Mapping of equine lentivirus receptor 1 residues critical for equine infectious anemia virus envelope binding

The equine lentivirus receptor 1 (ELR1), a member of the tumor necrosis factor receptor (TNFR) protein family, has been identified as a functional receptor for equine infectious anemia virus (EIAV). Toward defining the functional interactions between the EIAV SU protein (gp90) and its ELR1 receptor, we mapped the gp90 binding domain of ELR1 by a combination of binding and functional assays using the EIAV SU gp90 protein and various chimeric receptor proteins derived from exchanges between the functional ELR1 and the nonbinding homolog, mouse herpesvirus entry mediator (murine HveA). Complementary exchanges of the respective cysteine-rich domains (CRD) between the ELR1 and murine HveA proteins revealed CRD1 as the predominant determinant of functional gp90 binding to ELR1 and also to a chimeric murine HveA protein expressed on the surface of transfected Cf2Th cells. Mutations of individual amino acids in the CRD1 segment of ELR1 and murine HveA indicated the Leu70 in CRD1 as essential for functional binding of EIAV gp90 and for virus infection of transduced Cf2Th cells. The specificity of the EIAV SU binding domain identified for the ELR1 receptor is fundamentally identical to that reported previously for functional binding of feline immunodeficiency virus SU to its coreceptor CD134, another TNFR protein. These results indicate unexpected common features of the specific mechanisms by which diverse lentiviruses can employ TNFR proteins as functional receptors.     

Immune responses and viral replication in long-term inapparent carrier ponies inoculated with equine infectious anemia virus

Persistent infection of equids by equine infectious anemia virus (EIAV) is typically characterized by a progression during the first year postinfection from chronic disease with recurring disease cycles to a long-term asymptomatic infection that is maintained indefinitely. The goal of the current study was to perform a comprehensive longitudinal analysis of the course of virus infection and development of host immunity in experimentally infected horses as they progressed from chronic disease to long-term inapparent carriage. We previously described the evolution of EIAV genomic quasispecies (C. Leroux, C. J. Issel, and R. C. Montelaro, J. Virol. 71:9627-9639, 1997) and host immune responses (S. A. Hammond, S. J. Cook, D. L. Lichtenstein, C. J. Issel, and R. C. Montelaro, J. Virol. 71:3840-3852, 1997) in four experimentally infected ponies during sequential disease episodes associated with chronic disease during the first 10 months postinfection. In the current study, we extended the studies of these experimentally infected ponies to 3 years postinfection to characterize the levels of virus replication and development of host immune responses associated with the progression from chronic disease to long-term inapparent infection. The results of these studies revealed over a 10(3)-fold difference in the steady-state levels of plasma viral RNA detected during long-term inapparent infection that correlated with the severity of chronic disease, indicating different levels of control of virus replication during long-term inapparent infections. Detailed analyses of antibody and cellular immune responses in all four ponies over the 3-year course of infection revealed a similar evolution during the first year postinfection of robust humoral and cellular immunity that then remained relatively constant during long-term inapparent infection. These observations indicate that immune parameters that have previously been correlated with EIAV vaccine protection fail to provide reliable immune correlates of control of virus replication or clinical outcome in experimental infections. Thus, these data emphasize the differences between immunity to virus exposure and immune control of an established viral infection and further emphasize the need to develop and evaluate novel immunoassays to define reliable immune correlates to vaccine and infection immunity, respectively.     

Endocytosis and a low-pH step are required for productive entry of equine infectious anemia virus

Recently, it has become evident that entry of some retroviruses into host cells is dependent upon a vesicle-localized, low-pH step. The entry mechanism of equine infectious anemia virus (EIAV) has yet to be examined. Here, we demonstrate that wild-type strains of EIAV require a low-pH step for productive entry. Lysosomotropic agents that inhibit the acidification of internal vesicles inhibited productive entry of EIAV. The presence of ammonium chloride (30 mM), monensin (30 microM), or bafilomycin A (50 nM) in the medium dramatically decreased the number of EIAV antigen-positive cells. We found that a low-pH step was required for EIAV infection of tissue culture cell lines as well as primary cells, such as endothelial cells and monocyte-derived macrophages. The ammonium chloride treatment did not reduce virion stability, nor did the treatment prevent virion binding to cells. Consistent with a requirement for a low-pH step, virion infectivity was enhanced more than threefold by brief low-pH treatment following binding of viral particles to permissive cells. A superinfecting variant strain of EIAV, vMA-1c, did not require a low-pH step for productive infection of fibroblasts. However, lysosomotropic agents were inhibitory to vMA-1c infection in the other cell types that vMA-1c infected but did not superinfect, indicating that the entry pathway used by vMA-1c for superinfection abrogates the need for the low-pH step.     

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.     

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).     

Analysis of equine humoral immune responses to the transmembrane envelope glycoprotein (gp45) of equine infectious anemia virus.

Defined segments of the transmembrane envelope glycoprotein (gp45) of equine infectious anemia virus were expressed as TrpLE fusion proteins and examined for their reactivity in Western immunoblots against a diverse panel of equine immune sera. The most immunogenic region of gp45 was localized to its amino terminus, positioned between the hydrophobic fusion and the transmembrane domains. A series of overlapping synthetic peptides were used in enzyme-linked immunosorbent assays to define an immunodominant epitope within this region. In contrast, the carboxy-terminal half of gp45 displayed both weak and variable immunoreactivity with equine immune sera.     

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.     

Maturation of the cellular and humoral immune responses to persistent infection in horses by equine infectious anemia virus is a complex and lengthy process.

Equine infectious anemia virus (EIAV) provides a natural model system by which immunological control of lentivirus infections may be studied. To date, no detailed study addressing in parallel both the humoral and cellular immune responses induced in horses upon infection by EIAV has been conducted. Therefore, we initiated the first comprehensive characterization of the cellular and humoral immune responses during clinical progression from chronic disease to inapparent stages of EIAV infection. Using new analyses of antibody avidity and antibody epitope conformation dependence that had not been previously employed in this system, we observed that the humoral immune response to EIAV required a 6- to 8-month period in which to fully mature. During this time frame, EIAV-specific antibody evolved gradually from a population characterized by low-avidity, nonneutralizing, and predominantly linear epitope specificity to an antibody population with an avidity of moderate to high levels, neutralizing activity, and predominantly conformational epitope specificity. Analyses of the cell-mediated immune response to EIAV revealed CD4+ and CD8+ major histocompatibility complex-restricted, EIAV-specific cytotoxic T-lymphocyte (CTL) activity apparent within 3 to 4 weeks postinfection, temporally correlating with the resolution of the primary viremia. After resolution of the initial viremia, EIAV-specific CTL activity differed greatly among the experimentally infected ponies, with some animals having readily detectable CTL activity while others had little measurable CTL activity. Thus, in contrast to the initial viremia, it appeared that no single immune parameter correlated with the resolution of further viremic episodes. Instead, immune control of EIAV infection during the clinically inapparent stage of infection appears to rely on a complex combination of immune system mechanisms to suppress viral replication that effectively functions only after the immune system has evolved to a fully mature state 6 to 8 months postinfection. These findings strongly imply the necessity for candidate EIAV and other lentivirus vaccines to achieve this immune system maturation for efficacious immunological control of lentivirus challenge.     

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.     

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.     

Computational modeling suggests dimerization of equine infectious anemia virus Rev is required for RNA binding

Background

The lentiviral Rev protein mediates nuclear export of intron-containing viral RNAs that encode structural proteins or serve as the viral genome. Following translation, HIV-1 Rev localizes to the nucleus and binds its cognate sequence, termed the Rev-responsive element (RRE), in incompletely spliced viral RNA. Rev subsequently multimerizes along the viral RNA and associates with the cellular Crm1 export machinery to translocate the RNA-protein complex to the cytoplasm. Equine infectious anemia virus (EIAV) Rev is functionally homologous to HIV-1 Rev, but shares very little sequence similarity and differs in domain organization. EIAV Rev also contains a bipartite RNA binding domain comprising two short arginine-rich motifs (designated ARM-1 and ARM-2) spaced 79 residues apart in the amino acid sequence. To gain insight into the topology of the bipartite RNA binding domain, a computational approach was used to model the tertiary structure of EIAV Rev.

Results

The tertiary structure of EIAV Rev was modeled using several protein structure prediction and model quality assessment servers. Two types of structures were predicted: an elongated structure with an extended central alpha helix, and a globular structure with a central bundle of helices. Assessment of models on the basis of biophysical properties indicated they were of average quality. In almost all models, ARM-1 and ARM-2 were spatially separated by >15 Å, suggesting that they do not form a single RNA binding interface on the monomer. A highly conserved canonical coiled-coil motif was identified in the central region of EIAV Rev, suggesting that an RNA binding interface could be formed through dimerization of Rev and juxtaposition of ARM-1 and ARM-2. In support of this, purified Rev protein migrated as a dimer in Blue native gels, and mutation of a residue predicted to form a key coiled-coil contact disrupted dimerization and abrogated RNA binding. In contrast, mutation of residues outside the predicted coiled-coil interface had no effect on dimerization or RNA binding.

Conclusions

Our results suggest that EIAV Rev binding to the RRE requires dimerization via a coiled-coil motif to juxtapose two RNA binding motifs, ARM-1 and ARM-2.

     

Functional roles of equine infectious anemia virus Gag p9 in viral budding and infection

Previous studies utilizing Gag polyprotein budding assays with transfected cells reveal that the equine infectious anemia virus (EIAV) Gag p9 protein provides a late assembly function mediated by a critical Y(23)P(24)D(25)L(26) motif (L-domain) to release viral particles from the plasma membrane. To elucidate further the role of EIAV p9 in virus assembly and replication, we have examined the replication properties of a defined series of p9 truncation and site-directed mutations in the context of a reference infectious molecular proviral clone, EIAV(uk). Characterization of these p9 proviral mutants revealed new functional properties of p9 in EIAV replication, not previously elucidated by Gag polyprotein budding assays. The results of these studies demonstrated that only the N-terminal 31 amino acids of a total of 51 residues in the complete p9 protein were required to maintain replication competence in transfected equine cells; proviral mutants with p9 C-terminal truncations of 20 or fewer amino acids remained replication competent, while mutants with truncations of 21 or more residues were completely replication defective. The inability of the defective p9 proviral mutations to produce infectious virus could not be attributed to defects in Gag polyprotein expression or processing, in virion RT activity, or in virus budding. While proviral replication competence appeared to be associated with the presence of a K(30)K(31) motif and potential ubiquitination of the EIAV p9 protein, mutations of these lysine residues to methionines produced variant proviruses that replicated as well as the parental EIAV(uk) in transfected ED cells. Thus, these observations reveal for the first time that EIAV p9 is not absolutely required for virus budding in the context of proviral gene expression, suggesting that other EIAV proteins can at least in part mediate late budding functions previously associated with the p9 protein. In addition, the data define a function for EIAV p9 in the infectivity of virus particles, indicating a previously unrecognized role for this Gag protein in EIAV replication.