DNA sequence of the Bryan high-titer strain of Rous sarcoma virus: extent of env deletion and possible genealogical relationship with other viral strains

Raji cells, collected at various times from a synchronized culture, were gently lysed, and the high-molecular-weight DNA was enriched ca. 10-fold for latent Epstein-Barr virus (EBV) genomes by equilibrium density gradient centrifugation in neutral CsCl. The heavy-density DNA pool, which included more than 90% of the total intracellular EBV DNA sequences, was further fractionated by velocity sedimentation on neutral glycerol gradients, and material from fractions containing potential EBV DNA replicative forms was examined in the electron microscope. Early in the cellular S phase, when the EBV DNA content was found to be doubling in parallel with host chromosome replication, half of the 50- to 55-micron circular EBV genomes were observed to have two or more DNA branch points or forks. Most molecules were in a relaxed theta configuration, indicative of the Cairns mode of DNA replication. In the supercoiled state, the two daughter strands of the partially replicated molecules were seen to be wrapped around each other. Two theta structures had more than two DNA forks, indicating that DNA replication can initiate more than once on the same DNA molecule. Late in the S phase, the EBV DNA sedimenting at positions where theta structures were found with early S phase samples was composed of catenated dimers rather than partially replicated genomes. It is concluded that the circular EBV genomes, which are the major intracellular form in latently infected cells, are maintained as independent replicons and are not synthesized from an integrated template.     

Viral glycoprotein synthesis studies in an established line of Japanese quail embryo cells infected with the Bryan high-titer strain of Rous sarcoma virus.

Although a glycoprotein with an approximate molecular weight of 43,000 is associated with purified virions of the Bryan high-titer strain of Rous sarcoma virus propagated on R(-)Q cells, these virions lack gp85, the major glycoprotein of the avian tumor virus envelope. As measured by immune precipitation with a specific antiserum, gp85 does not accumulate to detectable levels in R(-)Q cells.     

Generation of a water-soluble oligomeric ectodomain of the Rous sarcoma virus envelope glycoprotein.

Sequences encoding the transmembrane domain of the Rous sarcoma virus envelope (Env) glycoprotein were deleted and replaced with sequences that signal addition of a glycosyl phosphatidylinositol (GPI) membrane anchor. Stable NIH 3T3 cell lines expressing either the wild-type transmembrane-anchored Env or the Env chimera with a GPI tail were established. The GPI-anchored envelope glycoprotein is expressed, oligomerized, and transported to the cell surface in a manner identical to that of its wild-type transmembrane-anchored counterpart. The GPI-linked protein is quantitatively removed from the cell surface by treatment with phosphatidylinositol phospholipase C. The phosphatidylinositol phospholipase C-released, water-soluble Env glycoprotein ectodomain retains the wild-type oligomeric structure and provides a useful tool for studying the subgroup-specific binding and fusion activities of a prototypic retroviral Env glycoprotein.     

Deletion mutant of the Bratislava-77 strain of Rous sarcoma virus containing a fusion of the group-specific antigen and envelope genes.

The genetic compositions of two independently derived preparations of the Bratislava-77 strain (B77) of Rous sarcoma virus were analyzed after each was passaged seven or more times in duck embryo fibroblasts. RNase, T1-resistant oligonucleotide fingerprint analysis of virion RNA from both preparations of duck-passaged B77 revealed the presence of two large noncontiguous deletions. Approximately 75% of the RNAs contained a deletion which spans oligonucleotides 304 to 4 on the viral genome (about 3,500 nucleotides) and encompasses all of the B77 polymerase gene. More than 90% of the RNAs also contained a deletion which spans src-specific oligonucleotides 6 and 5(about 2,200 nucleotides) and is identical to the deletion observed in transformation-defective B77. Virion RNA from duck-passaged B77 also contained two oligonucleotides (D1 and D2) not observed in the RNA of B77 virus grown on chicken embryo fibroblasts. Analysis of the virion RNA of duck-passaged B77 by denaturing agarose gel electrophoresis revealed four major subunits with molecular weights of 3.40 x 10(6), 2.65 x 10(6), 2.25 x 10(6), and 1.55 x 10(6). Whereas the 3.40- and 2.65-megadalton (Mdal) RNA species comigrated with the nondefective and transformation-defective RNAs of B77 propagated on chicken embryo fibroblasts, no counterparts to the 2.25- and 1.55-Mdal RNAs were observed in the RNA of B77 grown on chicken embryo fibroblasts. Oligonucleotide fingerprint analysis of these RNA species revealed that the 2.65-Mdal RNA contains the src-specific deletion and that 2.25-Mdal RNA contains the polymerase region deletion; both of these deletions were observed in the 1.55-Mdal RNA, which was the major RNA subunit species detected in duck-passaged B77. The new oligonucleotides (D1 and D2) observed in the duck-passaged virus were present in the 2.25- and 1.55-Mdal RNA species in vitro and in vivo and directs the synthesis of a 130,000-dalton protein (p130). p130 contains antigenic determinants specific for p27 (gag gene) and gp85 (env gene) but does not contain sequences which cross-react with antisera directed against the alpha beta form of RNA-dependent DNA polymerase (pol gene). This RNA, therefore, is generated by a fusion of the gag and env genes of Rous sarcoma virus B77.     

Identification of a transformation-specific protein induced by a Rous sarcoma virus.

Using antisera obtained from rats bearing Schmidt-Ruppin strain Rous sarcoma virus-induced tumors, we have idnetified a protein with an apparent molecular weight of 56,000 daltons and an isoelectric point of 6.3 in extracts of chick embryo fibroblasts transformed by a wild-type nondefective Rous sarcoma virus (Schmidt-Ruppin strain). This protein was not found in cells infected by trnasformation-defective mutants with either a partial or complete deletion of the src gene, nor in cells infected by a nontransforming avian leukosis virus. The 56,000 dalton molecular weight protein was found to be synthesized at both the permissive and nonpermissive temperatures in cells infected by either of two conditionallethal mutants that are temperature-sensitive in cell transformation. The amount of this protein, however, accumulated in cells infected by these temperature-sensitive mutants, relative to the structural polypeptides, differed significnatly from that seen with the nondefective virus. Pulsechase experiments indicate that the protein is extremely unstable, with a half-life of about 20 min, and does not serve as a precursor to any of the detectable virion polypeptides. Furthermore, incubation of the rat antiserum with purified, disrupted virus did not affect its immunoreactivity to this particular protein. We conclude that this 56,000 dalton molecular weight protein is a nonstructural protein specific to cells transformed by Rous sarcoma virus.     

The membrane-binding domain of the Rous sarcoma virus Gag protein.

The Gag protein of Rous sarcoma virus (RSV) can direct particle assembly and budding at the plasma membrane independently of the other virus-encoded products. A previous deletion analysis has suggested that the first 86 amino acids of RSV Gag constitute a large membrane-binding domain that is absolutely required for these processes. To test this hypothesis, we inserted these residues in place of the N-terminal membrane-binding domain of the pp60v-src, a transforming protein whose biological activity requires plasma membrane localization. The ability of the Src chimera to induce cellular transformation suggests that the RSV sequence indeed contains an independent, functional domain.     

Evidence for a second function of the MA sequence in the Rous sarcoma virus Gag protein.

During retrovirus assembly, Gag proteins bind to the inner leaflet of the plasma membrane to initiate the budding process. The molecular basis of this protein-lipid interaction is poorly understood. For the human, immunodeficiency virus type 1 Gag protein, we recently reported that the membrane-binding domain resides within the N-terminal 31 amino acids and consists of two components: myristate and a cluster of basic residues, which together promote membrane binding in vitro and budding in vivo (W. Zhou, L. J. Parent, J. W. Wills, and M. D. Resh, J. Virol. 68:2556-2569, 1994). The positively charged residues associate electrostatically with acidic phospholipids to stabilize membrane binding, while myristate provides membrane-binding energy via hydrophobic interactions. Here we demonstrate that the human immunodeficiency virus type 1 Gag membrane-binding domain can fully replace the membrane-targeting function of the N-terminal 100 residues of the non-myristylated Rous sarcoma virus (RSV) Gag protein. To further explore the importance of myristate and basic residues in membrane binding, we developed a gain-of-function assay whereby budding was restored to defective mutants of RSV Gag. Detailed mutational analysis revealed that the position, number, and context of charged residues are crucial to budding. Myristate provides additional membrane-binding energy, which is critical when a Gag protein is near the threshold of stable membrane association. Finally, viruses with altered matrix (MA) proteins that are noninfectious, even though they produce particles with high efficiency, were identified. Thus, we present the first evidence that the RSV MA sequence plays two distinct roles, membrane binding during particle assembly and a second, as yet undefined function required for viral infectivity.     

Biosynthesis of an unglycosylated envelope glycoprotein of Rous sarcoma virus in the presence of tunicamycin.

Cells stably infected with Rous sarcoma virus were treated with tunicamycin to prevent the glycosylation of the precursor (pr92gp) to the two viral envelope glycoproteins gp85 and gp35. Pretreatment of the cells for 4 h with the antibiotic resulted in a 90% reduction in [3H]mannose incorporation into total cellular glycoproteins, intracellular viral glycoproteins, and released virus particles. Protein synthesis and virus particle formation were not significantly affected by the treatment. A new polypeptide made in the presence of the drug was identified by immunoprecipitation of pulse-labeled cell lysates with monospecific anti-gp85 and anti-gp35 sera. This polypeptide, migrating on sodium dodecyl sulfate-polyacrylamide gels as a molecule of 62,000 daltons (pr62), contained no [3H]mannose, was labeled with [S35]methionine and [3H]arginine, could not be chased into the higher-molecular-weight glycosylated form, and contained the same [3H]arginine tryptic peptides as pr92gp. The unglycosylated pr62 was still detectable 2 h after the pulse labeling of the cells. The lack of glycosylation of pr62 did not seem to reduce its stability. No clear evidence for the incorporation of this molecule or its cleavage products into viral particles could be obtained. To code for an envelope polypeptide of 62,000 daltons, only about 1,500 nucleotides or 15% of the total coding capacity of the virus are needed.     

Phosphorylation and metabolism of the transforming protein of Rous sarcoma virus.

p60src, the transforming protein of Rous sarcoma virus, was found to contain 0.5 to 0.9 mol of total phosphate per mol of polypeptide. The protein is known to be phosphorylated at two sites, a serine in the amino-terminal domain and a tyrosine in the carboxy-terminal domain. Because our indirect analysis suggests that the serine is phosphorylated to approximately twice the extent of the tyrosine, we estimate that p60src contains approximately 0.3 to 0.6 mol of phosphoserine and 0.2 to 0.3 mol of phosphotyrosine per mol of polypeptide. p60src was found to represent approximately 0.02% of the total incorporated radioactivity in Rous sarcoma virus-transformed chick cells labeled with [35S]methionine for 48 h. This corresponds to approximately 500,000 molecules of p60src per cell. Pulse-chase experiments revealed that the half-life of p60src ranged from 2 to 7 h, depending on the strain of virus examined. The P60src of the Schmidt-Ruppin strain was significantly more stable than that of the Prague strain.     

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.     

Structure and self-association of the Rous sarcoma virus capsid protein

BACKGROUND: The capsid protein (CA) of retroviruses, such as Rous sarcoma virus (RSV), consists of two independently folded domains. CA functions as part of a polyprotein during particle assembly and budding and, in addition, forms a shell encapsidating the genomic RNA in the mature, infectious virus. RESULTS: The structures of the N- and C-terminal domains of RSV CA have been determined by X-ray crystallography and solution nuclear magnetic resonance (NMR) spectroscopy, respectively. The N-terminal domain comprises seven alpha helices and a short beta hairpin at the N terminus. The N-terminal domain associates through a small, tightly packed, twofold symmetric interface within the crystal, different from those previously described for other retroviral CAs. The C-terminal domain is a compact bundle of four alpha helices, although the last few residues are disordered. In dilute solution, RSV CA is predominantly monomeric. We show, however, using electron microscopy, that intact RSV CA can assemble in vitro to form both tubular structures constructed from toroidal oligomers and planar monolayers. Both modes of assembly occur under similar solution conditions, and both sheets and tubes exhibit long-range order. CONCLUSIONS: The tertiary structure of CA is conserved across the major retroviral genera, yet sequence variations are sufficient to cause change in associative behavior. CA forms the exterior shell of the viral core in all mature retroviruses. However, the core morphology differs between viruses. Consistent with this observation, we find that the capsid proteins of RSV and human immunodeficiency virus type 1 exhibit different associative behavior in dilute solution and assemble in vitro into different structures.     

Synthesis and processing of viral glycoproteins in two nonconditional mutants of Rous sarcoma virus.

We have studied the pattern of glycoprotein synthesis in two nonconditional mutants of Rous sarcoma virus. One mutant, SE33, produces no viral particles but synthesizes Pr92env, which is cleaved intracellularly to mature glycoproteins. The second mutant, SE521, encodes a gPr92env which is not cleaved to gp85 or gp37 and therefore produces virions with the phenotype of Bryan RSV(-) or NY8. Neither of these mutants have detectable genomic deletions. The study of these mutants has led to the following conclusions. (i) In the absence of particle production or p15 synthesis, gPr92env can be cleaved to the mature glycoprotein which is found on the cell surface. (ii) Noncleaved gPr92env is not packaged into virions but is found on the cell surface. (iii) gPr92env alone can account for subgroup specific viral interference. (iv) gPr92env is probably transported to the cell surface before additional glycosylation or cleavage to mature virion glycoprotein. The nonprocessed precursor of SE521 appears to be glycosylated normally, and thus far we have been unable to determine the basis for the defect in this mutant.     

Further evidence for the existence of B-lymphocyte subpopulations.

We have studied the homing properties of B lymphocytes by using ⁵¹Cr-labeled lymphoid cells obtained from athymic, nu/nu mice, and animals made T-lymphocyte deficient by thymectomy and lethal irradiation followed by reconstitution with syngeneic bone marrow. Comparison was made to the patterns of distribution observed when cell preparations containing normal numbers of T and B lymphocytes were migrated. A small but significant percentage of labeled lymphocytes from lymph nodes, spleen, Peyer's Patches, and bone marrow of T-cell-deficient animals was shown to be lymph node seeking. Secondary transfers of lymph node cells from primary recipients caused enrichment of this lymph node-seeking population. Treatment of T-lymphocyte-deficient lymphoid cell preparations with neuraminidase reduced the percentages of cells homing to the lymph nodes. The data showed that B lymphocytes exhibit unique homing properties when injected into normal recipients. In addition, direct comparison of the homing patterns of B lymphocytes prepared from spleen and lymph nodes of athymic mice revealed differences suggesting that these lymphoid organs contained unique mixtures of at least two different kinds of B cell. The evidence supports the notion that the B-lymphocyte populations contain at least two subpopulations, one of which possesses the ability to home to lymph nodes.     

Heterogeneous RNA of the Prague strain of Rous sarcoma virus caused by undiluted passages.

The size of genomic RNA in PR-RSV A passaged in chick embryo fibroblasts (CEF) or quail embryo fibroblasts (QEF) was determined by gel electrophoresis. The results showed that 3 undiluted passages resulted in heterogeneity of RNA. The heterogeneity of the smaller incomplete RNAs in the virus stock was decreased by diluted passage or cloning, but RNA of the b subunit size and a subunit RNA of complete genome size were relatively stable. These heterogeneous RNAs were characterized by hybridization analysis. The RNAs from 4 peaks hybridized with both cDNAtotal and cDNAsrc to appreciable extents, indicating that they were derived from viral RNA and that at least some of them contained the src sequence. This finding and the failure to isolate a td mutant from the undiluted-passaged virus stock or from some subclones that had a and b subunits of RNA indicate that the td virus was only a minor constituent of the incomplete virus population caused by undiluted passages. Some viruses with incomplete RNA in the virus stock could produce foci with the aid of td B77 or RAV-60. The emergence of rd viruses by undiluted passages was indicated.