Genomic variation and segregation of equine infectious anemia virus during acute infection.

Equine infectious anemia virus (EIAV) is a lentivirus that infects and persists in the monocyte/macrophage populations of blood and tissues. We employed polymerase chain reaction to investigate the distribution and the level of genome variability of EIAV DNA in different tissues of a horse infected with a highly virulent variant of the Wyoming strain of the virus. Long terminal repeat, gag, and pol primer pairs were used to direct the amplification of EIAV DNA from the peripheral blood mononuclear cells and from cells, presumably the macrophage subtypes, of the kidney, spleen, liver, lymph node, and cerebellum and periventricular regions of the brain. Polymerase chain reaction analysis of four domains within the envelope showed that viral subtypes with particular envelope domains segregated to different tissues as defined by the presence or absence of a given type of domain. Collectively, these results show that virus variants in the env gene home to different tissues, presumably because of selection for tissue-specific envelope determinants.     

Rapid emergence of novel antigenic and genetic variants of equine infectious anemia virus during persistent infection.

Previous results from our laboratory have demonstrated that equine infectious anemia virus displays structural variations in its surface glycoproteins and RNA genome during passage and chronic infections in experimentally infected Shetland ponies (Montelaro et al., J. Biol. Chem. 259:10539-10544, 1984; Payne et al., J. Gen. Virol. 65:1395-1399, 1984). The present study was undertaken to obtain an antigenic and biochemical characterization of equine infectious anemia virus isolates recovered from an experimentally infected pony during sequential disease episodes, each separated by intervals of only 4 to 8 weeks. The virus isolates could be distinguished antigenically by neutralization assays with serum from the infected pony and by Western blot analysis with a monoclonal antibody against the major surface glycoprotein gp90, thus demonstrating that novel antigenic variants of equine infectious anemia virus predominate during each clinical episode. The respective virion glycoproteins displayed different electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels, indicating structural variation. Tryptic peptide and glycopeptide maps of the viral proteins of each virus isolate revealed biochemical alterations involving amino acid sequence and glycosylation patterns in the virion surface glycoproteins gp90 and gp45. In contrast, no structural variation was observed in the internal viral proteins pp15, p26, and p9 from any of the four virus isolates. Oligonucleotide mapping experiments revealed similar but unique RNase T1-resistant oligonucleotide fingerprints of the RNA genomes of each of the virus isolates. Localization of altered oligonucleotides for one virus isolate placed two of three unique oligonucleotides within the predicted env gene region of the genome, perhaps correlating with the structural variation observed in the envelope glycoproteins. Thus these results support the concept that equine infectious anemia virus is indeed capable of relatively rapid genomic variations during replication, some of which result in altered glycoprotein structures and antigenic variants which are responsible for the unique periodic disease nature observed in persistently infected animals. The findings of envelope specific differences in isolates of visna virus and of human T-cell lymphotropic virus III (acquired immune deficiency syndrome-related virus) suggest that this variation may be a common characteristic of the subfamily Lentivirinae.     

Structural proteins of equine infectious anemia virus.

Equine infectious anemia virus was found to be comprised of fourteen polypeptides of molecular weight ranging from 10,000 to 79,000. Eighty percent of the virion protein was accounted for by five polypeptides, including two non-glycosylated components (p29 and p13) comprising one-half of the virion protein and three glycoproteins (gp77/79, gp64, and gp40).     

Characterization of RNA from equine infectious anemia virus.

The genome of equine infectious anemia virus, a nononcogenic retrovirus, has been characterized by velocity sedimentation, electrophoresis in polyacrylamide gels, buoyant density in CS2SO4, and susceptibility to nuclease digestion. The nucleic acid of purified virus was resolved by sedimentation analysis into a fast-sedimenting genome component, which comprises about two-thirds of the virion RNA, and a slow-sedimenting RNA, which is probably comprised of host-derived tRNA and a trace amount of 5S RNA. The fast-sedimenting RNA had a sedimentation coefficient of 62S and a molecular weight of 5.4 X 10(6) to 5.6 X 10(6), as determined by sedimentation velocity and electrophoretic mobility. Upon heat denaturation, [3H]uridine-labeled 62S RNA dissociated into material comprised of 90 to 95% single-stranded species, sedimenting predominantly at 34S, with a molecular weight of 2.7 X 10(6) to 2.9 X 10(6) and 5 to 10% 4S RNA. The 62S RNA was predominantly single-stranded but contained double-stranded regions, as indicated by partial resistance to RNase IA and SI nuclease and by a lower buoyant density in CS2SO4 than that of the single-stranded 34S RNA derived by heat denaturation. These data indicated that the viral genome consisted of two 34S subunits of single-stranded RNA held in a high-molecular-weight complex with 4S RNA by a mechanism involving a small degree of base pairing. Thus, the structure of equine infectious anemia virus RNA is similar to that of other retroviruses.     

Regulation of equine infectious anemia virus expression

Equine infectious anemia virus (EIAV) is an ungulate lentivirus that is related to human immunodeficiency virus (HIV). Much of the understanding of lentiviral gene regulation comes from studies using HIV. HIV studies have provided insights into molecular regulation of EIAV expression; however, much of the regulation of EIAV expression stands in stark contrast to that of HIV. This review provides an overview of the current state of knowledge of EIAV regulation by comparing and contrasting EIAV gene regulation to HIV. The role of EIAV gene regulation is discussed in relation to EIAV pathogenesis.     

RNA-dependent DNA polymerase associated with equine infectious anemia virus.

Equine infectious anemia (EIAV) is shown to have an associated RNA-instructed DNA polymerase similar in its cofactor requirements and reaction conditions to the RNA tumor virus DNA polymerases. Demonstrating this DNA polymerase activity requires a critical concentration of a nonionic detergent, all four deoxyribonucleoside triphosphates, and a divalent metal ion. The reaction is sensitive to RNase, and a substantial fraction of the FNA synthesized is complementary to viral RNA. The detection of a complex of tritium-labeled polymerase product DNA-template RNA, which sedimented at 60S to 70S, provided evidence that EIAV contains high-molecular-weight RNA. These results, obtained with both virus propagated in cell culture and virus from the serum of an experimentally infected horse, indicate that EIAV may properly be considered a member of the family Retroviridae. They may also be pertinent to the mechanism(s) of viral persistence and periodic recrudescence of disease in chronically infected horses.     

Chemical and immunological characterizations of equine infectious anemia virus gag-encoded proteins.

The viral core proteins (p15, p26, p11, and p9) of equine infectious anemia virus (EIAV) (Wyoming strain) were purified by reverse-phase high-pressure liquid chromatography. Each purified protein was analyzed for amino acid content, N-terminal amino acid sequence, C-terminal amino acid sequence, and phosphoamino acid content. The results of N- and C-terminal amino acid sequence analysis of each gag protein, taken together with the nucleotide sequence of the EIAV gag gene (R. M. Stephens, J. W. Casey, and N. R. Rice, Science 231:589-594, 1986), show that the order of the proteins in the precursor is p15-p26-*-p11-p9, where a pentapeptide also found in the virus is represented by the asterisk. The data are in complete agreement with the predicted structure of the gag polyprotein and show the peptide bonds cleaved during proteolytic processing. The N-terminus of p15 is blocked to Edman degradation. The p11 protein is identical to the nucleic acid-binding protein of EIAV previously isolated (C. W. Long, L. E. Henderson, and S. Oroszlan, Virology 104:491-496, 1980). High-titer rabbit antiserum was prepared against each purified protein. These antisera were used to detect the putative gag precursor (Pr55gag) and intermediate cleavage products designated Pr49 (p15-p26-*-p11), Pr40 (p15-p26), and Pr35 (p26-*-p11) in the virus and in virus-infected cells. High-titer antisera to EIAV p15 and p26 showed cross-reactivity with the homologous protein of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus.     

Late domain-dependent inhibition of equine infectious anemia virus budding

The Gag proteins of a number of different retroviruses contain late or L domains that promote the release of virions from the plasma membrane. Three types of L domains have been identified to date: Pro-Thr-Ala-Pro (PTAP), Pro-Pro-X-Tyr, and Tyr-Pro-Asp-Leu. It has previously been demonstrated that overexpression of the N-terminal, E2-like domain of the endosomal sorting factor TSG101 (TSG-5') inhibits human immunodeficiency virus type 1 (HIV-1) release but does not affect the release of the PPPY-containing retrovirus murine leukemia virus (MLV), whereas overexpression of the C-terminal portion of TSG101 (TSG-3') potently disrupts both HIV-1 and MLV budding. In addition, it has been reported that, while the release of a number of retroviruses is disrupted by proteasome inhibitors, equine infectious anemia virus (EIAV) budding is not affected by these agents. In this study, we tested the ability of TSG-5', TSG-3', and full-length TSG101 (TSG-F) overexpression, a dominant negative form of the AAA ATPase Vps4, and proteasome inhibitors to disrupt the budding of EIAV particles bearing each of the three types of L domain. The results indicate that (i) inhibition by TSG-5' correlates with dependence on PTAP; (ii) the release of wild-type EIAV (EIAV/WT) is insensitive to TSG-3', whereas this C-terminal TSG101 fragment potently impairs the budding of EIAV when it is rendered PTAP or PPPY dependent; (iii) budding of all EIAV clones is blocked by dominant negative Vps4; and (iv) EIAV/WT release is not impaired by proteasome inhibitors, while EIAV/PTAP and EIAV/PPPY release is strongly disrupted by these compounds. These findings highlight intriguing similarities and differences in host factor utilization by retroviral L domains and suggest that the insensitivity of EIAV to proteasome inhibitors is conferred by the L domain itself and not by determinants in Gag outside the L domain.     

Viral DNA in horses infected with equine infectious anemia virus.

The amount and distribution of viral DNA were established in a horse acutely infected with the Wyoming strain of equine infectious anemia virus (EIAV). The highest concentration of viral DNA were found in the liver, lymph nodes, bone marrow, and spleen. The kidney, choroid plexus, and peripheral blood leukocytes also contained viral DNA, but at a lower level. It is estimated that at day 16 postinoculation, almost all of the viral DNA was located in the tissues, with the liver alone containing about 90 times more EIAV DNA than the peripheral blood leukocytes did. Assuming a monocyte-macrophage target, each infected cell contained multiple copies of viral DNA (between 6 and 60 copies in liver Kupffer cells). At day 16 postinoculation, most of the EIAV DNA was not integrated into host DNA, but existed in both linear and circular unintegrated forms. In contrast to acute infection, viral DNA was not detectable in tissues from asymptomatic horses with circulating antibody to EIAV.