Phosphoproteins: structural components of oncornaviruses

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 ...

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Montelaro¹,

Lohrey²,

Parekh³

et al. 1982

“…The ppl2 of 292 field isolate was also used to determine peptide homology between the nonphosphorylated and variously phosphorylated ppl2 species. The pp12 purified from 292 virus, labeled with 3H-amino acids and [32P]phosphate (13,14), was resolved by DEAE-cellulose chromatography (18) into three major peaks. One was non-phosphorylated, and the other two varied in the degree of phosphorylation (data not shown).…”

Section: ~~~~~~~~~-mentioning

confidence: 99%

“…Structural proteins of type C RNA tumor viruses consist of a major high-molecular-weight glycoprotein and several low-molecular-weight non-glycosylated proteins. One of the non-glycosylated proteins has been shown to be the major structural phosphoprotein of the virion (13,14). In viruses of lower mammalian species, including mice, rats, cats, minks, and pigs, the 12,000-dalton protein (p12) is always phosphorylated (ppl2) (13, 14; our unpublished data).…”

mentioning

confidence: 93%

RNA tumor virus phosphoproteins: primary structural analysis and identification of phosphopeptides

Pal¹,

Bryant²,

Roy-Burman³

1978

Self Cite

Two-dimensional tryptic peptide mapping was used to compare the peptide sequences of the phosphoprotein (pp12) of cloned ecotropic and amphotropic wild mouse leukemia viruses, strains 1504 and 292. The maps of two ecotropic isolates were very similar to one another, as were the maps of two amphotropic isolates. There was also extensive similarity between the maps of this protein from ecotropic and amphotropic viruses, although characteristic peptide differences were readily recognized. These differences were consistent with the general type specificity of oncovirus phosphoproteins. The pp12 of the field isoalte of 292 virus contained five phosphopeptides, and the non-phosphorylated and variously phosphorylated species of this pp12 showed identical peptide maps, indicating differential phosphorylation of a single polypeptide.

“…Phosphorylated proteins similar to NS have been found in other RNA and DNA viruses (8,20,22) and are often associated with the viral nucleic acid (10,16). There is an indication that the degree of phosphorylation may play a regulatory role in viral replication.…”

mentioning

confidence: 99%

Transcriptional activities of different phosphorylated species of NS protein purified from vesicular stomatitis virions and cytoplasm of infected cells

Kingsford¹,

Emerson²

1980

113

Vesicular stomatitis virus contains a phosphorylated NS protein which is necessary along with L protein and RNP template for transcription of mRNA. To further define the structure of the NS protein and its function in transcription and replication, virion NS was purified and separated into two different phosphorylated forms (NSI and NSII) on DEAE-cellulose columns. Cytoplasmic preparations of NS contained one phosphorylated species which eluted from the column in the same place as the virion NSI. When electrophoresed in sodium dodecyl sulfate-polyacrylamide gels containing urea, NSI and NSII each resolved into two components, whereas cell NS migrated as a single band. NSI and cell NS exhibited little activity in a reconstituted transcription assay, whereas the more highly phosphorylated NSII was very active in the same system. Addition of NSI or cell NS to a transcription system containing NSII resulted in even higher levels of activity, indicating that the various NS species might have different enzymatic functions.