The first and third uORFs in RSV leader RNA are efficiently translated: implications for translational regulation and viral RNA packaging.

Rous sarcoma virus (RSV) RNA leader contains three short upstream open reading frames. We have shown recently that both uORFs 1 and 3 influence in vivo translation of the downstream gag gene and are involved in the virus RNA packaging process. In this report, we have studied the translational events occurring at the upstream AUGs in vivo. We show that (i) the first and third AUGs are efficient translational initiation sites; (ii) ribosomes reinitiate efficiently at AUG3; and (iii) deletions in the intercistronic distance between uORF1 and 3 (which is well conserved among avian strains) prevent ribosome initiation at AUG3, thus increasing translation efficiency at the downstream AUGgag. The roles of the uORFs in translation and packaging are discussed.

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 stoichiometric 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 intracellular 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.

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.

Effect of rearrangements and duplications of the Cys-His motifs of Rous sarcoma virus nucleocapsid protein.

It has been widely documented that the nucleocapsid protein p12 (NC) of Rous sarcoma virus (RSV) has a role in the encapsidation and maturation of the virus genomic RNA during particle formation, and particularly important appear to be the Cys-His motifs of this protein. Since some retroviruses only have one such motif, we have investigated the significance of the two distinct Cys-His motifs of RSV NC. The analysis of the phenotype of virus NC mutants with precise rearrangements or duplications of the motifs highlights the following features. (i) The two motifs are not functionally equivalent. (ii) The order and number of Cys-His motifs are less important for RSV NC than the presence of two distinct motifs for both the encapsidation of virus genomic RNA and maintenance of the integrity of the RNA after particle formation. (iii) The proximal motif has a distinct function in the virus replication cycle other than RNA encapsidation and dimerization. (iv) The presence of three Cys-His motifs reduces virus infectivity and leads to high-frequency deletion events (of one of the motifs) after infection: the resulting RNA species encode a wild type-like NC protein restoring full infectivity to the progeny virus particles. Additionally, the data suggest that this occurs only after infection. The deletion probably arises by intramolecular displacement of the replication complex between repeat sequences.

Role of the open reading frames of Rous sarcoma virus leader RNA in translation and genome packaging.

The Rous sarcoma virus (RSV) RNA leader sequence carries three open reading frames (uORFs) upstream of the AUG initiator of the gag gene. We studied, in vivo, the role of these uORFs by changing two or three nucleotides of the three AUGs or by deleting the first uORF. Our results show that (i) unlike most previously characterized uORFs, which decrease translation, the first uORF (AUG1) of RSV acts as an enhancer of translation, since absence of the first AUG decreased translation; AUG3 also modulates translation, probably by interfering with scanning ribosomes as described for other upstream ORFs, and mutation of AUG2 had no effect on translation. (ii) Mutation of each of the upstream AUGs lowered the infectivity of progeny virions. (iii) Unexpectedly, mutation of AUG1 and/or AUG3 dramatically reduced RNA packaging by 50-to 100-fold, unlike mutation of AUG2 which did not alter RNA packaging efficiency. Additional mutants in the vicinity of uORF1 and uORF3 were constructed in order to elucidate the mechanism by which uORFs affect RNA packaging: a translation model requiring uORFs 1 and 3, and involving ribosome pausing at AUG 3 is discussed.

Specificity of Rous sarcoma virus nucleocapsid protein in genomic RNA packaging.

Site-directed mutagenesis has shown that the nucleocapsid (NC) protein of Rous sarcoma virus (RSV) is required for packaging and dimerization of viral RNA. However, it has not been possible to demonstrate, in vivo or in vitro, specific binding of viral RNA sequences by NC. To determine whether specific packaging of viral RNA is mediated by NC in vivo, we have constructed RSV mutants carrying sequences of Moloney murine leukemia virus (MoMuLV). Either the NC coding region alone, the psi RNA packaging sequence, or both the NC and psi sequences of MoMuLV were substituted for the corresponding regions of a full-length RSV clone to yield chimeric plasmid pAPrcMNC, pAPrc psi M, or pAPrcM psi M, respectively. In addition, a mutant of RSV in which the NC is completely deleted was tested as a control. Upon transfection, each of the chimeric mutants produced viral particles containing processed core proteins but were noninfectious. Thus, MoMuLV NC can replace RSV NC functionally in the assembly and release of mature virions but not in infectivity. Surprisingly, the full-deletion mutant showed a strong block in virus release, suggesting that NC is involved in virus assembly. Mutant PrcMNC packaged 50- to 100-fold less RSV RNA than did the wild type; in cotransfection experiments, MoMuLV RNA was preferentially packaged. This result suggests that the specific recognition of viral RNA during virus assembly involves, at least in part, the NC protein.

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 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.

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.

Point mutations in the proximal Cys-His box of Rous sarcoma virus nucleocapsid protein.

To extend our previous studies of the function of the Cys-His box of Rous sarcoma virus NC protein, we have constructed a series of point mutations of the conserved or nonconserved amino acids of the proximal Cys-His box and a one-amino-acid deletion. All mutants were characterized for production of viral proteins and particles, for packaging and maturation of viral RNA, for reverse transcriptase activity, and for infectivity. Our results indicated the following. (i) Mutations affecting the strictly conserved amino acids cysteine 21, cysteine 24, and histidine 29 were lethal; only the mutant His-29----Pro was still able to package viral RNA, most of it in an immature form. (ii) Mutation of the highly conserved glycine 28 to valine reduced viral RNA packaging by 90% and infectivity 30-fold, whereas mutant Gly-28----Ala was fully infectious. This suggests a steric hindrance limit at this position. (iii) Shortening the distance between cysteine 24 and histidine 29 by deleting one amino acid abolished the maturation of viral RNA and yielded noninfectious particles. (iv) Substitution of tyrosine 22 by serine lowered viral RNA packaging efficiency and yielded particles that were 400-fold less infectious; double mutant Tyr-22Thr-23----SerSer had the same infectivity as Tyr-22----Ser, whereas mutant Thr-23----Ser was fully infectious. (v) Changing glutamine 33 to a charged glutamate residue did not affect virus infectivity. Similarities and differences between our avian mutants and those in murine retroviruses are discussed.

The proteins associated with the soluble form of p36, the main target of the src oncogene product in chicken fibroblasts, are glycolytic enzymes.

One of the main targets of pp60v-src tyrosine kinase is a 34 to 39-kilodalton protein of chicken embryo fibroblasts called p36 or calpactin I. We have previously reported an association of the cytoplasmic fraction of p36 (10-20% of the total cellular p36) with three chicken polypeptides named p32, p48, and p54. We have now raised and affinity-purified antibodies against each of these proteins. This has allowed their identification: p32 is lactate dehydrogenase, p48 is enolase, and p54 is phosphoglucose isomerase. An association between p36 and two other known substrates of pp60v-src, the glycolytic enzymes enolase and lactate dehydrogenase, suggests a cellular organization of the various targets of the oncogene tyrosine kinases. Furthermore, a possible relationship between p36 and glycolysis is questioned.

Mutations in Rous sarcoma virus nucleocapsid protein p12 (NC): deletions of Cys-His boxes.

Rous sarcoma virus nucleocapsid protein p12 (NC) contains two conserved amino acid motifs, the Cys-His boxes, which constitute potential metal-binding domains. To try to understand the function of NC and of each of its Cys-His boxes during the viral life cycle, particularly in viral RNA packaging, we have used synthetic oligonucleotides to delete precisely either the proximal or the distal box, or both Cys-His boxes. The mutant DNAs were transfected into chicken embryo fibroblasts, and the virions produced in a transient assay were characterized biochemically for production of viral proteins and particles, RNA packaging, and infectivity. The results indicated the following. (i) The deletion of either the proximal or the distal box decreases the amount of viral RNA packaged in the particles and results in incomplete 70S dimer formation. (ii) The deletion of both boxes inhibits viral RNA packaging. (iii) The deletion of the proximal, but not the distal, box suppresses any detectable infectivity, while the deletion of the distal, but not the proximal, box lowers infectivity 100 to 200 times.

Association of three chicken proteins with the 34 kD target of Rous sarcoma virus tyrosine kinase.

Transformation of chicken embryo fibroblasts by avian retroviruses induces the tyrosine phosphorylation of a 34-39 kD cellular protein (p34). In vitro, p34 isolated from intestine interacts with F-actin in a Ca2+-dependent manner. We report here that, in the absence of Ca2+ chelators, three proteins co-purified with p34 extracted from a cytosolic or membrane fraction of chicken embryo fibroblasts; these two fractions account respectively for 10-20% and 50% of the total cellular p34. Isolated from the cytosoluble fraction of fibroblasts by sucrose gradient centrifugation and hydrophobic chromatography, p34 and the other proteins behaved as a homogeneous species upon non-denaturing gel electrophoresis, gel filtration, and CsCl density gradient centrifugation, thus indicating a strong association. Moreover, an analysis by electron microscopy following uranyl acetate staining revealed particles with a raspberry-like shape. This association was always disrupted by the calcium-chelating agent, EGTA.

A 20S particle ubiquitous from yeast to human.

We have purified and characterized a particle sedimenting at 20S from the postribosomal fraction of yeast, wheat germ, Drosophila melanogaster tissue culture cells, chicken embryo fibroblasts, rabbit reticulocyte lysate, and HeLa cells. Most of the protein constituents of the 20S particle have molecular weights of 20-35 kd and differ between species; however, some do have similar molecular weights and isoelectric points, suggesting they are related. Several low-molecular-weight RNAs, distinct from tRNAs, co-purify with the particle isolated from all these species and show increasingly more complex patterns ascending the arbitrary order from yeast to human (yeast, plant, insect, bird, and mammals). In Drosophila, we present evidence that these small RNAs are tightly associated with this 20S structure.

Rous sarcoma virus nucleic acid-binding protein p12 is necessary for viral 70S RNA dimer formation and packaging.

To study the function(s) of the Rous sarcoma virus nucleic acid-binding protein p12, we constructed mutants by using two restriction sites in the p12 proviral coding sequence of the Prague C strain to insert KpnI synthetic linkers. The two restriction sites are in the same reading frame, which allowed us to construct a deletion mutant lacking the two conserved Cys-His regions and a duplication mutant containing three intact Cys-His boxes. These mutant DNAs were transfected into chicken embryo fibroblasts, and the viral particles produced in a transient assay were characterized biochemically and for infectivity. Our results indicate that the Rous sarcoma virus nucleic acid-binding protein p12 is necessary for genomic RNA packaging but not for particle assembly and is implicated in the formation of a stable 70S dimeric RNA. Moreover, the fact that one mutant was apparently able to package normal 70S RNA but was not infectious suggests a role for p12 during the infection process.

Characterization of the prosome from Drosophila and its similarity to the cytoplasmic structures formed by the low molecular weight heat-shock proteins.

We have identified and characterized a ribonucleoprotein structure from the cytoplasm of Drosophila melanogaster tissue culture cells which is equivalent to the prosome, a recently described ribonucleoprotein particle of duck and mouse cells. During the recovery period following heat shock, the low mol. wt. heat-shock proteins form cytoplasmic ribonucleoprotein particles which co-purify with the Drosophila prosome. Both ribonucleoprotein particles share several structural properties but their protein constituents differ in their metabolism and cellular localization during the heat treatment. We also report the partial nucleotide sequences of several small RNA species associated with the Drosophila prosome. One of them has a strong sequence homology with the U6 mammalian small nuclear RNA.

It is Rous sarcoma virus protein P12 and not P19 that binds tightly to Rous sarcoma virus RNA.

The interactions between Rous Sarcoma virus (RSV) RNA and the viral proteins in the virus have been analysed by Sen & Todaro (1977) using ultraviolet light irradiation; they showed that the major protein ultraviolet light cross-linked to the viral RNA was P19 as identified by polyacrylamide gel electrophoresis. We report here that it is not viral protein P19 but P12 that binds tightly to RSV RNA upon ultraviolet light irradiation of the virus. Therefore, the binding sites of the viral protein along RSV RNA that we have characterized previously should be correctly attributed now to P12 and not P19.

High spontaneous mutation rate of Rous sarcoma virus demonstrated by direct sequencing of the RNA genome.

Direct and extensive sequencing of RSV RNA genome is reported. More than 10,000 nt of the T1 RNase resistant RSV RNA fragments (1) have been sequenced and shown to cover 3900 nt of RSV genome. The frequent sequence variations found indicate that RSV supports a very high incidence of spontaneous mutations in the course of replication, one very probable cause of the genetic diversity among the avian retroviruses. Sequences of the structured RSV RNAs allowed us also to precisely characterize the structured domains of the retroviral genome and show that the src gene is not structured.

A cellular protein phosphorylated by the avian sarcoma virus transforming gene product is associated with ribonucleoprotein particles.

In chick embryo fibroblasts transformed by Rous sarcoma virus (RSV) the tyrosine phosphorylation of a cellular protein of 34,000 daltons mol. wt. (34 kd) is greatly enhanced; this was shown to be catalyzed by the phosphotransferase activity of RSV transforming protein pp60src. We report here that in cytoplasmic extracts of both normal and transformed cells, in the presence of magnesium ions, the majority of the 34-kd protein is associated with large structures and that a fraction of 34 kd appears to be associated with ribonucleoprotein particles (RNPs). In addition, upon u.v. light cross-linking of RNA to protein in normal or transformed cells, an anti-34 kd serum immunoprecipitates RNA fragments of apparent low sequence complexity as detected by T1 fingerprint analysis. Our results indicate that the 34-kd protein may play a role in the cell at the level of RNPs.