mM EDTA (pH 7). The solution was adjusted to 0.3 M NaOH, heated to 100" for 20 min, and quick-cooled in ice. After neutralization, the DNA was precipitated with ethanol at -2O", centrifuged at 16,300 g, and resuspended in 3 mM EDTA (pH 7). The DNA was then annealed with r2P]cDNA as described by Morris et al. (1977) except cell DNA was 6.1 mg/ml in the annealing mix instead of 3.5 mg/ml. Single-stranded DNA was assayed with Sl nuclease as previously described (Leong et al., 1972) .

Cell-free protein synthesis. RNA was preparatively isolated from formamidepolyacrylamide gels as described above. Preparation of micrococcal nuclease-pretreated rabbit reticulocyte lysates and conditions for cell-free protein synthesis were essentially as described by Pelham and Jackson (1976) . Following incubation, samples were prepared for electrophoresis on 10% polyacrylamide-SDS gels (Laemmli, 1970) by treatment of the incubation mixtures (25 ~1) with RNase A (final concentration, 4 pg/ml) for 15 min at room temperature followed by the addition of 25 ~1 dissociation buffer (7 mM Tris-phosphate, pH 6.8, 3.46 M mercaptoethanol, 30% glycerol, 6% SDS, and 0.006% bromphenol blue). Gels were dried and fluorographed according to the procedure of Bonner and Laskey (1974) .

The inhibition by actinomycin D of host cell DNA-dependent RNA synthesis has been reported to be noninhibitory for the replication of coronaviruses (Tyrrell et al., 1975) . In order to confirm that viral RNA could be detected in actinomycin D-treated, A59-infected L cells, the incorporation of [3H]uridine into acid-precipitable material was measured as a function of time following virus inoculation. The results, presented in Fig. 1 , demonstrated a sharp increase in RNA synthesis between 5 and 6 hr PI. During this time, virus-induced cell fusion was evident in approximately 20% (5 hr PI) to 80% (6 hr PI) of the total cell monolayer. The subsequent decline in RNA synthesis observed at 7 hr PI was likely attributable to loss of cells from the infected monolayer due to the observed detachment of cell syncytia from the plastic substrate.

Isolation of 32P-Labekd Viral 0.8 X lo6

Dalton RNA RNA extracted from %P-labeled, actinomycin D-treated, infected cells was poly(A) selected and subjected to polyacrylamide gel electrophoresis in formamide. Autoradiography of the gel showed the presence of several labeled RNA bands, the smallest and also most heavily labeled of which had an apparent molecular weight of 0.8 X 106. The estimated molecular weight of this species was determined by assigning a molecular weight of 1.75 X lo6 to 28 S mouse ribosomal RNA and 0.68 x lo6 to 18 S mouse ribosomal RNA (McMaster and Carmichael, 1977) . The 0.8 x lo6 dalton RNA was preparatively purified by excision and extraction of the gel, followed by re-poly(A) selection. An aliquot of the purified 0.8 X lo6 dalton RNA was examined for purity and intactness by reelectrophoresis. As can be seen in Fig.  2 , the purified RNA migrates as a single spot with only negligible contaminants. I&nt$icatim of the 0.8 X 16 Dalton RNA as Viral Messenger RNA for the Nucleocapsid Protein Translation of the purified 0.8 X lo6 dalton RNA in an in vitro, reticulocyte-derived translation system yielded a single viral polypeptide of 53K daltons (Fig. 3) . This polypeptide virtually comigrated with authentic viral nucleocapsid N protein present in an extract from ['?Qnethioninelabeled A59-infected L cells. Identity between the in vitro-synthesized 53K protein and the authentic viral N protein was confirmed by peptide mapping (data not shown). In addition, a polypeptide of approximately 42K daltons was consistently detected in the in vitro translation extract, whether RNA was added or not (Fig. 3) . This polypeptide is, therefore, likely to be the mRNA-independent 42K species orig- RNA from =P-labeled, A59-infected cells was separated by preparative gel electrophoresis and the 0.8 X lo6 dalton RNA band extracted from the gel (see Materials and Methods). An aliquot of this purified RNA was reexamined by gel electrophoresis; an autoradiogram is shown.

inally described by Pelham and Jackson (1976) and which has been observed by numerous other investigators using the reticulocyte-derived translation system.

We prepared single-stranded [=P]cDNA against isolated 0.8 X lo6 dalton RNA. This cDNA was then used, in an RNA transfer procedure, to identify intracellular A59specific RNA. As expected, this cDNA annealed predominantly with a 0.8 X lo6 dalton RNA species in A59-infected L cell RNA (Fig. 4, lane A) . However, the cDNA also showed significant annealing with five other RNA species with estimated molecular weights of 1.1, 1.4, 1.6, 3 and 4 X 106. (Fig. 4, lane A) . Similar results were obtained using poly(A)-selected A59-infected L cell RNA (Fig. 4, lane B) , indi-cating that all the observed RNA species, which share homology with the [=P]cDNA, were polyadenylated. These RNA species were visible in autoradiograms within several hours of exposure. In contrast to these results with infected L cell RNA, the cDNA showed no annealing with either uninfected L cell RNA (Fig. 4, lane C) or poly (A)-selected, uninfected L cell RNA (Fig. 4, lane D) . Furthermore, the cDNA showed no annealing with RNA which was poly(A) selected from as much as one mil- Nomenclature of viral proteins is as proposed by Sturman et al. (1980) except for PEI which we have shown (Cheley and Anderson, in press) to be a precursor polypeptide to El. The specificity of the cDNA prepared from 0.8 X lo6 dalton RNA was further tested by annealing it with tRNA, uninfected L cell RNA, poly(A)-selected, uninfected L cell RNA, and A59-infected L cell RNA. Actinomycin D was added to the A59-infected L cells to prevent the expression of endogenous L cell genes (including retrovirus genes). The results are shown in Table 1 . The tRNA hybridization data indicated that the [32P]cDNA showed no significant self annealing. In addition, the [32P]cDNA showed less than 10% annealing with either uninfected, or poly(A)-selected, uninfected L cell RNA. Thus, in agreement with the RNA transfer results, the cDNA had little if any homology with uninfected L cell RNA. In contrast, the cDNA annealed completely with A59-infected L cell RNA with a C&of 5 X low2 (using data from Table 1 ). This result, using the calculation of Varmus et al. (1973) , indicated that greater than 1000 genome equivalents were present per infected cell and was consistent with the observed rapid appearance of the major virus-specific RNA bands in the RNA transfer procedures (Fig. 4) .

All A59-infected cell RNA was prepared from cells infected in the presence of actinomycin D to inhibit expression of host cell genes. In addition, an aliquot of the [32P]cDNA was annealed with uninfected L cell DNA. This annealing was performed under conditions (0.6 M NaCl, 68", Cot = 1.6 X 104, DNA/cDNA ratio = 9 X 10') that would allow single copy DNA to anneal to within 90% or more of completion (Morris et al., 1977) . However, no annealing above background was observed between the [32P] cDNA and the L cell DNA. It is thus clear from the RNA transfer and the hybridization procedures that the major RNA bands identified by the [32P]cDNA are indeed virus specific.

The results of the present study provide definitive proof for the presence of six major polyadenylated, viral RNAs in MHV-infected cells. The smallest of these RNAs, a species of 0.8 X lo6 daltons, encodes the viral nucleocapsid protein. Moreover, and most significantly, this RNA contains sequences which are also contained within the five larger RNA species. Our results are thus analogous with the findings of Stern and Kennedy (1980a, b) that cells infected with the avian coronavirus, IBV, also contain a "nested set" of (Birnstiel et d, 1972) . b L cells infected with A59 virus in the presence of actinomycin D (see Methods). ' Not tested. ' C,t calculated using the starting amount of RNA before poly(A) selection (500 pg).

subgenomic RNAs. Taken together, the evidence suggests that coronaviruses, as exemplified by MHV and IBV, code for five to six polyadanylated subgenomic RNAs whose molecular weights lie within the range of approximately 0.8-4 X 106. The existence of homologous sequences within a set of viral RNAs suggests parallels with certain other positive-strand RNA viruses. In the replication of the alphavirus, Semliki Forest virus, structural and nonstructural polypeptides are translated from a 26 S RNA and a 42 S RNA, respectively. A copy of 26 S RNA sequence is present within the 42 S RNA molecule, although translation of the 42 S RNA appears to yield only nonstructural polypeptides (Glanville et aZ., 1976) . It has been suggested in studies with Semliki Forest virus (Glanville et al., 1976) as well as certain plant viruses (Shih and Kaesberg, 1973; Hunter et al., 1976) and polyoma virus (Hunter and Gibson, 1978 ) that translational initiation of eukaryotic mRNAs may occur at only a single site near the 5' terminus, and that additional coding regions located further downstream are not translated. Thus, the transcription of a set of viral mRNAs which share 3' end sequence homology but differ in length may be a common effective mechanism to ensure translational initiation of all viral genes by the host cell.

Contemporary analyses of MHV show the presence of three major size classes of structural proteins (Anderson et al., 1979; Bond et al., 1979; Sturman, 1977; Wege et al., 1979) as well as a polyadenylated, single-stranded, RNA genome of approximately 5.4 X lo6 daltons (Lai and Stohlman, 1978; Wege et al., 1978) . Using reported molecular weight estimates for the three major viral structural proteins of A59 (Sturman et cd., 1980) , i.e., a 18OK dalton glycoprotein, a 50K nucleocapsid protein, and a 23K protein, it can be calculated that somewhat less than half of the total coding capacity of the genome is occupied by sequences encoding these structural polypeptides. The remainder of the genome would presumably comprise nonstructural protein-encoding regions as well as any noncoding regions. Experiments performed on the JHM strain of MHV (Siddell et aZ., 1980) suggest that at least two viral structural polypeptides are encoded by separate mRNAs rather than by a single polycistronic mRNA as in the case of other positive-strand viruses such as alphaviruses (Mowshowitz, 1973) or enteroviruses such as poliovirus (Jacobson and Baltimore, 1968) . Our own studies with MHV (Cheley and Anderson, in press) have shown that each of the three major structural polypeptides is translationally initiated in an independent rather than in a sequential manner; this result is again indicative of the existence of separate viral mRNAs for each structural protein.

Assuming, therefore, three mRNAs for structural polypeptides and an approximately equal number for nonstructural proteins, the present finding of six major polyadenylated viral RNAs in MHV-infected cells is not inconsistent with the total number of mRNAs expected. We are currently attempting to verify this idea by characterization of the in vitro-directed translation products from each of the subgenomic RNA species.

Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes

Comparison of polypeptides of two strains of murine hepatitis virus

Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose

Kinetic complexity of RNA molecules

Pathogenic murine coronaviruses. II. Characterization of virus-specific proteins of murine coronaviruses JHMV and A59V. Virologl/ 94

A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels

Cellular synthesis and modification of murine hepatitis viral polypeptides

Integration of the DNA of mouse mammary tumor virus in virus-infected normal and neoplastic tissue of the mouse

vivo and in vitro models of demyelinating diseases

Isolation of Halle measles virus-specific RNA from BGMK cells and preparation of complementary DNA

Gel electrophoresis of avian leukosis and sarcoma viral RNA in formamide: Comparison with other viral and cellular RNA species

Initiation of translation directed by 42s and 26s RNAs from Semliki Forest Virus in vitro

Messenger RNA for the coat protein of tobacco mosaic virus

Characterization of the mRNA's for the polyoma virus capsid proteins VPl, VP2 and VP3

Polypeptide cleavages in the formation of poliovirus proteins

Cleavage of structural proteins during the assembly of the head of bacteriophage T4

RNA of mouse hepatitis virus

Virus specific ribonucleic acid in cells producing Rous sarcoma virus: Detection and characterization

Biological properties of avian coronavirus RNA

Genome of infectious bronchitis virus

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

The characterisation of the virion RNA of avian infectious bronchitis virus

Comparison of mouse mammary tumor virus-specific DNA in inbred, wild and asian mice, and in tumors and normal organs from inbred mice

Identification of polysomal RNA in BHK cells infected by Sindbis virus

An efficient mRNA-dependent translation system from reticulocyte lysates

Production of unintegrated mouse mammary tumor virus DNA in infected rat hepatoma cells is a secondary action of dexamethasone

Nucleotide sequence complexities, molecular weights and poly (A) content of the vesicular stomatitis virus mRNA species

The origin of altered cell lines from mouse, monkey and man as indicated by chromosome and transplantation studies

Presence of infectious polyadenylated RNA in the coronavirus avian bronchitis virus

Mapping of linear and circular forms of mouse mammary tumor virus DNA with restriction endonucleases: Evidence for a large specific deletion occurring at high frequency during circularization

Translation of brome mosaic viral ribonucleic acid in a cell-free system derived from wheat embryo

Coronavirus JHM: Cell-free synthesis of structural protein ~60

Coronavirus multiplication strategy. I. Identification and characterization of virus-specified RNA

Coronavirus multiplication strategy. II. Mapping the avian infectious bronchitis virus intracellular RNA species to the genome

Messenger RNA for myosin polypeptides: Isolation from single myogenic cell cultures

Characterization of a coronavirus. I. Structural proteins: Effects of preparative conditions on the migration of protein in polyacrylamide gels

Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid

X-Ray intensifying screens greatly enhance the detection by autoradiography of the radioactive isotopes =P and '=I

The nucleic acid of infectious bronchitis virus

COronaviridae: Second report

Transcription of mouse mammary tumor virus genes in tissues from high and low tumor incidence mouse strains

The ribonucleic acid of infectious bronchitis virus

Genomic RNA of the murine coronavirus JHM

Structural polypeptides of the murine coronavirus JHM

We are grateful to H. Shida for advice and assistance with the cell-free translation system. We also thank R. Hartwick for his help with the photography. This work was supported by grants from the Medical Research Council of Canada, Grant MA-5941 to R. Anderson and Grant MA-7321 to V. L. Morris.