Virions Of Rous Sarcoma Virus Research Articles

Cryo-EM structure of the Rous sarcoma virus octameric cleaved synaptic complex intasome

Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous sarcoma virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes.

The Discovery of Reverse Transcriptase.

In 1970 the independent and simultaneous discovery of reverse transcriptase in retroviruses (then RNA tumor viruses) by David Baltimore and Howard Temin revolutionized molecular biology and laid the foundations for retrovirology and cancer biology. In this historical review we describe the formulation of the controversial provirus hypothesis by Temin, which ultimately was proven by his discovery of reverse transcriptase in Rous sarcoma virus virions. Baltimore arrived at the same discovery through his studies on replication of RNA-containing viruses, starting with poliovirus and then moving to vesicular stomatitis virus, where he discovered a virion RNA polymerase. Subsequent studies of reverse transcriptase led to the elucidation of the mechanism of retrovirus replication, the discovery of oncogenes, the advent of molecular cloning, the search for human cancer viruses, and the discovery and treatment of HIV/AIDS.

RSV Capsid Polymorphism Correlates with Polymerization Efficiency and Envelope Glycoprotein Content: Implications that Nucleation Controls Morphogenesis

We used cryo-electron tomography to visualize Rous sarcoma virus, the prototypic alpharetrovirus. Its polyprotein Gag assembles into spherical procapsids, concomitant with budding. In maturation, Gag is dissected into its matrix, capsid protein (CA), and nucleocapsid moieties. CA reassembles into cores housing the viral RNA and replication enzymes. Evidence suggests that a correctly formed core is essential for infectivity. The virions in our data set range from ∼105 to ∼175 nm in diameter. Their cores are highly polymorphic. We observe angular cores, including some that are distinctively “coffin-shaped” for which we propose a novel fullerene geometry; cores with continuous curvature including, rarely, fullerene cones; and tubular cores. Angular cores are the most voluminous and densely packed; tubes and some curved cores contain less material, suggesting incomplete packaging. From the tomograms, we measured the surface areas of cores and, hence, their contents of CA subunits. From the virion diameters, we estimated their original complements of Gag. We find that Rous sarcoma virus virions, like the human immunodeficiency virus, contain unassembled CA subunits and that the fraction of CA that is assembled correlates with core type; angular cores incorporate ∼80% of the available subunits, and open-ended tubes, ∼30%. The number of glycoprotein spikes is variable (∼0 to 118) and also correlates with core type; virions with angular cores average 82 spikes, whereas those with tubular cores average 14 spikes. These observations imply that initiation of CA assembly, in which interactions of spike endodomains with the Gag layer play a role, is a critical determinant of core morphology.

A substitution in rous sarcoma virus integrase that separates its two biologically relevant enzymatic activities.

Retroviral integrase prepares viral DNA for integration by removing 2 nucleotides from each end of unintegrated DNA in a reaction referred to as processing. However, it has been known since the processing assay was first described that avian integrases frequently nick 3 nucleotides, as well as 2 nucleotides, from viral DNA ends when reaction mixtures contain Mn2+. We now report that specificity for the biologically relevant "-2" site is enhanced when the serine at amino acid 124 of Rous sarcoma virus (RSV) integrase is replaced by alanine, valine, glycine, lysine, or aspartate. The protein with a serine-to-aspartate substitution exhibited especially high fidelity for the correct site, as evidenced by a ratio of -2 nicks to -3 nicks that was more than 40-fold greater than that for the wild-type enzyme in reactions with Mn2+. Even with Mg2+, the substituted proteins exhibited greater specificity than the wild type, especially the S124D protein. Moreover, this protein was more efficient than the wild type at processing viral DNA ends. Unexpectedly, however, the S124D protein was significantly impaired at catalyzing the insertion of viral DNA ends in reactions with Mn2+ and joining was undetectable in reactions with Mg2+. Thus, the S124D protein has separated the processing and joining activities of integrase. Similar results were found for human immunodeficiency virus integrase with the analogous substitution. No proteins with comparable properties have been described. Moreover, RSV virions containing integrase with the S124D mutation were unable to replicate in cell cultures. Together, these data suggest that integrase has evolved to have submaximal processing activity so that it can also catalyze DNA joining.

Requirements for minus-strand transfer catalyzed by Rous sarcoma virus reverse transcriptase.

We have examined the specific minus-strand transfer reactions that occur after the synthesis of minus strong-stop DNA and nonspecific strand switching on homopolymeric poly(rA) templates with different types of Rous sarcoma virus (RSV) reverse transcriptases. Three different types of reverse transcriptases can be isolated from virions of RSV: heterodimeric alphabeta and homodimeric alpha and beta. The mechanism of minus-strand transfer was examined using a model primer-template substrate corresponding to the 5'- and 3'-terminal RNA regions of the RSV genome. The results reveal that the RNase H activity of RSV reverse transcriptases is required for minus-strand transfer. Less than 2% of strand transfer of the extended product is detectable with RNase H-deficient enzymes. We could show that the alpha homodimer lacking the integrase domain can perform strand transfer almost as efficiently as the alphabeta and alphaPol heterodimers. In contrast, the activities of beta and Pol for minus-strand transfer are reduced. Furthermore, a two- to fivefold increase in minus-strand transfer activities was observed in the presence of human immunodeficiency virus type 1 nucleocapsid protein.

Mass determination of rous sarcoma virus virions by scanning transmission electron microscopy.

The internal structural protein of retroviruses, Gag, comprises most of the mass of the virion, and Gag itself can give rise to virus-like particles when expressed in appropriate cells. Previously the stoichiometry of Gag in virions was inferred from indirect measurements carried out 2 decades ago. We now have directly determined the masses of individual particles of the prototypic avian retrovirus, Rous sarcoma virus (RSV), by using scanning transmission electron microscopy. In this technique, the number of scattered electrons in the dark-field image integrated over an individual freeze-dried virus particle on a grid is directly proportional to its mass. The RSV virions had a mean mass of 2.5 x 10(8) Da, corresponding to about 1,500 Gag molecules per virion. The population of virions was not homogeneous, with about one-third to two-thirds of the virions deviating from the mean by more than 10% of the mass in two respective preparations. The mean masses for virions carrying genomes of 7.4 or 9.3 kb were indistinguishable, suggesting that mass variability is not due to differences in RNA incorporation.

V-Src enhances phosphorylation at Ser-282 of the Rous sarcoma virus integrase.

The Rous sarcoma virus (RSV) integrase (IN) and the beta polypeptide (beta) of the reverse transcriptase are posttranslationally modified by phosphorylation on Ser at amino acid position 282 of IN. When IN was immunoprecipitated from RSV (Prague A strain) virions, approximately 30 to 40% of the IN molecules were phosphorylated. When IN was immunoprecipitated from a v-src deletion mutant (delta Mst-A) of RSV or from avian myeloblastosis virus (AMV), the percentage of IN molecules that were phosphorylated was significantly reduced. This reduction in phosphorylation of IN between virions was verified by [35S]Met-[35S]Cys or 32P labeling of IN, followed by immunoprecipitation analysis using antisera directed to the amino or carboxyl terminus of IN. In delta Mst-A or AMV, a nonphosphorylated, slightly truncated (at the carboxyl terminus) polypeptide was the major species of IN. The enhanced phosphorylation of IN does not appear to be a general function of transformed cells, since enhanced phosphorylation was not detected in AMV derived from viremic chickens or from a v-src deletion mutant of RSV propagated in a chemically transformed quail cell line, QT6. From these data, we conclude that v-Src is necessary for efficient phosphorylation of IN and beta.

Phosphotyrosine-containing 120,000-dalton protein coimmunoprecipitated with pp60 v-src from rous sarcoma virus-transformed mammalian cells

Serum from rabbits bearing tumors (TBR serum) induced by the Rous sarcoma virus (RSV) was originally developed to identify the RSV arc gene protein-pp60 v-src. It is also capable of directly or indirectly immunoprecipitating a number of proteins besides pp60 v-src from lysates of RSV-transformed cells. This report describes a highly phosphorylated protein of approximately 120,000 Da (pp120) which is specifically immunoprecipitated from RSV-transformed mammalian cells by TBR sera and monospecific antibodies against pp60 v-src However, it was not immunoprecipitated by TBR serum from RSV-transformed chicken embryo fibroblasts. Phosphoamino acid analysis revealed that pp120 contains phosphoserine and phosphotyrosine in relative amounts similar to that found in pp60 v-src Various experimental results indicate that pp120 is not structurally related to RSV virion proteins or to the putative pp60 v-src substrate-vinculin. Furthermore, proteolytic peptide mapping and immunoblotting experiments indicate that although pp120 is immunoprecipitated by various anti-pp60 v-src antibodies, it does not appear to be related to the RSV transforming protein. These results suggest that pp120 is distinct from previously reported substrates of pp60 v-src and may exist in association with the transforming protein in mammalian cell lysates.

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.

Identification of a packaged cellular mRNA in virions of rous sarcoma virus.

A novel messenger activity has been identified by in vitro translation of the 70S virion RNAs of a variety of avian leukosis and avian sarcoma viruses. When the 70S virion RNA complex was heat dissociated and the polyadenylated RNA was fractionated on neutral sucrose gradients, a polypeptide of 34,000 daltons (34K) was observed in the translation products of 18S polyadenylic acid-containing virion RNA. Aside from the p60(src)-related subgenomic messenger activities, this was the only prominent messenger activity that sedimented at <20S. It was determined that the 34K protein was not virally coded because (i) messenger activity for the 34K protein was not generated by mild alkaline hydrolysis of 35S genomic RNA, (ii) the 34K proteins synthesized in response to different virion RNAs had identical tryptic peptide maps, and (iii) the tryptic peptide map of the 34K protein coded for by virion RNA was identical to that of a major in vitro translation product of 34,000 daltons made from 18S uninfected chick cell polyadenylated RNA. The 18S RNA was shown to be contained within virion particles, rather than part of a cellular structure copurifying with virus preparations, by demonstrating the presence of 34K messenger activity in virion cores made from detergent-disrupted virus. This cellular mRNA, however, was not observed in the virion RNAs of Rous-associated virus types 0 and 2 avian leukosis viruses and therefore is not packaged by all avian retroviruses. Since no other cellular message has been detected by this assay, it seems likely that the 34K mRNA found in 70S virion RNA is the result of selective packaging of an abundant host cell mRNA by certain avian retroviruses.

Product of in vitro translation of the Rous sarcoma virus src gene has protein kinase activity

In vitro translation of Rous sarcoma virus virion RNA resulted in the synthesis of a protein kinase which, when immunoprecipitated with antitumor serum, phosphorylated the immunoglobulin heavy chain. Even though in vitro translation of virion RNA resulted in the synthesis of a number of polypeptides which were recognized by antitumor serum, control experiments demonstrated that an immunoprecipitable protein kinase activity was found only when an immunoprecipitable p60src, the polypeptide product of the src gene, was synthesized. A protein kinase with similar properties was therefore intimately associated with p60src which was synthesized in vitro in the reticulocyte lysate, just as it is with p60src which is obtained from transformed chick and mammalian cells. It is therefore highly unlikely that this association is artifactual. ts NY68 is a mutant of Rous sarcoma virus which is able to transform cells at 36 but not at 41 degrees C. In vitro translation of ts NY68 virion RNA at 30 degrees C resulted in efficient synthesis of immunoprecipitable p60src, but very inefficient synthesis of an immunoprecipitable protein kinase. The p60src obtained by in vitro translation of wild-type virion RNA was more than 20-fold more active as a protein kinase than was that obtained from ts NY68 RNA. The correlation in the case of ts NY68 of a deficiency in protein kinase activity with an inability to transform cells at high temperature suggests that the protein kinase activity associated with p60src is indeed critical to cellular transformation.

In vitro Synthesis of Infectious DNA Copies of the Rous Sarcoma Virus Genome

Detergent-disrupted virions of Rous sarcoma virus can synthesize in vitro an infectious viral DNA. Infectivity of the DNA was determined in transfection assays at the endpoint dilution. It was found that the specific infectivity of the crude endogenous reaction product was 34 infectious units/microgram. The endogenous reaction yielded up to 8.5 x 10(-7) infectious units per biologically active input virion, i.e., nearly 0.1 -1 infectious molecules. The possible structure of these molecules is discussed.

Multiple forms of sarc gene proteins from Rous sarcoma virus RNA.

In a previous study we were able to identify two proteins of 25,000 and 18,000 daltons that were made from RNA of transforming virions of Rous sarcoma virus (RSV) and that were missing from the translation products of a transformation-defective deletion mutant of RSV. In the present study we have separated RSV virion RNA on sucrose gradients and have determined that the two putative sarc gene products are synthesized as doublets from an mRNA of approximately 18 S. There also appear to be several other sizes of virion mRNA that direct the synthesis of other viral proteins. These data are discussed in terms of the structure of the RSV genome. In addition to the 25,000- and 18,000-dalton doublets, there also is a 60,000-dalton protein whose synthesis is directed by 18S viral RNA from transforming virion of RSV. Peptide mapping has shown that the 60,000- and 25,000-dalton doublet are structurally related. In addition, the use of two-dimensional gel electrophoresis has allowed us to resolve both bands of the 25,000-dalton doublet into several differently charged species.

In vitro translation yields a possible Rous sarcoma virus src gene product.

In vitro translation of Rous sarcoma virus (RSV) virion RNA in the messenger-dependent reticulocyte lysate system yielded polypeptides that were not synthesized by translation of RNA from a transformation-defective deletion mutant of RSV. These RSV-specific products migrated on sodium dodecyl sulfate/polyacrylamide gels as two doublets of approximately 25,000 and 17,000 daltons. Synthesis of these proteins was not sensitive to inhibition by m7GTP; however, synthesis of the 76,000-dalton precursor of the internal structural proteins was sensitive to inhibition by m7GTP. Tryptic peptide maps showed the 25,000- and 17,000-dalton proteins to be related to one another but to be distinct from the 76,000-dalton protein. The 25,000-dalton protein was translated only from a polyadenylylated RNA of approximately 2500 nucleotides, whereas the 76,000-dalton protein was translated from 38S RNA, corresponding to the entire viral genome. A 180,000-dalton protein was also synthesized from 38S RSV virion RNA. From the absence of the 25,000- and 17,000-dalton proteins in the translation products of transformation-defective RSV RNA and the size of their RNA templates, we conclude that these proteins may be derived from coding sequences within the RSV src gene.

Cell-free synthesis of two proteins unique to RNA of transforming virions of Rous sarcoma virus.

We have utilized a reticulocyte lysate system to translate the 35S RNA of Rous sarcoma virus. Autoradiograms of the protein products separated on sodium dodecyl sulfate/polyacrylamide gels reveal a heterogeneous mixture of proteins of sizes ranging from 13,000 to 180,000 daltons. In comparing the translational products from 35S RNA of Prague B Rous sarcoma virus with those formed from the RNA of a transformation-defective deletion mutant derived from Prague B, we have found that two proteins, 25,000 and 18,000 daltons, are missing from the latter. Neither of these proteins is immunoprecipitated by monospecific antisera against the structural proteins of avian RNA tumor viruses. The combined atomic mass of 43,000 daltons corresponds to the amount of genetic coding capacity (40,000-50,000 daltons in terms of protein products) deleted from the RNA of the transformation-defective viruses. We propose that these proteins are coded for by the putative oncogene (onc) or sarc (src) gene and that one or both of them may be responsible for the oncogenic transformation caused by these viruses in infected cells.

Presence of ribonucleotide sequences complementary to rous sarcoma virus (RSV) RNA in chicken cells infected with RSV

RNA--RNA molecular hybridization between [125I] RNA from Rous sarcoma virus virions and RNAs isolated from various subcellular fractions, i.e. nuclei, mitochondria, free and membrane-bound polyribosomes, from tumors induced by RSV in chickens resulted in the formation of RNAase-resistant hybrids only with the RNA of mitochondria and membrane-bound polysomes. The origin of complementary regions in the RNAs from these organelles is discussed.

New procedure for the direct analysis of in vitro reverse transcription of Rous sarcoma virus RNA.

Based on the observation that in vitro transcription of Rous sarcoma virus (RSV) RNA by avian myeloblastosis virus DNA polymerase renders the RNA PROGRESSIVELY MORE SENSITIVE TO Escherichia coli RNase H digestion, a new procedure for the in situ analysis of this process has been developed. In vitro transcription products of 32P-labeled RSV RNA are first treated with RNase H, the resistant fraction is then digested to completion with RNase T1, and the oligonucleotides are analyzed by a fingerprint technique. By using the established order of these oligonucleotides along the RNA molecule, a comparison of the yields of each oligonucleotide, before and after transcription, allows qualitative and quantitative in situ analyses of the transcription process. Using this new procedure, we find that upon transcription of purified RSV RNA, DNA synthesis occurs mainly at three sites, one near the 5' end and two near the center of the subunit RNA molecule, and that most of these RNA molecules are competent templates for limited transcription at these specific sites. We also show that purified RSV 70S RNA contains a low-molecular-weight DNA hybridized to a nucleotide sequence near the center of the subunit molecule. Furthermore , we find that the low-molecular-weight nucleic acid fraction extracted from purified RSV virions contains DNA that can hybridize to RSV 70S RNA and that the virion DNA in such hybrids can function as a primer for an extensive in vitro reverse transcription.

Cell-free translation of virion RNA from nondefective and transformation-defective Rous sarcoma viruses

Nondefective and transformation-defective virion subunit RNAs from two strains of Rous sarcoma virus (RSV) were translated in cell-free systems derived from Krebs IIA ascites cells, wheat germ, and L-cells. In each case the predominant viral-specific product was a polypeptide of molecular weight 76,000 that is related to the internal viral group-specific antigens, as judged by immunoprecipitation with monospecific antisera and tryptic peptide fingerprinting. No difference could be detected between the translation products of 35S RNA from nondefective and transformation-defective RSV virions, nor of 35S RNA from different strains of RSV. The 76,000-molecular-weight polypeptide synthesized in response to 35S RNA in vitro was labeled with formyl-methionine from initiator tRNA. Models for viral protein synthesis are discussed in the light of these results, and arguments positioning the group-specific antigen gene at the 5' end of the 35S RNA are presented.

The methionyl transfer RNAs of Rous sarcoma virus

Virions of Rous sarcoma virus contain the two major classes of methionyl tRNA present in uninfected chick embryo fibroblasts. The formylated species, N-formyl methionyl tRNA, appears to be selectively associated with the viral 70 S RNA genome.

Transcription of the Rous sarcoma virus genome in vitro and in vivo.

RNA-directed DNA synthesis by detergent-disrupted virions of Rous sarcoma virus (RSV) initiates by the covalent attachment of pdA to the 3'-terminal rA of a 4S RNA hydrogen-bonded to the 70S RNA template. This 4S "primer" has structural features of tRNA and can be aminoacylated with methionine. Synthesis and integration of provirus DNA can be monitored in both permissive (duck) and nonpermissive (mouse) cells acutely infected with RSV. The results of these studies, as well as data obtained with RSV-infected mammalian cells which have reverted from a transformed to a pheno-typically normal state, indicate that integration of viral genes into the host chromosome is not sufficient cause for transformation. Pertinent features of virus-specific RNA-directed DNA synthesis in vitro and in vivo are reviewed and compared.