High-frequency recombination within the gag gene of Rous sarcoma virus.

We isolated 28 recombinants of Rous sarcoma virus at early (24 h) and late (7 days) times after infection. These recombinants were selected for wild type in the pol and src genes and analyzed for their env and gag phenotypes. We were unable to show strong linkage between any two markers, including two markers within a single gene (gag).     

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.     

Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain of gag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.     

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.     

Functional chimeras of the Rous sarcoma virus and human immunodeficiency virus gag proteins.

The Gag protein encoded by Rous sarcoma virus (RSV) is the only viral product required for the process of budding whereby virus particles are formed at the plasma membrane. Deletion analysis of this Gag molecule has revealed several regions (assembly domains) that are important for budding. One of these domains is located at the amino terminus and is needed for membrane binding. Another is located within the carboxy-terminal third of the protein. Though there is little sequence homology among the Gag proteins of unrelated retroviruses, it seemed possible that their assembly domains might be functionally conserved, and to explore this idea, numerous Gag chimeras were made. The results indicate that the first 10 amino acids of the human immunodeficiency virus (HIV) Gag protein can suppress the block to budding caused by deletions in the RSV MA sequence, much as described previously for the first 10 residues from the Src oncoprotein (J.W. Wills, R.C. Craven, R. A. Weldon, Jr., T. D. Nelle, and C.R. Erdie, J. Virol. 65:3804-3812, 1991). In addition, the carboxy-terminal half of the HIV Gag protein was fused to a truncated RSV Gag molecule, mutant Bg-Bs, which is unable to direct core assembly. This chimera was able to produce particles at a rate identical to that of RSV and of a density similar to that of authentic virions. Deletion analysis of the carboxy-terminal chimera revealed two small regions within the HIV NC protein that were sufficient for endowing mutant Bg-Bs with these properties. Chimeras lacking both regions produced particles of a low density, suggesting that these sequences may be involved in the tight packing of Gag molecules during assembly. In a related set of experiments, replacement of the RSV protease with that of HIV resulted in premature processing within the RSV sequence and a block to budding. Particle assembly was restored when the HIV PR activity was inactivated by mutagenesis. Collectively, the data presented here illustrate the functional similarities of Gag proteins from unrelated retroviruses.     

Complementation studies with Rous sarcoma virus gag and gag-pol polyprotein mutants.

Avian retroviruses (with the notable exception of spleen necrosis virus) express their protease (PR) both in their gag and their gag-pol polyprotein precursors, in contrast to other retroviruses, notably, the mammalian retroviruses, in which PR is encoded in the gag-pol polyprotein or in a separate reading frame as a gag-pro product. The consequence is that the avian PR is expressed in stoichiometric rather than catalytic amounts. To investigate the significance of the particular genome organization of the avian retrovirus prototype Rous sarcoma virus, we developed an assay that measures complementation between the gag and the gag-pol polyproteins by expressing them from two different plasmids in transfected cells. By using this assay, we showed that the protease PR from the gag-pol polyprotein is capable of autocatalytic self-cleavage and -activation when coexpressed with a protease-deficient gag protein and that the PR domain has a role in viral particle assembly. Furthermore, this complementation assay can be used to investigate the role of the gag domain in the gag-pol polyprotein by determining whether it can rescue a defect in the gag polyprotein. We report here the results of such an experiment, which studied a mutation in the N terminus of the gag gene.     

Role of the Rous sarcoma virus p10 domain in shape determination of gag virus-like particles assembled in vitro and within Escherichia coli

Purified retrovirus Gag proteins can assemble in vitro into virus-like particles (VLPs) in the presence of RNA. It was shown previously that a Rous sarcoma virus Gag protein missing only the protease domain forms spherical particles resembling immature virions lacking a membrane but that a similar protein missing the p10 domain forms tubular particles. Thus, p10 plays a role in spherical particle formation. To further study this shape-determining function, we dissected the p10 domain by mutagenesis and examined VLPs assembled within Escherichia coli or assembled in vitro from purified proteins. The results identified a minimal contiguous segment of 25 amino acid residues at the C terminus of p10 that is sufficient to restore efficient spherical assembly to a p10 deletion mutant. Random and site-directed mutations were introduced into this segment of polypeptide, and the shapes of particles formed in E. coli were examined in crude extracts by electron microscopy. Three phenotypes were observed: tubular morphology, spherical morphology, or no regular structure. While the particle morphology visualized in crude extracts generally was the same as that visualized for purified proteins, some tubular mutants scored as spherical when tested as purified proteins, suggesting that a cellular factor may also play a role in shape determination. We also examined the assembly properties of smaller Gag proteins consisting of the capsid protein-nucleocapsid protein (CA-NC) domains with short N-terminal extensions or deletions. Addition of one or three residues allowed CA-NC to form spheres instead of tubes in vitro, but the efficiency of assembly was extremely low. Deletion of the N-terminal residue(s) abrogated assembly. Taken together, these results imply that the N terminus of CA and the adjacent upstream 25 residues play an important role in the polymerization of the Gag protein.     

Repositioning basic residues in the M domain of the Rous sarcoma virus gag protein

The first 86 residues of the Rous sarcoma virus (RSV) Gag protein form a membrane-binding (M) domain that directs Gag to the plasma membrane during budding. Unlike other retroviral Gag proteins, RSV Gag is not myristylated; however, the RSV M domain does contain 11 basic residues that could potentially interact with acidic phospholipids in the plasma membrane. To investigate this possibility, we analyzed mutants in which basic residues in the M domain were replaced with asparagines or glutamines. The data show that neutralizing as few as two basic residues in the M domain blocked particle release and prevented Gag from localizing to the plasma membrane. Though not as severe, single neutralizations also diminished budding and, when expressed in the context of proviral clones, reduced the ability of RSV to spread in cell cultures. To further explore the role of basic residues in particle production, we added lysines to new positions in the M domain. Using this approach, we found that the budding efficiency of RSV Gag can be improved by adding pairs of lysines and that the basic residues in the M domain can be repositioned without affecting particle release. These data provide the first gain-of-function evidence for the importance of basic residues in a retroviral M domain and support a model in which RSV Gag binds to the plasma membrane via electrostatic interactions.     

Rous sarcoma virus does not induce sarcoma in early chick embryos by lack of the v-src gene expression.

The Schmidt-Ruppin or the B77 strain of Rous sarcoma virus (RSV) was inoculated into limb buds of 4.5-days-old avian embryos. No sarcoma but blister formation was observed in those RSV-inoculated embryos. Protein kinase activity of pp60v-src in RSV-inoculated embryos, even in the site of virus inoculation, was the same as that in mock-infected embryos. This indicated that the expression of the v-src gene did not attain superiority over that of the c-src gene in RSV-inoculated embryos. The v-src gene was detected in every DNA from tissues of RSV-inoculated embryos but not in DNAs from tissues of RSV-inoculated chicken except for the DNA from Rous sarcoma. Those results confirmed that the lack of sarcoma induction in early avian embryos by RSV was due to the lack of the expression of the v-src gene which was present in the target cells.     

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.     

Cell surface changes and Rous sarcoma virus gene expression in synchronized cells

We have investigated whether cell surface changes associated with growth control and malignant transformation are linked to the cell cycle. Chicken embryo cells synchronized by double thymidine block were examined for cell-cycle-dependent alterations in membrane function (measured by transport of 2-deoxyglucose, uridine, thymidine, and mannitol), in cell surface morphology (examined by scanning electron microscopy), and in the ability of tumor virus gene expression to induce a transformation-specific change in membrane function. We reach the following conclusions: (a) The high rate of 2-deoxyglucose transport seen in transformed cells and the low rates of 2-deoxyglucose and uridine transport characteristic of density-inhibited cells do not occur in normal growing cells as they traverse the cell cycle. (b) Although there are cell cycle-dependent changes in surface morphology, they are not reflected in corresponding changes in membrane function. (c) Tumor virus gene expression can alter cell membrane function at any stage in the cell cycle and without progression through the cell cycle.     

Fine mapping and characterization of the Rous sarcoma virus Pr76gag late assembly domain.

The p2 region of the Rous sarcoma virus (RSV) Gag polyprotein contains an assembly domain, which is required late in replication for efficient budding of virus-like particles from cells (J. W. Wills, C. E. Cameron, C. B. Wilson, Y. Xiang, R. P. Bennett, and J. Leis, J. Virol. 68:6605-6618, 1994). This domain, referred to as the L domain, was previously mapped to the 11 amino acids of p2b. Through the analysis of a series of deletion and substitution mutations, the L domain has now been fine mapped to a highly conserved amino acid sequence, PPPPYV of p2b. Sequences flanking PPPPYV motif can be deleted without any effect on budding. Defects caused by L-domain deletions can be rescued by placing a wild-type copy of the sequence at several other positions in RSV Gag. A proline-rich P(S/T)APP motif is found in many retroviral Gag polyproteins; the motif found in the p6 region of human immunodeficiency virus type 1 has been implicated in late functions of the virus. Substitution of the RSV L domain with this motif in a 10-amino-acid sequence derived from visna leukemia virus results in wild-type release of virus particles from cells. In contrast, the slightly different sequences from Gibbon ape leukemia virus, Moloney leukemia virus, PSAPP alone, or a proline-rich SH3 binding sequence do not efficiently rescue RSV L-domain mutations.