Effect of p15-associated protease from an avian RNA tumor virus on avian virus-specific polyprotein precursors.

A proteolytic activity is associated with structural protein p15 in avian RNA tumor viruses. Its effect on the known intracellular viral polyprotein precursors obtained by immunoprecipitation was investigated. Cleavage of Pr76gag resulted in the sequential appearance of p15, p27, and p19. The intracellular precursor Pr180gag-pol was also cleaved by p15, whereas the intracellular glycoprotein precursors of avian RNA tumor viruses, Pr92env, remained unaffected by p15 under all conditions tested. The specificities of the antibodies used to precipitate the precursors influenced the pattern of intermediates and cleavage products obtained by p15 treatment. If virus harvested from the the Prague strain of Rous sarcoma virus, subgroup C-transformed cells at 15-min intervals was incubated at 37 degrees C for further maturation, RNA-dependent DNA polymerase activity showed an optimum of DNA synthesis with 70S viral RNA or synthetic template-primers after short incubation periods. The presence of additional p15 during incubation resulted in a shift of the enzyme activity peak toward earlier time points. Virus harvested at 3-h intervals contained significant amounts of Pr180gag-pol and Pr76gag. The addition of p15 resulted in the cleavage of Pr180gag-pol and Pr76gag, but only a few distinct low-molecular-weight polypeptides appeared. Treatment of purified RNA-dependent DNA polymerase with p15 in vitro resulted in a disappearance of the beta subunit and an enrichment of the alpha subunit. In addition, a polypeptide of 32 x 10(3) molecular weight was generated. The cleavage pattern observed differed from the one obtained by trypsin treatment.     

Amino-terminal amino acid sequence of p10, the fifth major gag polypeptide of avian sarcoma and leukemia viruses.

We have identified p10 as a fifth gag protein of avian sarcoma and leukemia viruses. Amino-terminal protein sequencing of this polypeptide purified from the Prague C strain of Rous sarcoma virus and from avian myeloblastosis virus implies that it is encoded within a stretch of 64 amino acid residues between p19 and p27 on the gag precursor polypeptide. For p10 from the Prague C strain of Rous sarcoma virus the first 30 residues were found to be identical with the predicted amino acid sequence from the Prague C strain of Rous sarcoma virus DNA sequence, whereas for p10 from avian myeloblastosis virus the protein sequence for the same region showed two amino acid substitutions. Amino acid composition data indicate that there are no gross composition changes beyond the region sequenced. The amino terminus of p10 is located two amino acid residues past the carboxy terminus of p19, whereas its carboxy terminus probably is located immediately adjacent to the first amino acid residue of p27.     

Primary structure of p19 species of avian sarcoma and leukemia viruses.

The internal structural proteins of avian sarcoma and leukemia viruses are derived from a precursor polypeptide that is the product of the viral gag gene. The N-terminal domain of the precursor gives rise to p19, a protein that interacts with the lipid envelope of the virus and that may also interact with viral RNA. The C terminus of p19 from the Prague C strain of Rous sarcoma virus was previously assigned to a tyrosine residue 175 amino acids from the N terminus. We have used metabolic labeling and carboxypeptidase digestion to show that the C terminus of p19 is actually tyrosine 155. This implies the existence of a sixth gag protein 22 amino acids in length and located between p19 and p10 on the gag precursor. The p19 species of some recombinant avian sarcoma viruses and of the defective endogenous virus derived from the ev-1 locus migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as if they were about 4,000 daltons smaller than p19. We have elucidated the structure of these forms, called p19 beta, by analysis of the proteins and determination of the DNA sequence of the p19 region of the gag gene from ev-1 and ev-2. Esterification of carboxyl groups completely suppressed the differences in migration of p19 and p19 beta. Peptide mapping showed the altered mobility to be determined by sequences in the C-terminal cyanogen bromide fragment of the proteins. We conclude from the DNA sequence that a single glutamate-lysine alteration is responsible for the altered electrophoretic mobility.     

Rous sarcoma virus expression in Saccharomyces cerevisiae: processing and membrane targeting of the gag gene product.

In avian cells, the product of the gag gene of Rous sarcoma virus, Pr76gag, has been shown to be targeted to the plasma membrane, to form virus particles, and then to be processed into mature viral gag proteins. To explore how these phenomena may be dependent upon cellular (host) factors, we expressed the Rous sarcoma virus gag gene in a lower eucaryote, Saccharomyces cerevisiae, and studied the behavior of the gag gene product. We show here that Pr76gag is processed in yeast cells and that this processing is specific, since it is abolished in a mutant in which the active site of the gag protease has been destroyed. In this mutant, the uncleaved precursor is found associated with the yeast plasma membrane, yet no virus particles were detected in cells or in the culture medium. From our results, we can speculate either that in yeast cells, a host protease initiates Pr76gag processing in the cytosol or that in avian cells, an inhibitor prevents the processing until the viral particle is formed.     

Feline sarcoma virus-coded polyprotein: enzymatic cleavage by a type C virus-coded structural protein.

A purified 15,000-molecular-weight (Mr) Prague strain Rous sarcoma virus gag gene-coded structural protein, p15, was shown to enzymatically cleave the previously described 130,000 Mr feline sarcoma virus-coded polyprotein, Pr130. Cleavage products included proteins ranging in molecular weight from 12,000 to 110,000. The specificity of this cleavage reactivity was indicated by the fact that, under similar conditions, neither purified type C viral structural proteins nor nonviral proteins such as bovine serum albumin were cleaved to significant extents. Moreover, feline leukemia virus Pr65gag was efficiently cleaved, resulting in the generations of proteins of 30,000 (p30), 15,000 (p15), 12,000 (p12), and 10,000 (p10) Mr. Using enzymatically (p15) treated feline sarcoma virus Pr130 as starting material, we were able to purify a major 72,000 Mr cleavage product and to show it to contain the previously described feline sarcoma virus-coded nonstructural component.     

Isolation of monoclonal antibodies against avian oncornaviral protein p19

For the production of monoclonal antibodies against pp60src and the gag precursor protein Pr76gag, the spleens of mice bearing tumors that had been induced by avian sarcoma virus Schmidt-Ruppin D-transformed cells were used. One hybridoma culture produced antibodies that were directed against the p19 portion of the gag precursor. However, no antibodies directed against pp60src could be detected in any of the hybridoma supernatants. The anti-p19-producing hybridoma culture was cloned twice in soft agar, and a stable clone was used for the production of high-titer ascites fluid in mice. The monoclonal antibodies belonged to the immunoglobulin G subclass 2b. The antibodies precipitated Pr76gag and the processed virion-associated p19, as well as the 75,000-molecular-weight gag fusion protein from avian erythroblastosis virus-transformed bone marrow cells. Also, viral ribonucleoprotein complexes were specifically precipitable, indicating that they contain p19 molecules.     

Creation and expression of myristylated forms of Rous sarcoma virus gag protein in mammalian cells.

Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, has long been known to be capable of infecting and transforming mammalian cells; however, such transformed cells do not release virus particles. The RSV gag product (Pr76gag) produced in these cells is not released into the culture medium or proteolytically processed to release mature products. Thus, the behavior of Pr76gag in mammalian cells is much like that of mammalian retroviral Gag proteins which have been altered so as to block the addition of myristic acid at residue 2 (Gly). Because the RSV gag product does not possess a myristic acid addition site, we hypothesized that the creation of one by oligonucleotide-directed mutagenesis might permit particles to be released from mammalian cells. Two myristylated forms of Pr76 were created. In Pr76myr1, the first 10 amino acids have been exchanged for those of p60v-src, which are known to be sufficient for myristylation. In Pr76myr2, the Glu at the second residue has been substituted with Gly. The alleles encoding the modified and wild-type forms of Pr76 have been expressed at high levels in mammalian (CV-1) cells by using an SV40-based vector. Surprisingly, we have found that expression of high levels of the unmodified (wild-type) product, Pr76myr0, results in low levels of particle formation and precursor processing. This indicates that myristic acid is not the sole determinant for targeting. However, the addition of myristic acid to Pr76myr1 or Pr76myr2 resulted in a fivefold enhancement in Gag function. In all aspects examined, the behavior of myristylated Pr76 was identical to that of the authentic product produced in avian cells. We also show that processing is mediated by the gag-encoded protease and that removal of the amino terminus to create Pr76gagX results in an inability to form particles or be processed. This suggests that proper targeting is prerequisite for activation of the RSV protease in mammalian cells.     

gag-Related polypeptides encoded by replication-defective avian oncoviruses.

The content of viral structural (gag) protein sequences in polypeptides encoded by replication-defective avian erythroblastosis virus (AEV) and myelocytomatosis virus MC29 was assessed by immunological and peptide analyses. Direct comparison with gag proteins of the associated helper viruses revealed that MC29 110K polypeptide contained p19, p12, and p27, whereas the AEV 75K polypeptide had sequences related only to p19 and p12. Both of these polypeptides contained some information that was unrelated to gag, pol, or env gene products. In addition, no homology was detected between these unique peptides of MC29 110K and AEV 75K. The AEV 75K polypeptide shared strain-specific tryptic peptides with the p19 encoded by its naturally occurring helper virus; this observation suggests that gag-related sequences in 75K were originally derived from the helper viral gag gene. Digestion of oxidized MC29 110K and AEV 75K proteins with the Staphylococcus aureus V8 protease generated a fragment which comigrated with N-acetylmethionylsulfoneglutamic acid, a blocked dipeptide which is the putative amino-terminal sequence of structural protein p19 and gag precursor Pr76gag. This last finding is evidence that the gag sequences are located at the N-terminal end of the MC29 110K and AEV 75K polypeptides.     

Role of the gag polyprotein precursor in packaging and maturation of Rous sarcoma virus genomic RNA.

Rous sarcoma virus nucleocapsid protein (NC) has been shown by site-directed mutagenesis to be involved in viral RNA packaging and in the subsequent maturation of genomic RNA in the progeny viral particles. To investigate whether NC exerts these activities as a free protein or as a domain of the polyprotein precursor Pr76gag, we have constructed several mutants unable to process Pr76gag and analyzed their properties in a transient-transfection assay of chicken embryo fibroblasts, the natural host of Rous sarcoma virus. A point mutation in the protease (PR) active site completely prevents Pr76gag processing. The full-length Pr76gag polyprotein is still able to package viral RNA, but cannot mature it. A shorter gag precursor polyprotein lacking the C-terminal PR domain, but retaining that of the NC protein, is however, unable even to package viral RNA. This indicates that the NC protein can participate in packaging viral RNA only as part of a full-length Pr76gag and that the PR domain is, indirectly or directly, also involved in RNA packaging. These results also demonstrate that processing of Pr76gag is necessary for viral RNA dimerization.     

Characterization of ribosome binding on Rous sarcoma virus RNA in vitro.

We determined the sites at which ribosomes form initiation complexes on Rous sarcoma virus RNA in order to determine how initiation of Pr76gag synthesis at the fourth AUG codon from the 5' end of Rous sarcoma virus strain SR-A RNA occurs. Ribosomes bind almost exclusively at the 5'-proximal AUG codon when chloride is present as the major anion added to the translational system. However, when chloride is replaced with acetate, ribosomes bind at the two 5'-proximal AUG codons, as well as at the initiation site for Pr76gag. We confirmed that the 5'-proximal AUG codon is part of a functional initiation site by identifying the seven-amino acid peptide encoded there. Our results suggest that (i) translation in vitro of Rous sarcoma virus virion RNA results in the synthesis of at least two polypeptides; (ii) the pattern of ribosome binding observed for Rous sarcoma virus RNA can be accounted for by the modified scanning hypothesis; and (iii) the interaction between 40S ribosomal subunits or 80S ribosomal complexes is stronger at the 5'-proximal AUG codon than at sites farther downstream, including the initiation site for the major viral proteins.     

In vivo modification of retroviral gag gene-encoded polyproteins by myristic acid.

It has recently been shown by mass spectral analysis (Henderson et al., Proc. Natl. Acad. Sci. U.S.A. 80:339-343, 1983) that the p15gag protein of murine leukemia viruses contains a novel post-translational modification, an amino-terminal myristyl (tetradecanoyl) amide. In this report we show that p15gag is the only structural protein to contain this fatty acid. In addition, the gag precursor polyproteins of type B, C, and D retroviruses have been examined for the presence of myristic acid by metabolic labeling and immunoprecipitation studies. In a panel of mammalian type C retroviruses we found that the precursor polyprotein Pr65gag homologs, but not the glycosylated forms (gPr80gag homologs), were specifically labeled after a 5-min incubation of infected cells with [3H]myristic acid. The gag precursor polyprotein was also labeled in mouse mammary tumor virus and in Mason-Pfizer monkey virus, but Pr76gag of Rous sarcoma virus failed to incorporate [3H]myristate. Under similar conditions, [3H]palmitate was not found to be incorporated into any viral gag proteins. Thus, myristylation appears to be a common feature of mammalian type B, C, and D retroviruses but not of avian retroviruses.     

Effects of inhibitors of lipid synthesis on the replication of Rous sarcoma virus. A specific effect of cerulenin on the processing of major non-glycosylated viral structural proteins.

The effects of two inhibitors of lipid biosynthesis on the replication of Rous sarcoma virus Prague C strain in chick embryo fibroblasts have been examined in media containing delipidated serum. 25-Hydroxycholestetate into sterols, had no effect on the formation of infectious virions or on the synthesis and processing of intracellular virion proteins. Cerulenin strongly inhibited [1(-14C)]acetate incorporation into fatty acids and partially inhibited its incorporation into sterols in chick embryo cells. Rous sarcoma virus production as measured by focus formation and by the production of [35S]methionine-labeled virions was strongly inhibited within 5 h after cerulenin addition to infected cultures. Examinatin of extracts of these cells revealed the accumulation of the 76 000 dalton precursor (Pr76) of the major non-glycosylated virion structural proteins, p27, p19, p15 and p12. The failure to process the 76 000 dalton precursor was coincident in time with the decrease in viron production. Neither whole serum nor mixtures of fatty acids plus cholesterol were able to reverse the effects of cerulenin.     

Effects of inhibitors of lipid synthesis on the replication of rous sarcoma virus. A specific effect of cerulenin on the processing of major non-glycosylated viral structural proteins

The effects of two inhibitors of lipid biosynthesis on the replication of Rous sarcoma virus Prague C strain in chick embryo fibroblasts have been examined in media containing delipidated serum. 25-Hydroxycholesterol, which markedly inhibits the incorporation of [1-¹⁴C]acetate into sterols, had no effect on the formation of infectious virions or on the synthesis and processing of intracellular virion proteins. Cerulenin strongly inhibited [1-¹⁴C]acetate incorporation into fatty acids and partially inhibited its incorporation into sterols in chick embryo cells. Rous sarcoma virus production as measured by focus formation and by the production of [³⁵S]methionine-labeled virions was strongly inhibited within 5 h after cerulenin addition to infected cultures. Examination of extracts of these cells revealed the accumulation of the 76 000 dalton precursor (Pr76) of the major non-glycosylated virion structural proteins, p27, p19, p15 and p12. The failure to process the 76 000 dalton precursor was coincident in time with the decrease in viron production. Neither whole serum nor mixtures of fatty acids plus cholesterol were able to reverse the effects of cerulenin.     

Quantitative separation of murine leukemia virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products.

The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag cleavage products (p15, p12, p30, and p10 plus p10') were found in equimolar amounts and p15(E)s were equimolar with p2(E)s. The stoichiometry of gag to env cleavage products was 4:1. These data are consistent with the proposal that proteolytic processing of precursor polyproteins occurs after virus assembly and that the C-terminal portion of Pr15(E) [i.e., p15(E)-p2(E)] is located on the inner side of the lipid bilayer of the virus.     

Identification of a ribosome-binding site for a leader peptide encoded by Rous sarcoma virus RNA.

A new method for identifying ribosome-binding sites was developed to determine whether AUG codons in the 5'-terminal RNA sequence of Rous sarcoma virus were used to initiate protein synthesis. We found that when translation is inhibited, the major ribosome-binding site on Rous sarcoma virus RNA is at the 5'-proximal AUG codon, even though the primary translational product from this RNA, Pr76gag, is encoded behind the fourth AUG codon 331 bases downstream from the observed initiation site. These results suggest that ribosomes can initiate translation on Rous sarcoma virus RNA at more than one site, thereby producing a seven-amino-acid peptide, as well as the gag gene polyprotein precursor of Mr 76,000.     

Localization of lipid-protein and protein-protein interactions within the murine retrovirus gag precursor by a novel peptide-mapping technique

In HTG2 hamster cells infected with the replication-defective Gazdar murine sarcoma virus only immature virus particles are formed, with the uncleaved gag precursor Pr65 as the only major protein in the virion. We have investigated the structure of these particles by using in situ cross-linking followed by chemical and enzymatic cleavages of Pr65 to localize sites of lipid-protein and protein-protein interactions. Lipid-protein cross-links were localized within a 10-kDa fragment in the p15 region of Pr65. Homotypic protein-protein cross-links between Pr65 units were localized within the p15 regions and also within the p10 regions of Pr65. Similar data for processed gag proteins in Rauscher murine leukemia virus, a prototype of a mature C-type virus, suggest that these interactions of the gag precursor are not altered during maturation. To identify the sites of cross-linking within Pr65, we have developed a two-dimensional peptide mapping technique that is based on nearest neighbor analysis of fragments released by cyanogen bromide treatment of partial cleavage products in gel slices. In conjunction with cross-linking, the peptide mapping technique is a powerful means for localizing specific interactions on a polypeptide backbone.     

Correlation of RNA binding affinity of avian oncornavirus p19 proteins with the extent of processing of virus genome RNA in cells.

We purified the p19 proteins from the Prague C strain of Rous sarcoma virus, avian myeloblastosis virus, B77 sarcoma virus, myeloblastosis-associated virus-2(0), and PR-E 95-C virus and measured their binding affinities for 60S viral RNA by the nitrocellulose filter binding technique. The apparent association constants of the p19 proteins from Rous sarcoma virus Prague C, avian myeloblastosis virus, and B77 sarcoma virus for homologous and heterologous 60S RNAs were similar (1.5 x 10(11) to 2.6 x 10(11) liters/mol), whereas those of myeloblastosis-associated virus-2(0) and PR-E 95-C virus were 10-fold lower. The sizes and relative amounts of the virus-specific polyadenylic acid-containing RNAs in the cytoplasms of cells infected with Rous sarcoma virus Prague C, myeloblastosis-associated virus-2(0), and PR-E 95-C virus were determined by fractionating the RNAs on agarose gels containing methylmercury hydroxide, transferring them to diazobenzyloxymethyl paper and hybridizing them to a 70-nucleotide complementary DNA probe. In cells infected with Rous sarcoma virus Prague C we detected 3.4 x 10(6)-, 1.9 x 10(6)-, and 1.1 x 10(6)-dalton RNAs, in PR-E 95-C virus-infected cells we detected 3.4 x 10(6)-, 1.9 x 10(6)- and 0.7 x 10(6)-dalton RNAs, and in cells infected with myeloblastosis-associated virus-2(0) we detected 3 x 10(6)- and 1.3 x 10(6)-dalton RNAs. Each of these RNA species contained RNA sequences derived from the 5' terminus of genome-length RNA, as evidenced by hybridization with the 5' 70-nucleotide complementary DNA. The ratios of subgenomic mRNA's to genome-length RNAs in cells infected with myeloblastosis-associated virus-2(0) and PR-E 95-C virus were three- to five-fold higher than the ratio in cells infected with Rous sarcoma virus Prague C. These results suggest that more processing of viral RNA in infected cells is correlated with lower binding affinities of the p19 protein for viral RNA, and they are consistent with the hypothesis that the p19 protein controls processing of viral RNA in cells.     

Molecular characterization of gag proteins from simian immunodeficiency virus (SIVMne).

A simian immunodeficiency virus (SIV) designated SIVMne was isolated from a pig-tailed macaque with lymphoma housed at the University of Washington Regional Primate Research Center, Seattle. To better establish the relationship of SIVMne to other immunodeficiency viruses, we purified and determined the partial amino acid sequences of six structural proteins (p1, p2, p6, p8, p16, and p28) from SIVMne and compared these amino acid sequences to the translated nucleotide sequences of SIVMac and human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). A total of 125 residues of SIVMne amino acid sequence were compared to the predicted amino acid sequences of the gag precursors of SIV and HIVs. In the compared regions 92% of the SIVMne amino acids were identical to predicted residues of SIVMac, 83% were identical to predicted residues of HIV-2, and 41% were identical to predicted residues of HIV-1. These data reveal that the six SIVMne proteins are proteolytic cleavage products of the gag precursor (Pr60gag) and that their order in the structure of Pr60gag is p16-p28-p2-p8-p1-p6. Rabbit antisera prepared against purified p28 and p16 were shown to cross-react with proteins of 60, 54, and 47 kilodaltons present in the viral preparation and believed to be SIVMne Pr60gag and intermediate cleavage products, respectively. SIVMne p16 was shown to contain covalently bound myristic acid, and p8 was identified as a nucleic acid-binding protein. The high degree of amino acid sequence homology between SIVs and HIV-2 around proven proteolytic cleavage sites in SIV Pr60gag suggests that proteolytic processing of the HIV-2 gag precursor is probably very similar to processing of the SIV gag precursor. Peptide bonds cleaved during proteolytic processing of the SIV gag precursor were similar to bonds cleaved during processing of HIV-1 gag precursors, suggesting that the SIV and HIV viral proteases have similar cleavage site specificities.