Rous Sarcoma Virus Gag Gene Research Articles

Site-Directed Mutagenesis of the P2 Region of the Rous Sarcoma Virus gag Gene: Effects on Gag Polyprotein Processing

The Pr76Gag and Pr180Gag-Pol polyprotein precursors of Rous sarcoma virus contain a 22-amino-acid spacer peptide, called p2, located between the amino acid sequences of the mature Gag proteins MA and p10. This spacer peptide is present in stolchiometric amounts in the virion, albeit cleaved into two parts, but its function is unknown. The primary sequence of this peptide includes a region that is highly conserved among retroviruses, consisting of four prolines followed by tyrosine. We have Investigated the role of p2, particularly the polyproline motif, in the virus life cycle by site-directed mutagenesis. Mutations in this region result in the intracellular accumulation of a truncated Gag precursor, due either to a block in the intracellular processing of the precursor or to the premature activation of the viral protease. Since in cells infected by Rous Sarcoma Virus there is no significant intracellullar processing of the Gag polyprotein precursor, our data suggest that the p2 domain plays a role in controlling the activation of the protease. These mutations also result in a reduction in virus particle release, probably as a direct consequence of the aberrant precursor processing since a construct In with both p2 and the protease active site were mutated did not exhibit aberrant processing of the Gag polyproteins and formed particles with an efficiency similar to that of the wild type. This indicates that it is the viral protease that is responsible for the aberrant processing and suggests that the p2 region is not required for assembly. Although the virus genomic RNA packaged Into virions produced by the p2 mutants is more susceptible to degradation, it appears that the p2 domain does not have a direct role in RNA packaging and protection.

Characterization of Rous sarcoma virus intronic sequences that negatively regulate splicing

Retroviruses splice only a fraction of their primary RNA transcripts to subgenomic mRNA. The unspliced RNA is transported to the cytoplasm, where it serves as genomic RNA as well as mRNA for the gag and pol genes. Deletion of sequences from the Rous sarcoma virus gag gene, which is part of the intron of the subgenomic mRNAs, was previously observed to result in an increase in the ratio of spliced to unspliced RNA. These sequences, which we termed a negative regulator of splicing (NRS), can be moved to the intron of a heterologous gene resulting in an accumulation of unspliced RNA in the nucleus. We have used such constructs, assayed by transient expression in chicken embryo fibroblasts, to define the minimal sequences necessary to inhibit splicing. Maximal NRS activity was observed with a 300-nt fragment containing RSV nts 707–1006; two noncontiguous domains within this fragment, one of which contains a polypyrimidine tract, were both found to be essential. The NRS element was active exclusively in the sense orientation in two heterologous introns tested and in both avian and mammalian cells. Position dependence was also observed, with highest activity when the NRS was inserted in the intron near the 5′ splice site. The NRS element was also active at an exon position 136 nts upstream of the 5′ splice site but not at sites further upstream. In addition, it did not affect the splicing of a downstream intron.

Nonsense codons within the Rous sarcoma virus gag gene decrease the stability of unspliced viral RNA.

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. In contrast, the levels of spliced viral RNAs were not affected in our transient expression assays in chicken cells. Experiments using the transcription inhibitor dactinomycin showed that mutant unspliced RNAs were degraded more rapidly than wild-type RNA. Furthermore, mutant RNAs could be partially stabilized by coexpression of wild-type gag proteins in trans; however, intact gag proteins were not required to maintain the stability of RNAs which did not contain premature termination codons. Thus, termination codons seemed to destabilize the RNA not because of their effect on gag protein function but instead because they disrupted the process of translating the gag region of the RNA. Analysis of double-mutant constructs containing both deletions and termination codons within the gag gene also suggested that the stability of the unspliced RNA was affected by a cis-acting interaction between the RNA and ribosomes.

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.

Proposed gag-encoded transcriptional activator is not necessary for Rous sarcoma virus replication or transformation

It has been reported that gene expression directed by the long terminal repeat of Rous sarcoma virus (RSV) is trans activated by a protein encoded in an alternate reading frame within the RSV gag gene (S. Broome and W. Gilbert, Cell 40:537-546, 1985). We have made specific mutations to test the role of the putative transcriptional activator in RSV replication. Termination codons were created within the alternate reading frame coding for the trans activator, and the mutations were introduced into an infectious RSV plasmid. We were unable to demonstrate specific trans activation of the RSV long terminal repeat by either wild-type or mutant RSV plasmids in transient cotransfection assays. Experiments using mutant or wild-type RSV-infected chick embryo fibroblasts indicated that the proposed RSV transcriptional activator was not required for viral replication or transformation and did not increase steady-state levels of viral RNA.