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PubMed2006

The negative regulator of splicing element of Rous sarcoma virus promotes polyadenylation.

The Rous sarcoma virus gag gene contains a cis-acting negative regulator of splicing (NRS) element that is implicated in viral polyadenylation regulation. To study the mechanism of polyadenylation promotion at the viral poly(A) site located over 8 kb downstream, we performed in vitro polyadenylation analysis. RNA containing only the poly(A) site and flanking sequences in the 3' long terminal repeat (LTR) was not polyadenylated detectably in vitro; however, if the transcript contained the NRS upstream of the LTR, polyadenylation was observed. Insertion of the viral env 3' splice site sequence between the NRS and the LTR did not alter the level of polyadenylation appreciably. We conclude that the NRS promotes polyadenylation in vitro and can do so without formation of a splicing complex with a 3' splice site. We then explored the roles of several cellular factors in NRS-mediated polyadenylation. Mutation of the binding sites of U1 and U11 snRNPs to the NRS did not affect polyadenylation, whereas hnRNP H strongly inhibited polyadenylation. We propose a model in which hnRNP H and SR proteins compete for binding to the NRS. Bound SR proteins may bridge between the NRS and the 3' LTR and aid in the recruitment of the 3'-end processing machinery.

Avian Sarcoma Viruses↗
PubMed1993

Analysis of deletions and thermosensitive mutations in Rous sarcoma virus gag protein p10.

Rous sarcoma virus protein p10 is a gag component of the virion present in stoichiometric amount but of unknown function. To characterize this protein, a series of mutants of p10 with linker insertions or deletions was generated by site-directed mutagenesis of a cloned proviral DNA. The deletions and two of the linkers insertions, which disrupted proline pairs, reduced the yield of virus particles upon transfection. These two linker insertion mutants were moreover thermosensitive for this phenotype, producing fewer virus particles at 41 degrees C than at 36 degrees C. Examination of the intracellular viral proteins demonstrated that for all mutants, the amount of gag precursor was similar to the wild-type level. Moreover, the amount of mature gag CA that could be detected by this analysis was similar between each of the mutants and the wild type. This finding suggests that the transport of gag to the membrane and the initial stages of maturation were not affected by the mutations. The virus particles contained normal amounts of active reverse transcriptase, showing that the gag-pol polyprotein was incorporated and cleaved properly. Viral RNA was quantitatively and qualitatively similar in mutant and wild-type virions. However, the infectivity of the mutants virions differed; one of the thermosensitive linker insertions that had no effect on particle production at 36 degrees C was nevertheless noninfectious at that temperature. Together, these data suggest that the p10 protein is involved in a late steps of virus maturation, possibly budding, and perhaps also in an early event of viral infection.

Amino Acid Sequence↗
PubMed2005

Detailed mapping of the nuclear export signal in the Rous sarcoma virus Gag protein.

The Rous sarcoma virus (RSV) Gag polyprotein undergoes transient nuclear trafficking as an intrinsic part of the virus assembly pathway. Nuclear export of Gag is crucial for the efficient production of viral particles and is accomplished through the action of a leptomycin B (LMB)-dependent nuclear export signal (NES) in the p10 domain (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc. Natl. Acad. Sci. USA 99:3944-3949, 2002). We have now mapped the nuclear export activity to the C-terminal portion of the p10 sequence and identified the four hydrophobic amino acids within this region that comprise a leucine-rich NES. Alteration of these hydrophobic residues resulted in the accumulation of Gag proteins within the nucleus and a budding defect greater than that obtained with LMB treatment of cells expressing the wild-type Gag protein (Scheifele et al., Proc. Natl. Acad. Sci. USA 99:3944-3949, 2002). In addition, export of Gag from the nucleus was found to be a rate-limiting step in virus-like particle production. Consistent with a role for the NES sequence in viral replication, this cluster of hydrophobic residues in p10 is conserved across a wide range of avian retroviruses. Furthermore, naturally occurring substitutions within this region in related viruses maintained nuclear export activity and remained sensitive to the activity of LMB. Using gain-of-function approaches, we found that the hydrophobic motif in p10 was sufficient to promote the nuclear export of a heterologous protein and was positionally independent within the Gag polyprotein. Finally, the export pathway was further defined by the ability of specific nucleoporin inhibitors to prevent the egress of Gag from the nucleus, thereby identifying additional cellular mediators of RSV replication.

Active Transport, Cell Nucleus↗
PubMed1972

Virus-specific ribonucleic acid in cells producing rous sarcoma virus: detection and characterization.

Cells producing Rous sarcoma virus contain virus-specific ribonucleic acid (RNA) which can be identified by hybridization to single-stranded deoxyribonucleic acid (DNA) synthesized with RNA-directed DNA polymerase. Hybridization was detected by either fractionation on hydroxyapatite or hydrolysis with single strand-specific nucleases. Similar results were obtained with both procedures. The hybrids formed between enzymatically synthesized DNA and viral RNA have a high order of thermal stability, with only minor evidence of mismatched nucleotide sequences. Virus-specific RNA is present in both nuclei and cytoplasm of infected cells. This RNA is remarkably heterogeneous in size, including molecules which are probably restricted to the nucleus and which sediment in their native state more rapidly than the viral genome. The nature of the RNA found in cytoplasmic fractions varies from preparation to preparation, but heterogeneous RNA (ca. 4-50S), smaller than the viral genome, is always present in substantial amounts.

Aspergillus↗
PubMed2004

Insertion of a classical nuclear import signal into the matrix domain of the Rous sarcoma virus Gag protein interferes with virus replication.

The Rous sarcoma virus Gag protein undergoes transient nuclear trafficking during virus assembly. Nuclear import is mediated by a nuclear targeting sequence within the MA domain. To gain insight into the role of nuclear transport in replication, we investigated whether addition of a "classical " nuclear localization signal (NLS) in Gag would affect virus assembly or infectivity. A bipartite NLS derived from nucleoplasmin was inserted into a region of the MA domain of Gag that is dispensable for budding and infectivity. Gag proteins bearing the nucleoplasmin NLS insertion displayed an assembly defect. Mutant virus particles (RC.V8.NLS) were not infectious, although they were indistinguishable from wild-type virions in Gag, Gag-Pol, Env, and genomic RNA incorporation and Gag protein processing. Unexpectedly, postinfection viral DNA synthesis was also normal, as similar amounts of two-long-terminal-repeat junction molecules were detected for RC.V8.NLS and wild type, suggesting that the replication block occurred after nuclear entry of proviral DNA. Phenotypically revertant viruses arose after continued passage in culture, and sequence analysis revealed that the nucleoplasmin NLS coding sequence was deleted from the gag gene. To determine whether the nuclear targeting activity of the nucleoplasmin sequence was responsible for the infectivity defect, two critical basic amino acids in the NLS were altered. This virus (RC.V8.KR/AA) had restored infectivity, and the MA.KR/AA protein showed reduced nuclear localization, comparable to the wild-type MA protein. These data demonstrate that addition of a second NLS, which might direct MA and/or Gag into the nucleus by an alternate import pathway, is not compatible with productive virus infection.

Active Transport, Cell Nucleus↗
PubMed1997

In vitro assembly of virus-like particles with Rous sarcoma virus Gag deletion mutants: identification of the p10 domain as a morphological determinant in the formation of spherical particles.

Retroviruses are unusual in that expression of a single protein, Gag, leads to budding of virus-like particles into the extracellular space. We have developed conditions under which virus-like particles are formed spontaneously in vitro from fragments of Rous sarcoma virus (RSV) Gag protein purified after expression in Escherichia coli. The CA-NC fragment of Gag was shown previously to assemble into hollow cylinders (S. Campbell and V. M. Vogt, J. Virol. 69:6487-6497, 1995). We have now extended these studies to larger Gag proteins. In every case examined, assembly into regular structures required RNA. A nearly full-length Gag missing only the C-terminal PR domain, as well as similar proteins missing in addition the N-terminal half of MA, the C-terminal half of MA, the entire MA sequence, or the entire p2 sequence, all assembled into spherical particles resembling RSV in size. By contrast, proteins missing p10 assembled into cylindrical particles like those formed by CA-NC alone. Thin section electron microscopy showed that each of these Gag proteins formed in the expressing E. coli cells particles similar in shape to those seen in vitro. We conclude from these results that neither the sequences required for membrane binding in vivo, near the N terminus of Gag, nor the sequences required for a late step in budding, in the p2 portion of Gag, are essential for formation of virus-like particles in this system. Furthermore, we postulate the existence of a shape-determining sequence in p10, which provides or facilitates interactions required for the growing particle to be constrained to a spherical shape.

Avian Sarcoma Viruses↗
PubMed1989

Evaluation of the cocarcinogenic effect of wounding in Rous sarcoma virus tumorigenesis.

Chickens given injections of Rous sarcoma virus form sarcomas at the site of inoculation (primary tumor) and at the site of experimentally introduced wounds (wound tumor). This latter finding provides a model system to study systematically the mechanisms underlying the cocarcinogenic effects of wounding. Our experiments show the following. (a) Chickens inoculated with a Rous sarcoma virus-derived, replication-defective virus construct fail to elaborate wound tumors in spite of aggressively growing primary tumors. We thus rule out metastasis as a mechanism and conclude that infectious virus is required for wound tumor formation; (b) using bromodeoxyuridine incorporation and immunofluorescence on frozen sections we demonstrate proliferation in the unwounded wing in cell types which are normally targets for Rous sarcoma virus infection and transformation and conclude that proliferation per se is not sufficient to induce wound tumors; (c) using immunohistochemistry for the viral protein p19gag we show that wounding induces virus expression in fibroblasts of newly forming granulation tissue 2 days after injury. We also demonstrate expression of viral mRNA in wound tumors by in situ hybridization with a v-src probe. We discuss the possibility of activation of integrated, silent virus or the preferential infection of a special target cell population as a result of wounding as well as the potential role of wound factors in transformation.

Animals↗
PubMed1974

Virus production and release, cell longevity, and cloning efficiency of chicken embryo fibroblasts infected with Rous sarcoma virus.

Continuous virus production is a characteristic of chicken embryo fibroblasts (CEF) infected and transformed by a nondefective Schmidt-Ruppin subgroup A Rous sarcoma virus. This virus production has been examined with particular attention to the amount of newly budded virus which remained cell-associated, and to the amount and degree of viral aggregation at the cell surface and in the fluid tissue culture medium. The total biologically active virus associated with a Schmidt-Ruppin subgroup A Rous sarcoma virus-infected CEF culture was divided almost equally between that portion of virus which was present in the fluid medium and that portion which was cell-associated. Various mechanical and enzymatic methods were used to remove cell-bound virus and to disperse aggregates of virus in the tissue culture medium to assess cell production of virus per hour accurately, which was determined as an average of 16.4 focus-forming units per cell per hour. With appropriate culture conditions, it was found that Schmidt-Ruppin subgroup A Rous sarcoma virus-infected and -transformed CEF replicated faster, could be passaged more times, and grew to higher cell densities than did normal CEF and CEF infected with a subgroup A Rous associated virus. Subgroup A Rous sárcoma virus-infected CEF cloned with much lower efficiency than did subgroup A Rous associated virus-infected CEF or normal CEF. Experiments employing a temperature-sensitive mutant of subgroup A Schmidt-Ruppin Rous sarcoma virus- and Rous associated virus-infected CEF indicated that the poor cloning efficiency of Schmidt-Ruppin subgroup A Rous sarcoma virus infected cells was not due to the constant production of virus but was probably related to some property associated with transformation of the cell by Rous sarcoma virus.

Animals↗
PubMed2001

Infection of nondividing cells by Rous sarcoma virus.

A direct comparison demonstrates that Rous sarcoma virus is capable of infecting aphidicolin-arrested cells 10-fold more efficiently than murine leukemia virus but less efficiently than human immunodeficiency virus. The efficiency of infection of nondividing cells by the three viruses correlates with the respective ability of each viral DNA to enter the nucleus.

Animals↗
PubMed1984

Alteration of neural cell adhesion molecule (N-CAM) expression after neuronal cell transformation by Rous sarcoma virus.

The effect of transformation by Rous sarcoma virus on the neural cell adhesion molecule N-CAM was assessed by immunoblotting, immunofluorescence staining, and an in vitro cell-cell aggregation assay using highly specific antibodies to the adhesion molecule. Expression of N-CAM was found to be temperature dependent in several rat cerebellar cell lines infected with a mutant Rous sarcoma virus that is temperature sensitive for transformation. At the nonpermissive temperature, these cells displayed significant quantities of N-CAM and aggregated rapidly by an N-CAM-mediated mechanism. However, when the cell lines were grown at the permissive temperature, they were morphologically transformed, contained much lower amounts of N-CAM, and aggregated poorly. A similar temperature dependence of N-CAM expression was not observed in cultured primary rat cerebellar cells nor in a chemically transformed neuronal cell line. In all of the cell lines, N-CAM occurred in the adult forms; the embryonic form has so far been observed in normal embryonic tissues and a few regions of the adult brain. The findings show that N-CAM prevalence at the cell surface can be modulated by transformation with clear-cut effects on cell-cell adhesion.

Animals↗
PubMed1977

Terminal redundancy and the origin of replication of Rous sarcoma virus RNA.

In vitro synthesis of Rous sarcoma virus DNA by the virion endogenous DNA polymerase activity is initiated on a tRNAtrp primer located near the 5' end of the genome. A major product of such synthesis is a piece of DNA 101 nucleotides long (strong stop DNA) which can be isolated covalently bound to the tRNA primer. Here we show that the strong stop DNA is complementary to the extreme 5' end of the genome. We also show that the 5' and 3' termini of the Rous sarcoma virus genome, excluding the cap and the poly(A), have the identical sequence. We propose that the function of this sequence is to facilitate elongation from the 3' end of DNA chains initiated elsewhere on the virus genome.

Avian Sarcoma Viruses↗
PubMed1995

A suboptimal src 3' splice site is necessary for efficient replication of Rous sarcoma virus.

Regulation of splicing of Rous sarcoma virus (RSV) RNA primary transcripts is necessary, as with other retroviruses, to allow for the accumulation of unspliced RNA and approximately equivalent amounts of spliced env and src mRNAs. Previous studies have indicated that the env 3' splice site is suboptimal because it has a nonconsensus branchpoint sequence and that this suboptimal splice site is required for virus replication (R. A. Katz and A. M. Skalka, 1990, Mol. Cell Biol. 10:696-704). We show in this report that the RSV src 3' splice site is also suboptimal. Mutagenesis of the src polypyrimidine-rich tract, which is interspersed with purines, to an uninterrupted 14-nt pyrimidine tract resulted in a three- to fourfold increase in the level of src splicing. Concomitant with this increase in src splicing, a cryptic 5' splice site within the env gene was activated. Splicing at this splice site is normally detected in nonpermissive mammalian cells were src splicing is elevated but occurs at low levels in permissive chicken embryo fibroblasts (CEF). In CEF, mutant viruses with the improved src 3' splice site replicated significantly slower than the wild-type virus. Transformation-defective revertants lacking the src 3' splice site were rapidly selected after passage of the chicken cells infected with the mutant virus. Thus, an inefficient src 3' splice site appears to be necessary for the efficient replication of RSV.

Animals↗
PubMed1986

A similar ribosomal protein S6 kinase activity is found in insulin-treated 3T3-L1 cells and chick embryo fibroblasts transformed by Rous sarcoma virus.

Insulin and transformation by Rous sarcoma virus stimulate the phosphorylation of ribosomal protein S6. Soluble fractions containing activated S6 protein kinase from insulin-treated cells and from transformed chick embryo fibroblasts were compared. Based upon several characteristics notably elution from DEAE-cellulose and sedimentation in glycerol gradients, these two S6 protein kinase activities appear to be similar enzymes. Thus insulin and retroviral transformation may activate the same enzyme to regulate the phosphorylation state of S6.

Animals↗
PubMed1989

Two interferon sensitive steps in the replication cycle of Rous sarcoma virus.

De novo infection of Rous sarcoma virus (RSV) strains of receptor subgroups A, B, C, and D is inhibited by low doses of chick interferon. Adsorption and penetration into the cell are not marginally impaired by interferon treatment. Since the level of proviral DNA synthesis is strongly reduced in the interferon-treated cell it is concluded that uncoating or reverse transcription of the viral genome is inhibited. This inhibition of proviral DNA synthesis is not caused by an arrest of CEF in the stationary phase. Chronic infection of SR-RSV-A, -B, -C, but not SR-RSV-D is also sensitive to interferon. Chick interferon treatment (50 u/ml) also had no inhibitory effect on the amount of transcripts of the RSV-specific oncogene src or the cellular oncogenes src and myc.

Animals↗