Heteroduplex Analysis of the Sequence Relationships Between the Genomes of Kirsten and Harvey Sarcoma Viruses, Their Respective Parental Murine Leukemia Viruses, and the Rat Endogenous 30S RNA

The sequence relations between Kirsten murine sarcoma virus (Ki-SV), Harvey murine sarcoma virus (Ha-SV), and a rat endogenous 30S RNA were studied by electron microscope heteroduplex analysis. The sequence relationships between the sarcoma viruses and their respective parental murine leukemia viruses (Kirsten and Moloney murine leukemia viruses), as well as between the two murine leukemia viruses, were also studied. The only observed nonhomology feature of the Kirsten murine leukemia virus/Moloney murine leukemia virus heteroduplexes was a substitution loop with two arms of equal length extending from 1.80 +/- 0.18 kilobases (kb) to 2.65 +/- 0.27 kb from the 3' end of the RNA. It is believed that this feature lies in the env gene region of the viral genomes. The Ha-SV and Moloney murine leukemia virus genomes (respective lengths, 6.0 and 9.0 kb) were homologous in a 1.0 +/- 0.05-kb region at the 3' end and possibly over a 200-nucleotide region at the 5' ends; otherwise, they were nonhomologous. Ha-SV and Ki-SV (length, 7.5 kb) were homologous in the first 4.36 +/- 0.37-kb region from the 3' end and in a 0.70 +/- 0.15-kb region at the 5' end. In between, there was a nonhomology region, possibly containing a short (0.23-kb) region of partial or total homology. The heteroduplex analysis between rat endogenous 30S RNA and Ki-SV shows that there are mixed regions of sequence homology and nonhomology at both the 5' and 3' ends. However, there is a large (4-kb) region of homology between Ki-SV and the rat 30S RNA in the center of the genomes, with only a small nonhomology hairpin feature. These studies help to define the regions of homology between the Ha-SV and Ki-SV genomes with each other and with the rat endogenous 30S RNA. These regions may be related to the sarcoma genicity of the viruses. In particular, the 0.7-kb region of homology of Ha-SV with Ki-SV at the 5' ends may be related to the formation of a 21,000-dalton phosphoprotein in cells transformed by either virus.     

Gene mapping in Pichinde virus: assignment of viral polypeptides to genomic L and S RNAs.

Previous studies have demonstrated that Pichinde virus encodes at least three primary translation products. Using wild-type Pichinde and Munchique viruses and a reassortant between the two, designated RE-2, we were able to assign polypeptides L, GPC, and NP to viral L and S RNAs. The RE-2 virus contains the L RNA of Pichinde virus and the S RNA of Munchique virus. Two-dimensional tryptic peptide mapping of L-[35S]methionine-containing peptides demonstrated that NP and GPC were identical in Munchique and RE-2 viruses, and both differed from the corresponding Pichinde virus tryptic profiles. On the basis of this, NP and GPC must be encoded by viral S RNA. Similar comparisons for L polypeptide demonstrated that L is a virus-specific polypeptide encoded by L RNA.     

Formation of a Sindbis virus nonstructural protein and its relation of 42S mRNA function.

Chicken embryo fibroblasts infected with an RNA- temperature-sensitive mutant (ts24) of Sindbis virus accumulated a large-molecular-weight protein (p200) when cells were shifted from the permissive to nonpermissive temperature. Appearance of p200 was accompanied by a decrease in the synthesis of viral structural proteins, but [35S]methionine tryptic peptides from p200 were different from those derived from a 140,000-molecular-weight polypeptide that contains the amino acid sequences of viral structural proteins. Among three other RNA- ts mutants that were tested for p200 formation, only one (ts21) produced this protein. The accumulation of p200 in ts24- and ts21-infected cells could be correlated with a shift in the formation of 42S and 26S viral RNA that led to an increase in the relative amounts of 42S RNA. These data indicate that p200 is translated from the nonstructural genes of the virion 42S RNA and further suggest that this RNA does not function effectively in vivo as an mRNA for the Sindbis virus structural proteins.     

Cell-free translation of virion RNA from nondefective and transformation-defective Rous sarcoma viruses.

Nondefective and transformation-defective virion subunit RNAs from two strains of Rous sarcoma virus (RSV) were translated in cell-free systems derived from Krebs IIA ascites cells, wheat germ, and L-cells. In each case the predominant viral-specific product was a polypeptide of molecular weight 76,000 that is related to the internal viral group-specific antigens, as judged by immunoprecipitation with monospecific antisera and tryptic peptide fingerprinting. No difference could be detected between the translation products of 35S RNA from nondefective and transformation-defective RSV virions, nor of 35S RNA from different strains of RSV. The 76,000-molecular-weight polypeptide synthesized in response to 35S RNA in vitro was labeled with formyl-methionine from initiator tRNA. Models for viral protein synthesis are discussed in the light of these results, and arguments positioning the group-specific antigen gene at the 5' end of the 35S RNA are presented.     

Translation and processing of mouse hepatitis virus virion RNA in a cell-free system.

The first event after infection with mouse hepatitis virus strain A59 (MHV-A59) is presumed to be the synthesis of an RNA-dependent RNA polymerase from the input genomic RNA. The synthesis and processing of this putative polymerase protein was studied in a cell-free translation system utilizing 60S RNA from MHV-A59 virions. The polypeptide products of this reaction included two major species of 220 and 28 kilodaltons. Kinetics experiments indicated that both p220 and p28 appeared after 60 min of incubation and that protein p28 was synthesized initially as the N-terminal portion of a larger precursor protein. When the cell-free translation products were labeled with N-formyl[35S]methionyl-tRNAi, p28 was the predominant radioactive product, confirming its N-terminal location within a precursor protein. Translation in the presence of the protease inhibitors leupeptin and ZnCl2 resulted in the disappearance of p28 and p220 and the appearance of a new protein, p250. This product, which approached the maximal size predicted for a protein synthesized from genomic RNA, was not routinely detected in the absence of inhibitors even under conditions which optimized the translation reaction for elongation of proteins. Subsequent chelation of ZnCl2 resulted in the partial cleavage of the precursor protein and the reappearance of p28. One-dimensional peptide mapping with Staphylococcus aureus V-8 protease confirmed the precursor-product relationship of p250 and p28. The results show that MHV virion RNA, like many other viral RNAs, is translated into a large polyprotein, which is cleaved soon after synthesis into smaller, presumably functional proteins. This is in marked contrast to the synthesis of other MHV proteins, in which minimal proteolytic processing occurs.     

Expression of a potyvirus non-structural protein in transgenic tobacco

A cDNA fragment encoding the cytoplasmic inclusion protein of tobacco vein mottling virus was inserted into the plant expression cassette of a Ti plasmid-based binary vector. The vector was transferred to Agrobacterium tumifaciens, and following a modified leaf disc procedure, transformed tobacco plants were obtained. Analysis of poly(A)+ RNA from transgenic plants revealed a novel RNA of approximately 2100 nucleotides possessing tobacco vein mottling virus sequences. Also, immunoprecipitation of protein extracts of [35S]methionine-labeled transformed callus using anti-cytoplasmic inclusion protein antiserum revealed a polypeptide of approximately 70 kDa. This size is consistent with that predicted from the inserted tobacco vein mottling virus coding sequences. Together these data demonstrate the expression of the cytoplasmic inclusion protein in the absence of viral infections.     

Respiratory syncytial virus proteins: identification by immunoprecipitation.

The proteins of respiratory syncytial virus have not been clearly identified due to the lability of the virus and difficulties in its purification. We have pulse-labeled respiratory syncytial virus with [35S]methionine and [35S]cysteine and analyzed cell lysates by polyacrylamide gel electrophoresis. Five 35S-labeled viral proteins ranging in molecular weight from 21,000 to 73,000 (VP73, VP44, VP35, VP28, and VP21) were easily discernable above background cellular proteins. Treatment of the infected cells with 0.15 M NaCl before labeling suppressed host cell protein synthesis and allowed clearer visualization of the five viral proteins by polyacrylamide gel electrophoresis. Three glycoproteins (VGP 92, VGP 50, and VGP 17) were also identified after labeling with [3H]glucosamine. Five of these polypeptides (VP51, VP44, VP35, VP28, and VGP92) were shown to be antigenically active because they could be immunoprecipitated with anti-respiratory syncytial virus antibody produced in New Zealand white rabbits, cotton rats, and humans before analysis by polyacrylamide gel electrophoresis.     

Analysis of structural polypeptides of purified human cytomegalovirus.

Human cytomegalovirus strain C87 was purified by the following procedures. (i) Extracellular virus was concentrated by centrifugation at 100,000 X g for 90 min and passed through a Bio-Rad Bio-Gel A-15m column. Most of the virus was recovered in the void volume. (ii) After two consecutive isopycnic potassium tartrate gradient centrifugations (20 to 50%), coinciding peaks of plaque titer, protein, and radioactivity were found at a density of from 1.20 to 1.21 g/cm3. To characterize the structural polypeptides of human cytomegalovirus and to establish relative purification criteria, virus was purified from two mixtures: (i) [35S]methionine-labeled extracellular virus mixed with an equal volume of unlabeled normal culture fluid; (ii) unlabeled extracellular virus mixed with an equal volume of [357a1methionine-labeled normal culture fluid. The extent of purification, as judged by the ratio of cellular to viral radioactivity, was 39-fold; i.e. about 2.5% of the protein in the purified virus preparation could be accounted for by host protein contamination. Electrophoresis of purified [35S]methionine-labeled virus on a polyacrylamide gel slab showed that there were at least 33 viral structural polypeptides (VPs), and their molecular weights ranged from 11,000 to 290,000. Autoradiograms obtained from electropherograms of purified [14C]glucosamine labeled virus showed six bands. Four of these were so broad that several VPs corresponded to each of the glycosylated bands. When heavy (two fractions close to 1.21 g/cm3) and light (two fractions close to 1.20 g/cm3) fractions of the PFU peak from the second potassium tartrate gradient were analyzed separately, the number of polypeptides observed was the same, but the relative amounts of some polypeptides differed. The major polypeptide, VP17, was found in greater amounts in the heavy fraction (35%) than in the light fraction (22%). The amount of DNA as a percentage of the weight of protein was 2% for the light fraction and 1% for the heavy fraction.     

Vaccinia virus morphogenesis is interrupted when expression of the gene encoding an 11-kilodalton phosphorylated protein is prevented by the Escherichia coli lac repressor.

A conditional lethal vaccinia virus mutant, which constitutively expresses the Escherichia coli lac repressor and has the lac operator controlling the F18R gene (the 18th open reading frame of the HindIII F fragment of the vaccinia virus strain WR genome) encoding an 11-kDa protein, was previously shown to be dependent on the inducer isopropyl-beta-D-thiogalactoside (IPTG) for replication (Y. Zhang and B. Moss, Proc. Natl. Acad. Sci. USA 88:1511-1515, 1991). Further studies indicated that the yield of infectious virus could be regulated by titration with IPTG and that virus production was arrested by IPTG removal at appropriate times. Under nonpermissive conditions, an 11-kDa protein reactive with antiserum raised to a previously described DNA-binding phosphoprotein (S. Y. Kao and W. R. Bauer, Virology 159:399-407, 1987) was not synthesized, indicating that the latter is the product of the F18R gene. In the absence of IPTG, replication of viral DNA and the subsequent resolution of concatemeric DNA molecules appeared normal. Omission of IPTG did not alter the kinetics of early and late viral protein synthesis, although the absence of the 11-kDa polypeptide was noted by labeling infected cells with [35S]methionine or [32P]phosphate. Pulse-chase experiments revealed that proteolytic processing of the major viral structural proteins, P4a and P4b, was inhibited under nonpermissive conditions, suggesting a block in virus maturation. Without addition of IPTG, the failure of virus particle formation was indicated by sucrose gradient centrifugation of infected cell lysates and by the absence of vaccinia virus-mediated pH-dependent cell fusion. Electron microscopic examination of infected cells revealed that immature virus particles, with aberrant internal structures, accumulated when synthesis of the 11-kDa DNA-binding protein was prevented.     

Purification and characterization of c-Ki-ras p21 from bovine brain crude membranes

In the present studies, we attempted to purify the native molecular forms of the c-ras proteins (c-ras p21s) from bovine brain crude membranes and separated at least three GTP-binding proteins (G proteins) cross-reactive with the antibody recognizing all of Ha-, Ki-, and N-ras p21s. Among them, one G protein with a Mr of about 21,000 was highly purified and characterized. The Mr 21,000 G protein bound maximally about 0.6 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 30 nM. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by pretreatment with N-ethylmaleimide. Mr 21,000 G protein hydrolyzed GTP to liberate Pi with a turnover number of about 0.01 min-1. Mr 21,000 G protein was not copurified with the beta gamma subunits of the G proteins regulatory for adenylate cyclase. Mr 21,000 G protein was not recognized by the antibody against the ADP-ribosylation factor for Gs. The peptide map of Mr 21,000 G protein was different from those of the G proteins with Mr values of 25,000 and 20,000, designated as smg p25A and rho p20, respectively, which we have recently purified from bovine brain crude membranes. The partial amino acid sequence of Mr 21,000 G protein was identical with that of human c-Ki-ras 2B p21. These results indicate that Mr 21,000 G protein is bovine brain c-Ki-ras 2B p21 and that c-Ki-ras 2B p21 is present in bovine brain membranes.     

Detection of a Precursor Polypeptide of the Rapidly-Synthesized 32,000-Dalton Thylakoid Protein in Spinach Chloroplasts

Spinach chloroplast RNA was translated in a wheat germ cell-freesystem in the presence of [³⁵S]methionine or [³H]lysine, andthe products were analyzed by SDS polyacrylamide gel electrophoresisand fluorography. A polypeptide with molecular mass of 2,000-Dalarger than the 32,000-Da thylakoid protein was detected asa major product labeled by [³⁵S]methionine but not by [³H]lysine.Peptide mapping of this polypeptide showed a pattern very closeto that of the 32,000-Da protein synthesized in isolated chloroplasts.A better separation of this polypeptide from the 32,000-Da proteinwas observed in the electrophoresis on polyacrylamide gel includingurea at 8 M. Pulse-labeling of the isolated chloroplasts showedthe occurrence of the larger molecular weight form, which wasconverted to the mature size by a chasing incubation with coldmethionine. These results suggested that the 32,000-Da proteinof spinach is translated primarily as a high molecular weightprecursor in the chloroplasts, as has been reported for otherplant species. (Received March 30, 1985; Accepted April 23, 1985)     

Translation of three mouse hepatitis virus strain A59 subgenomic RNAs in Xenopus laevis oocytes

We have purified the seven virus-specific RNAs which were previously shown to be induced in Sac(-) cells upon infection with mouse hepatitis virus strain A59 (W. J. M. Spaan, P. J. M. Rottier, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 108:424-434, 1981). The individual RNAs, prepared by agarose gel electrophoresis of the polyadenylated RNA fraction from infected cells, were obtained pure, except for the preparations of RNAs 4, 5, and 6, which contained some contamination of RNA 7. The RNAs were microinjected into Xenopus laevis oocytes, and after incubation of these cells in the presence of [35S]methionine, the proteins synthesized were analyzed by polyacrylamide gel electrophoresis. Whereas no translation products of RNAs 1, 2, 4, and 5 were detected, the synthesis of virus-specific polypeptides coded by RNAs 3, 6, and 7 was observed. RNA 7 (0.6 X 10(6) daltons) directed the synthesis of a 54,000-molecular-weight polypeptide which comigrated with viral nucleocapsid protein and which was immunoprecipitated by antiserum from mice that had been infected with the virus. RNA 6 (0.9 X 10(6) daltons) directed the synthesis of three polypeptides with molecular weights of 24,000, 25,500, and 26,500, which migrated with the same electrophoretic mobilities as three low-molecular-weight virion polypeptides. After injection of RNA 3 (3.0 X 10(6) daltons), a polypeptide with a molecular weight of about 150,000 was immunoprecipitated. This polypeptide had no counterpart in the virion, but comigrated with a virus-specific glycoprotein present in infected cells which is immunoprecipitated by a rabbit antiserum against the mouse hepatitis virus strain A59 structural proteins. This antiserum could also immunoprecipitate the translation products of RNAs 3, 6, and 7. These results indicate that RNAs 3, 6, and 7 encode viral structural proteins. The significance of the data with respect to the strategy of coronavirus replication is discussed.     

The use of immunosorption for the analysis of influenza virus RNA in intracellular viral ribonucleoproteins

Protein A-containing formaldehyde-fixed S. aureus (strain Cowan) was incubated with an antiviral serum or with a monospecific serum against NP protein, washed, and used as immunosorbent in order to isolate viral ribonucleoproteins (nucleocapsids) containing intact viral RNA from the extracts of influenza virus infected [3H]-uridine-labelled cells.     

Identification of the DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus.

The DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus were located on the viral genome. The formation of an enzyme-guanylate covalent intermediate labeled with [alpha-32P]GTP allowed the identification of the large subunit of the capping enzyme and was used to monitor the appearance of the enzyme during the infectious cycle. This assay confirmed that after vaccinia infection, a novel 84,000-molecular-weight polypeptide corresponding to the large subunit was rapidly synthesized before viral DNA replication. Hybrid-selected cell-free translation of early viral mRNA established that vaccinia virus encoded a polypeptide identical in molecular weight with the 32P-labeled 84,000-molecular-weight polypeptide found in vaccinia virions. Like the authentic capping enzyme, this virus-encoded cell-free translation product bound specifically to DNA-cellulose. A comparison of the partial proteolytic digestion fragments generated by V8 protease, chymotrypsin, and trypsin demonstrated that the 32P-labeled large subunit and the [35S]methionine-labeled cell-free translation product were identical. The mRNA encoding the large subunit of the capping enzyme was located 3.1 kilobase pairs to the left of the HindIII D restriction fragment of the vaccinia genome. Furthermore, the mRNA was determined to be 3.0 kilobases in size, and its 5' and 3' termini were precisely located by S1 nuclease analysis.     

Polyoma virus complementary RNA directs the in vitro synthesis of capsid proteins VP1 and VP2.

Polyoma virus complementary RNA, synthesized in vitro by using highly purified Escherichia coli RNA polymerase and nondefective form I polyoma DNA, was translated in a wheat germ cell-free system. Polypeptides were synthesized that comigrated on sodium dodecyl sulfate-polyacrylamide gels with the polyoma capsid proteins VP1 and VP2, although most of the cell-free products were of smaller molecular weights. The VP1-size protein specifically immunoprecipitated with anti-polyoma virus serum, and upon digestion by trypsin yielded [35S]methionine-labeled tryptic peptides that co-chromatographed with the [3H]methionine-labeled tryptic peptides of virion-derived VP1 on both cation-exchange and anion-exchange resins. The VP2-size in vitro product contained all the virion VP2 methionine-labeled tryptic peptides, as shown by cation- and anion-exchange chromatography and two-dimensional fingerprinting on cellulose. We conclude that full-length polyoma VP1 and VP2 are synthesized in response to complementary RNA and consequently that the viral capsid proteins VP1, VP2, and VP3 are entirely virus coded.     

Messenger Activity of Virion RNA for Avian Leukosis Viral Envelope Glycoprotein

An intracellular assay for viral envelope glycoprotein (env) messenger was employed to analyze the RNA from virus particles of Rous-associated virus type 2. For this assay RNA was microinjected into cells infected by the env-deficient Bryan strain of Rous sarcoma virus [RSV(-) cells]. Only when the injected RNA could be translated by the recipient cells to produce viral envelope glycoprotein was the env deficiency of the RSV(-) cells complemented, enabling them to release focus-forming virus. RNA in a 21S size fraction from the Rous-associated virus particle promoted the release of numerous focus-forming virus from RSV(-) cells, whereas the major 35S virion RNA species was inactive. The env messenger activity sedimented as a sharp peak with high specific activity. RNase T1-generated fragments of virion 35S RNA were unable to promote the release of infectious virus from RSV(-) cells. Consequently, the active molecule was most likely to be env messenger which had been encapsulated by the virus particle from the cytoplasm of infected cells. Approximately 95% of the env messenger within the virion was associated with the virion high-molecular-weight RNA complex. The temperature required to dissociate env messenger from the high-molecular-weight complex was indistinguishable from the temperature required to disrupt the complex itself. Virion high-molecular-weight RNA that was associated with env messenger sedimented slightly more rapidly than the bulk virion RNA; this was the strongest evidence that the 21S messenger had been encapsulated directly from the infected cells. These data are considered along with a related observation [concerning the prolonged expression of env messenger after injection into RSV(-) cells] to raise the possibility that virus-encapsulated env messenger can become expressed within subsequently infected cells.     

Defective mutant of Sindbis virus with a smaller-molecular-weight form of the E1 glycoprotein.

We have isolated from a single plaque a mutant of Sindbis virus characterized by an E1 glycoprotein with higher electrophoretic mobility. This higher mobility is not attributable to a different extent of glycosylation of the protein nor to an altered proteolytic maturation pathway of the polypeptide precursor, but is the result of a deletion occurring during the replication of the viral RNA. The 26S RNA (the messenger for the Sindbis structural proteins) extracted from cells infected with the mutant is about 0.75 x 10(5) daltons smaller than the 26S RNA from the parental strain. As a consequence, in cells infected with the mutant, an E1 glycoprotein is synthesized with a polypeptide chain about 70 amino acids shorter. The biological relevance of this naturally occurring deletion of the viral genome is discussed.