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

Abstract

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 ...

The immunopathogenesis of equine infectious anemia virus

“…The viral envelope is derived from host cell plasma membranes during the budding process essential to particle maturation (Gonda et al, 1978;Matheka et al, 1976;Weiland et al, 1977). The viral core contains a Mg2+-dependent reverse transcriptase enzyme Charman et al, 19761, a 5.5 x lo6 Da RNA genome (Charman et al, 1976) consisting of two identical subunits of single-stranded RNA (Cheevers et al, 19771, and four virus specific non-glycosylated core proteins (~15, ~26, pll, and p9) (Henderson et al, 1987;Montelaro et al, 1982). About l/3 of the core nucleic acid is thought to be tRNA (Char-man et al, 1976).…”

“…Other lentiviruses, including HIV-l, have a myristic acid residue linked to the amino terminus of the capsid precursor protein that is thought to aid insertion of the protein into the cell membrane during virus assembly and release (Veronese et al, 1988). The p15 protein is variably phosphorylated at serine and threonine residues (Montelaro et al, 1982).…”

“…The small pll protein is thought to be closely associated with viral RNA in the intact virion. It is highly basic, with 17% lysine and arginine residues, and has a relatively high glycine content (Montelaro et al, 1982). These amino acids are associated with the nucleic acid binding and flexible nature of ribonucleoproteins of other viruses and of histones (Montelaro et al, 1982;Oroszlan and Gilden, 1980).…”

Rapid detection of viral-specific antibodies by enzyme-linked immunosorbent assay (ELISA)

“…Procedures for the growth of the virus and purification of the p26 protein have been developed (Montelaro et al, 1982). Enzyme conjugated, equine virus-specific polyclonal or monoclonal antibody was used as the detecting molecule.…”

Equine Infectious Anemia

“…9 The emergence in the host of novel antigenic variants of these surface glycoproteins results in the recrudescing febrile reactions that are characteristic of the disease. The virus has a dense central core consisting of viral RNA,'o four nonglycosylated structural nucleocapsid proteins, 35,76 and the viral reverse transcriptase enzyme. , , 9 The viral RNA genome encodes three major product groups, designated: gag (group-associated antigen) coding for the major internal structural proteins; pol (polymerase) designating the reverse transcriptase enzyme, ribonuclease H, and a DNA-nicking enzyme to aid integration into the host cell genome; and env (envelope) to produce the envelope glycoproteins gp90 and gp45.…”