Subpopulations of equine infectious anemia virus Rev coexist in vivo and differ in phenotype.
ABSTRACT: Lentiviruses exist in vivo as a population of related, nonidentical genotypes, commonly referred to as quasispecies. The quasispecies structure is characteristic of complex adaptive systems and contributes to the high rate of evolution in lentiviruses that confounds efforts to develop effective vaccines and antiviral therapies. Here, we describe analyses of genetic data from longitudinal studies of genetic variation in a lentivirus regulatory protein, Rev, over the course of disease in ponies experimentally infected with equine infectious anemia virus. As observed with other lentivirus data, the Rev variants exhibited a quasispecies character. Phylogenetic and partition analyses suggested that the Rev quasispecies comprised two distinct subpopulations that coexisted during infection. One subpopulation appeared to accumulate changes in a linear, time-dependent manner, while the other evolved radially from a common variant. Over time, the two subpopulations cycled in predominance coincident with changes in the disease state, suggesting that the two groups differed in selective advantage. Transient expression assays indicated the two populations differed significantly in Rev nuclear export activity. Chimeric proviral clones containing Rev genotypes representative of each population differed in rate and overall level of virus replication in vitro. The coexistence of genetically distinct viral subpopulations that differ in phenotype provides great adaptability to environmental changes within the infected host. A quasispecies model with multiple subpopulations may provide additional insight into the nature of lentivirus reservoirs and the evolution of antigenic and drug-resistant variants.
Project description:The lentiviruses are associated with a wide range of chronic diseases in mammals. These include immunodeficiencies (such as HIV/AIDS in humans), malignancies, and lymphatic and neurological disorders in primates, felids, and a variety of wild and domesticated ungulates. Evolutionary analyses of the genomic sequences of modern-day lentiviruses have suggested a relatively recent date for their emergence, but the failure to identify any endogenous, vertically transmitted examples has meant that their longer term evolutionary history and origin remain unknown. Here we report the discovery and characterization of retroviral sequences belonging to a new lentiviral subgroup from the European rabbit (Oryctolagus cuniculus). These viruses, the first endogenous examples described, are >7 million years old and thus provide the first evidence for an ancient origin of the lentiviruses. Despite being ancient, this subgroup contains many of the features found in present-day lentiviruses, such as the presence of tat and rev genes, thus also indicating an ancient origin for the complex regulation of lentivirus gene expression. Although the virus we describe is defective, reconstruction of an infectious progenitor could provide novel insights into lentivirus biology and host interactions.
Project description:A primary mechanism of lentivirus persistence is the ability of these viruses to evolve in response to biological and immunological selective pressures with a remarkable array of genetic and antigenic variations that constitute a perpetual natural experiment in genetic engineering. A widely accepted paradigm of lentivirus evolution is that the rate of genetic variation is correlated directly with the levels of virus replication: the greater the viral replication, the more opportunities that exist for genetic modifications and selection of viral variants. To test this hypothesis directly, we examined the patterns of equine infectious anemia virus (EIAV) envelope variation during a 2.5-year period in experimentally infected ponies that differed markedly in clinical progression and in steady-state levels of viral replication as indicated by plasma virus genomic RNA assays. The results of these comprehensive studies revealed for the first time similar extents of envelope gp90 variation in persistently infected ponies regardless of the number of disease cycles (one to six) and viremia during chronic disease. The extent of envelope variation was also independent of the apparent steady-state levels of virus replication during long-term asymptomatic infection, varying from undetectable to 10(5) genomic RNA copies per ml of plasma. In addition, the data confirmed the evolution of distinct virus populations (genomic quasispecies) associated with sequential febrile episodes during acute and chronic EIA and demonstrated for the first time ongoing envelope variation during long-term asymptomatic infections. Finally, comparison of the rates of evolution of the previously defined EIAV gp90 variable domains demonstrated distinct differences in the rates of nucleotide and amino acid sequence variation, presumably reflecting differences in the ability of different envelope domains to respond to immune or other biological selection pressures. Thus, these data suggest that EIAV variation can be associated predominantly with ongoing low levels of virus replication and selection in target tissues, even in the absence of substantial levels of plasma viremia, and that envelope variation continues during all stages of persistent infection as the virus successfully avoids clearance by host defense mechanisms.
Project description:G-quadruplexes (G4s) are secondary structures of nucleic acids that epigenetically regulate cellular processes. In the human immunodeficiency lentivirus 1 (HIV-1), dynamic G4s are located in the unique viral LTR promoter. Folding of HIV-1 LTR G4s inhibits viral transcription; stabilization by G4 ligands intensifies this effect. Cellular proteins modulate viral transcription by inducing/unfolding LTR G4s. We here expanded our investigation on the presence of LTR G4s to all lentiviruses. G4s in the 5'-LTR U3 region were completely conserved in primate lentiviruses. A G4 was also present in a cattle-infecting lentivirus. All other non-primate lentiviruses displayed hints of less stable G4s. In primate lentiviruses, the possibility to fold into G4s was highly conserved among strains. LTR G4 sequences were very similar among phylogenetically related primate viruses, while they increasingly differed in viruses that diverged early from a common ancestor. A strong correlation between primate lentivirus LTR G4s and Sp1/NF?B binding sites was found. All LTR G4s folded: their complexity was assessed by polymerase stop assay. Our data support a role of the lentiviruses 5'-LTR G4 region as control centre of viral transcription, where folding/unfolding of G4s and multiple recruitment of factors based on both sequence and structure may take place.
Project description:Although CTL are critical for control of lentiviruses, including equine infectious anemia virus, relatively little is known regarding the MHC class I molecules that present important epitopes to equine infectious anemia virus-specific CTL. The equine class I molecule 7-6 is associated with the equine leukocyte Ag (ELA)-A1 haplotype and presents the Env-RW12 and Gag-GW12 CTL epitopes. Some ELA-A1 target cells present both epitopes, whereas others are not recognized by Gag-GW12-specific CTL, suggesting that the ELA-A1 haplotype comprises functionally distinct alleles. The Rev-QW11 CTL epitope is also ELA-A1-restricted, but the molecule that presents Rev-QW11 is unknown. To determine whether functionally distinct class I molecules present ELA-A1-restricted CTL epitopes, we sequenced and expressed MHC class I genes from three ELA-A1 horses. Two horses had the 7-6 allele, which when expressed, presented Env-RW12, Gag-GW12, and Rev-QW11 to CTL. The other horse had a distinct allele, designated 141, encoding a molecule that differed from 7-6 by a single amino acid within the alpha-2 domain. This substitution did not affect recognition of Env-RW12, but resulted in more efficient recognition of Rev-QW11. Significantly, CTL recognition of Gag-GW12 was abrogated, despite Gag-GW12 binding to 141. Molecular modeling suggested that conformational changes in the 141/Gag-GW12 complex led to a loss of TCR recognition. These results confirmed that the ELA-A1 haplotype is comprised of functionally distinct alleles, and demonstrated for the first time that naturally occurring MHC class I molecules that vary by only a single amino acid can result in significantly different patterns of epitope recognition by lentivirus-specific CTL.
Project description:Asymptomatic infection with simian immunodeficiency virus (SIV) has been demonstrated in African Sykes' monkeys (Cercopithecus mitis albogularis), and virus isolation confirmed infection with a novel SIV from Sykes' monkeys (SIVsyk). Macaques inoculated with SIVsyk became persistently infected but remained clinically healthy. We utilized polymerase chain reaction amplification to generate a full-length, infectious molecular clone of SIVsyk. The genome organization of SIVsyk is similar to that of the other primate lentiviruses, consisting of gag, pol, vif, vpr, tat, rev, env, and nef. A unique feature is the absence of the highly conserved NF-kappa B binding site in the long terminal repeat. SIVsyk is genetically equidistant from other primate lentiviruses. Thus, SIVsyk represents a new group that is distinct from the four previously recognized primate lentivirus groups: human immunodeficiency virus type 1 (HIV-1), SIV from sooty mangabeys (SIVsmm) and HIV-2, SIV from African green monkeys (SIVagm), and SIV from mandrills (SIVmnd). The genetic differences between SIVsyk and SIVagm, isolates derived from monkeys of the same genus, underscore the potential for other distinct SIVs which have yet to be isolated and characterized.
Project description:A cis-acting RNA regulatory element, the Rev-responsive element (RRE), has essential roles in replication of lentiviruses, including human immunodeficiency virus (HIV-1) and equine infection anemia virus (EIAV). The RRE binds the viral trans-acting regulatory protein, Rev, to mediate nucleocytoplasmic transport of incompletely spliced mRNAs encoding viral structural genes and genomic RNA. Because of its potential as a clinical target, RRE-Rev interactions have been well studied in HIV-1; however, detailed molecular structures of Rev-RRE complexes in other lentiviruses are still lacking. In this study, we investigate the secondary structure of the EIAV RRE and interrogate regulatory protein-RNA interactions in EIAV Rev-RRE complexes. Computational prediction and detailed chemical probing and footprinting experiments were used to determine the RNA secondary structure of EIAV RRE-1, a 555 nt region that provides RRE function in vivo. Chemical probing experiments confirmed the presence of several predicted loop and stem-loop structures, which are conserved among 140 EIAV sequence variants. Footprinting experiments revealed that Rev binding induces significant structural rearrangement in two conserved domains characterized by stable stem-loop structures. Rev binding region-1 (RBR-1) corresponds to a genetically-defined Rev binding region that overlaps exon 1 of the EIAV rev gene and contains an exonic splicing enhancer (ESE). RBR-2, characterized for the first time in this study, is required for high affinity binding of EIAV Rev to the RRE. RBR-2 contains an RNA structural motif that is also found within the high affinity Rev binding site in HIV-1 (stem-loop IIB), and within or near mapped RRE regions of four additional lentiviruses. The powerful integration of computational and experimental approaches in this study has generated a validated RNA secondary structure for the EIAV RRE and provided provocative evidence that high affinity Rev binding sites of HIV-1 and EIAV share a conserved RNA structural motif. The presence of this motif in phylogenetically divergent lentiviruses suggests that it may play a role in highly conserved interactions that could be targeted in novel anti-lentiviral therapies.
Project description:By screening 74 chordate genomes for endogenous lentiviruses using Pol sequences of exogenous lentiviruses as a reference, we identified a novel endogenous lentivirus in the genome of the ferret (Mustela putorius furo). Phylogenetic analysis suggested that the ferret endogenous lentivirus, denoted ELVmpf, diverged early in the evolution of the mammalian lentiviruses, although with a lack of resolution at key nodes. These data support the notion that lentiviruses have evolved on timescales of millions of years.
Project description:We have investigated the genetic evolution of three functionally distinct regions of the equine infectious anemia virus (EIAV) genome (env, rev, and long terminal repeat) during recurring febrile episodes in a pony experimentally infected with a well-characterized reference biological clone designated EIAV(PV). Viral populations present in the plasma of an EIAV(PV)-infected pony during sequential febrile episodes (18, 34, 80, 106, and 337 days postinfection) were amplified from viral RNA, analyzed, and compared to the inoculated strain. The comparison of the viral quasispecies showed that the inoculated EIAV(PV) quasispecies were all represented during the first febrile episode, but entirely replaced at the time of the second febrile episode, and that new predominant quasispecies were associated with each subsequent cycle of disease. One of the more surprising results was the in vivo generation of large deletion (up to 15 amino acids) in the principal neutralizing domain (PND) of gp90 during the third febrile episode. This deletion did not alter the competence for in vitro replication as shown by the analysis of a env chimeric clone with a partially deleted PND and did not altered the fitness of the virus in vivo, since this partially deleted envelope became the major population during the fourth febrile episode. Finally, we showed that the amino acid mutations were not randomly distributed but delineated eight variables regions, V1 to V8, with V3 containing the PND region. These studies provide the first detailed description of the evolution of EIAV genomic quasispecies during persistent infection and reveal new insights into the genetics and potential mechanisms of lentivirus genomic variation.
Project description:An infectious molecular clone of the Petaluma strain of feline immunodeficiency virus (FIV) was isolated from a recombinant bacteriophage library containing genomic DNA prepared from FIV-infected Crandall feline kidney (CRFK) cells. The integrated provirus has a total length of 9472 base pairs. Three long open reading frames corresponding to GAG, POL, and ENV gene coding frames are evident. In addition, an open reading frame overlaps the 3' end of POL, in the region that encodes viral infectivity factor in the primate viruses. Several short open reading frames are present in the intergenic region between POL and ENV and within ENV, which may serve as exons for production of TAT and REV equivalents in FIV. Alignment of the predicted amino acid sequences of the FIV proteins with those of other lentiviruses indicates that FIV did not arise recently from any other characterized lentivirus.
Project description:Rev-like proteins are post-transcriptional regulatory proteins found in several retrovirus genera, including lentiviruses, betaretroviruses, and deltaretroviruses. These essential proteins mediate the nuclear export of incompletely spliced viral RNA, and act by tethering viral pre-mRNA to the host CRM1 nuclear export machinery. Although all Rev-like proteins are functionally homologous, they share less than 30% sequence identity. In the present study, we computationally assessed the extent of structural homology among retroviral Rev-like proteins within a phylogenetic framework.We undertook a comprehensive analysis of overall protein domain architecture and predicted secondary structural features for representative members of the Rev-like family of proteins. Similar patterns of ?-helical domains were identified for Rev-like proteins within each genus, with the exception of deltaretroviruses, which were devoid of ?-helices. Coiled-coil oligomerization motifs were also identified for most Rev-like proteins, with the notable exceptions of HIV-1, the deltaretroviruses, and some small ruminant lentiviruses. In Rev proteins of primate lentiviruses, the presence of predicted coiled-coil motifs segregated within specific primate lineages: HIV-1 descended from SIVs that lacked predicted coiled-coils in Rev whereas HIV-2 descended from SIVs that contained predicted coiled-coils in Rev. Phylogenetic ancestral reconstruction of coiled-coils for all Rev-like proteins predicted a single origin for the coiled-coil motif, followed by three losses of the predicted signal. The absence of a coiled-coil signal in HIV-1 was associated with replacement of canonical polar residues with non-canonical hydrophobic residues. However, hydrophobic residues were retained in the key 'a' and 'd' positions, and the ?-helical region of HIV-1 Rev oligomerization domain could be modeled as a helical wheel with two predicted interaction interfaces. Moreover, the predicted interfaces mapped to the dimerization and oligomerization interfaces in HIV-1 Rev crystal structures. Helical wheel projections of other retroviral Rev-like proteins, including endogenous sequences, revealed similar interaction interfaces that could mediate oligomerization.Sequence-based computational analyses of Rev-like proteins, together with helical wheel projections of oligomerization domains, reveal a conserved homogeneous structural basis for oligomerization by retroviral Rev-like proteins.