Localized sequence heterogeneity in the long terminal repeats of in vivo isolates of equine infectious anemia virus

Abstract

The role of in vivo long terminal repeat (LTR) sequence variation of the lentivirus equine infectious anemia virus (EIAV) has not been explored. In this study, we investigated the heterogeneity found in the LTR sequences from seven EIAV-seropositive horses: three horses with clinical disease and four horses without any detectable signs of disease. LTR sequences were targeted in this study because the LTR U3 enhancer region of tissue culture-derived isolates has been identified as one of the few hypervariable regions of the EIAV genome. Furthermore, LTR variation may regulate EIAV expression in vivo. Both intra-and interanimal sequence variations were investigated. The intra-animal variation was low in seropositive, healthy horses (on average 0.44%). Intra-animal variation was consistently higher in clinically ill horses (0.99%), suggesting that greater numbers of quasispecies of EIAV are present when active virus replication is ongoing. Interanimal comparisons of consensus sequences generated from each horse demonstrated that the enhancer region is a hotspot of sequence variation in vivo. Thirty-seven of the 83 nucleotides that compose the U3 enhancer region were variable between the different in vivo-derived LTRs. The remainder of the LTR that was analyzed was more conserved, 8 of 195 nucleotide positions being variable. Results of electrophoretic mobility shift assays demonstrated that some nucleotide substitutions that occurred in the enhancer region eliminated or altered transcription factor binding motifs that are known to be important for EIAV LTR expression. These data suggested that the selective pressures exerted on the EIAV LTR enhancer sequences are different from those exerted on the remainder of the LTR. Our findings are consistent with the possibility that enhancer sequence hypervariability can alter expression of the virus in tissue macrophages and therefore contribute to clinical disease in infected horses.

Biological Characterization of Rev Variation in Equine Infectious Anemia Virus

“…Genetic mutations in the viral env gene are associated with the occurrence of antigenic-variant viruses, and the role of antigenic variation in EIAV persistence has been extensively studied (14, 20,29,32,35). Additional clusters of genetic variation are found in the virus long terminal repeat and in the region of gp45/Rev overlap (1,4,21,27). The biological significance of variation in these regions is not clear; however, genetic changes which alter levels of viral gene expression may be important factors in viral pathogenesis in vivo.…”

Characterization of Rev variants selected during chronic and inapparent stages of equine infectious anemia virus infection

“…1) exhibits great genetic variation, and the error rate of RT has been shown to be as high as HIV-1 ( 6). The quasispecies nature of EIA V has been demonstrated for the genes env and rev, as well as the untranslated long terminal repeats (LTR) (1,8,54,61,86). Mutations within env have been shown to contribute to the emergence of neutralization-escape variants (54,87).…”

Genetic variation and evolution of equine infectious anemia virus rev quasispecies during long term persistent infection

“…The quasispecies nature of EIAV has been well documented (5,7,53,59). While genetic variation occurs throughout the EIAV genome, three regions of hypervariability have been observed: the viral long terminal repeat (LTR), the surface envelope (SU), and a region where the major exon of Rev is completely overlapped by the transmembrane protein (TM) (3,7,57). The antigenic variation caused by genetic variation in structural proteins is a wellstudied mechanism of EIAV persistence.…”

“…Recent studies suggest that genetic variation in the EIAV LTR and other regulatory proteins may contribute to viral persistence through down-regulation of viral gene expression (7,53,57). The LTR contains the only viral promoter region and the transcription start site for the virus.…”

“…In vivo variation has been observed in the EIAV LTR which altered transcription factor binding sites (7). Ablation of transcription factor binding sites may contribute both to changes in the level of virus gene expression during the course of infection and changes in cell tropism (57). Genetic variation in the regulatory gene, rev, has been observed both in vivo and in vitro (3,53).…”