Polyriboadenylate Sequences at the 3′-Termini of Ribonucleic Acid Obtained from Mammalian Leukemia and Sarcoma Viruses

We have recently found that Moloney murine leukemia virus assembles within cytoplasmic vacuoles of chronically infected NIH/3T3 cells rather than at their surface (submitted for publication). In the present study we found that if these cells were treated with interferon (IF) for 24 to 48 h the intracellular virus particles accumulated at a two- to threefold-higher level than that observed in untreated cells. Nevertheless, despite this accumulation, no difference between IF-treated and untreated cells was observed in the amount of the total cytoplasmic viral RNA or in its 35S or 21S species. When cellular virions were sedimented from the cytoplasmic fraction, a markedly higher amount of viral RNA was detected in the viral pellet of IF-treated cells than was detected in untreated cells, whereas the amount of viral RNA left in the virus-free cytoplasm of IF-treated cells was much lower than that in the untreated cells. Furthermore, the amount of the cytoplasmic polyriboadenylic acid-containing viral RNA was also remarkably higher in the IF-treated cells. Viral polyribosomes appeared to be fully functional in IF-treated cells, since no effect of IF on viral protein synthesis could be detected. Analysis of the nuclear viral RNA showed no difference between IF-treated and untreated cells after 24 h of IF treatment. Both contained a comparable amount of 35S viral RNA. However, at 48 h a significant accumulation of viral RNA was observed in the nucleus of the IF-treated cells as compared with the untreated cells, although in both cases only 35S species were evident. This accumulation appeared to activate a degradation process which destroyed nuclear viral RNA, since a dramatic shift toward smaller-sized molecules of viral RNA and a remarkable reduction in its amount were observed after 72 h of IF treatment.

Section: Resultssupporting

confidence: 55%

Section: Resultsmentioning

confidence: 99%

Characterization of intracellular viral RNA in interferon-treated cells chronically infected with murine leukemia virus

Salzberg¹,

Bakhanashvili²,

Bari³

et al. 1980

“…(Most of this work was submitted by G. F. Okasinski in partial fulfillment of the requirements for the Ph.D. degree, Michigan State Univ., East Lansing, 1976. This was presented in part at the 75th Annual Meeting of the American Society for Microbiology, 27 April-2 May 1975, New York, N.Y., and at the Cold Spring Harbor meeting on RNA Tumor Viruses, 28 May-1 June 1975, Cold Spring Harbor, N.Y.)…”

Section: Downloaded Frommentioning

confidence: 99%

“…Oncornaviruses contain a high-molecularweight genome composed of 28-358 subunits with a molecular weight of approximately 3 x 10 (2, 7, 9). These RNA subunits contain 3' poly(A) sequences (7,20,27). The apparent ability ofthese subunits to serve as mRNA in in vitro protein-synthesizing systems (24,30,35), combined with the presence on polyribosomes of viral-specific RNA with a molecular weight similar to that of genomic subunits (12,16,32), suggests that oncornavirus mRNA is very similar to genomic subunits.…”

mentioning

confidence: 99%

Analysis of intracellular feline leukemia virus proteins. I. Identification of a 60,000-dalton precursor of feline leukemia virus p30

Okasinki¹,

Velicer²

1976

The synthesis and release of feline leukemia virus p30 was studied using a permanently infected feline thymus tumor cell line. Disrupted cells were divided into two subcellular fractions, a cytoplasmic extract (CE) representing cellular material soluble in 0.5% NP-40 and a particulate fraction (PF) insoluble in 0.5% NP-40, but soluble in 0.2% deoxycholate and 0.5% NP-40. Intracellular feline leukemia virus p30 was isolated from infected cells by immune precipitation with antiserum to p30 and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the precipitated proteins. Cells labeled for 3 h with [15S]methionine contained equal amounts of p30 in both the CE and the PF. p30 synthesis was estimated to be 0.8% of the total host cell protein synthesis. Immune precipitates from cells pulse labeled for 2.5 min contained a labeled 60,000-dalton polypeptide (Pp6O) in the PF and a polypeptide in the CE that comigrated with feline leukemia virus p30 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When cells were chased after a pulse label, there was a rapid loss of Pp60 in the PF and an accumulation of p30 in the CE within 30 min followed by distribution of p30 in both the PF and the CE. Estimation of intracellular and extracellular p30 levels during a 0.5-to 24-h chase period suggested that most ofthe newly synthesized p30 was incorporated into extracellular virus. Tryptic peptide analysis of labeled Pp6O and p30 demonstrated the presence of 13 of 15 p30 peptides within the Pp6O molecule. The tryptic peptide analysis in concert with the pulse-chase labeling data provides strong evidence that Pp6O is a precursor of p30.

“…Poly(A) has been previously shown to be located at the 3' ends of RNA from Moloney leukemia and sarcoma viruses (11,12). The presence ofpoly(A) on all of the 1.8 x 10"-daltQn subunit molecules from M-MSV(MLV) (Fig.…”

Section: Resultsmentioning

confidence: 96%

Sequence homology between Moloney murine sarcoma virus and Moloney leukemia virus RNA

Bondurant¹,

Stoltzfus²,

Mitchell³

1976

The Moloney murine sarcoma-leukemia virus lM-MSV(MuLV)], propagated at high multiplicity of infection (MOI), was demonstrated previously to contain a native genome mass of 4 x 10" daltons as contrasted to a mass of 7 x 10' daltons for Moloney murine leukemia virus (M-MuLV). The 4 x 10'-dalton class of RNA from M-MSV(MuLV) was examined for base sequence homology with DNA complementary to the 7 x 106-dalton M-MuLV RNA genome. Approximately 86% of the M-MSV(MuLV) was protected from RNase digestion by hybridization, whereas 95% of M-MuLV was protected under identical conditions. These results indicate that the small RNA class of high-MOI M-MSV(MuLV) contains little (perhaps 10%) genetic information not present in M-MuLV. Virtually all of the 1.8 x 106-dalton subunits of M-MSV(MuLV) RNA contained regions of poly(A) since 94% of the RNA bound to oligo(dT) cellulose in 0.5 M KCI. This suggests that the formation of the 1.8 x 10'-dalton subunits occurs before their packaging into virions and does not result from hydrolysis of intact 3.5 x 106-dalton subunits by a virion-associated nuclease. Earlier work has demonstrated that preparations of Moloney sarcoma-leukemia virus [M-MSV(MuLV)] passed at high multiplicity of infection (MOI) contain high-molecular-weight RNA molecules representing a heterogeneous distribution of sizes (3, 10, 13). Two major native size classes with apparent molecular weights of 6.5 x 10" (corresponding to the single, high-molecular-weight species of RNA obtained from MSV-free M-MuLV) and 4.0 x 10" were distinguishable by gel electrophoresis in such M-MSV(MuLV) populations (3, 13). Serial passage of the virus at high MOI resulted in the predominance of the 4.0 x 106"-dalton class (3) and a relatively high MSV/MuLV ratio (approximately 0.5) (13). Although the native RNA profiles were heterogeneous, subunit RNA from the virus was quite homogeneous and had an apparent molecular weight of 1.8 x 10", a value that is approximately one-half that observed (3.4 x 10") for the subunits of RNAs from M-MuLV (free of MSV activity) or from M-MSV(MuLV) that was propagated at much lower MOIs (13). Maisel et al. (8) have also compared the denatured RNAs of Kirsten MuLV, Kirsten MSV(MuLV), M-MuLV, M-MSV(MuLV), and spleen-focus-forming Friend virus. In all cases studied, the sarcoma virus complexes yielded RNA subunit classes that