Novel p19-related protein in Rous-associated virus type 61: implications for avian gag gene order

We have reinvestigated the ability of the matrix protein (MA) (p19gag) of avian sarcoma and leukemia viruses to interact with RNA. Previous reports claimed on the one hand that MA can bind tightly and with a high degree of specificity to avian sarcoma and leukemia virus RNA in vitro and on the other that it cannot bind to RNA at all. We found that MA purified by any of several methods does bind to RNA, as measured by its ability to cause retention of radioactive RNA on nitrocellulose membranes in a filtration assay. However, this interaction is weak and lacks specificity. The interaction of MA with RNA was barely detectable by classical sedimentation analysis, and from this observation we estimate that the intrinsic MA-RNA association constant is ca. 10(3) M-1, at least 3 orders of magnitude smaller than the constant describing the interaction of the viral nucleocapsid protein (NC) (p12gag) with RNA, ca. 10(6) M-1. Separately purified phosphorylated and nonphosphorylated MA species bound RNA equally. We also found that MA can bind to DNA with an affinity similar to that for RNA. The large quantitative discrepancy between our results and earlier published reports can be traced in part to methods of data analysis.

Section: Resultsmentioning

confidence: 99%

RNA-binding properties of the matrix protein (p19gag) of avian sarcoma and leukemia viruses

Steeg

Vogt

1990

“…Purified EIAV and protein standards (Std) were included as references; the molecular weights are indicated on the left (94K, molecular weight of 94,000). About 20 ,ug of each protein pool, 75 ,ug of EIAV, and 60 ,ug of the protein standard mixture were prepared for and analyzed by SDS-PAGE as described previously (34,43,44). evidently as aggregates (38).…”

Section: Resultsmentioning

confidence: 99%

“…SDS-PAGE. The procedures used for sample preparation, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) on slab or cylindrical polyacrylamide gels, and analysis of gels by staining or fluorography have been described previously (33,43,44). However, the gels used in this study contained 1 M urea, which enhanced the resolution of the virion low-molecular-weight proteins compared with the standard gel system.…”

mentioning

confidence: 99%

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Isolation and comparative biochemical properties of the major internal polypeptides of equine infectious anemia virus

Montelaro¹,

Lohrey²,

Parekh³

et al. 1982

We describe procedures for the large-scale production of equine infectious anemia virus (EIAV) and for the isolation of the four major non-glycosylated virion proteins, designated p26, p15, pll, and p9. Comparisons of the purified proteins by peptide mapping procedures and by enzyme-linked immunosorbent assays demonstrated the unrelatedness of the four proteins. The characteristic properties of each purified protein were examined by determining isoelectric points and amino acid compositions. We found that EIAV p26 and p9 focus at pl values of 6.2 and 5.0, respectively, and that these proteins contain no unusual amino acids. In contrast, EIAV p15 reproducibly displayed a heterogeneous isoelectric focusing pattern, with major pl values ranging from 5.7 to 8.3. This charge variation evidently correlated with different levels of phosphorylated serine or threonine or both, which could be detected by an amino acid analysis of purified p15. EIAV pll apparently focused at a pI of >10, reflecting its high content of basic amino acids. Moreover, localization experiments indicated that all four nonglycosylated proteins constitute the internal components of the virus, with all of the virion pll closely associated with the viral RNA genome. Thus, our results demonstrated that EIAV, a lentivirus, contains structural polypeptides which are analogous to the structural polypeptides described previously in prototype C oncoviruses.Equine infectious anemia is unique among virus-induced diseases in that clinical symptoms and associated bursts of viremia occur in sequential episodes separated by several weeks or months (13, 23). The available evidence indicates that the periodic nature of this disease is due to the sequential production and release of novel antigenic strains of equine infectious anemia virus (EIAV) which temporarily escape host immunosurveillance systems (13, 23-26). Thus, EIAV provides a dynamic system for investigating the interaction between host immune mechanisms and antigens involved in a persistent virus infection. However, a prerequisite for studying this system is a thorough description of the antigens of the causative virus.EIAV matures by budding from cytoplasmic membranes (31, 48), displays a complex morphology characteristic of type C viruses (16), and contains a reverse transcriptase (2) and a high-molecular-weight (60S to 70S) RNA genome composed of subunits (11). Based on these properties, EIAV has been tentatively classified as a member of the family Retroviridae (9, 13, 23, 46). However, serological comparisons have failed to detect any relatedness be-tween EIAV and a number of retroviruses (9, 46), and some ultrastructural studies (16) have suggested that EIAV resembles visna virus, a member of the lentivirus subfamily, more closely than any member of the oncovirus subfamily. These observations and the initial descriptions of EIAV and visna virus polypeptides have led to the suggestion that the protein composition of lentiviruses may be distinct from the protein compositions of prototype C mammalian ...

“…This problem is exacerbated by the propensity of the p10 protein to leach out of gels even after fixation (21) and by the nonuniform or incomplete cleavage of MA. Depending on the strain of virus and on the virus preparation, MA may carry C-terminal extensions including p2a, p2b, or p10 or may be cleaved by a host cell protease to yield a polypeptide shorter than MA (22,24,33). Thus, it is possible that the apparent modest underrepresentation of MA in virions (Fig.…”

mentioning

confidence: 99%

Mass Determination of Rous Sarcoma Virus Virions by Scanning Transmission Electron Microscopy

Vogt

Simon

1999

122

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 × 108 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.