Equine Infectious Anemia Virus Rev Research Articles

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

BackgroundThe 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.ResultsThe 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0115-7) contains supplementary material, which is available to authorized users.

Molecular and Biological Characterization of Equine Infectious Anemia Virus Rev

Equine infectious anemia virus (EIAV) is one of the most divergent members of the lentivirus subfamily of retroviruses and is considered a useful comparative model for molecular studies of lentivirus replication. The Rev protein of EIAV is functionally homologous with other lentiviral Revs and facilitates export of incompletely spliced viral mRNAs through a Crm1-dependent pathway. The trans- and cis-acting elements that mediate EIAV Rev function are similar to, but distinct from, the well-characterized elements in human immunodeficiency virus (HIV-1), the prototypical Rev protein. In addition, the EIAV rev sequence is highly variable in vivo, and changes in Rev phenotype correlate with changes in clinical stages of EIAV infection. This review summarizes the molecular biology of EIAV Rev-RRE interactions and the consequences of Rev variation in vivo. A comparative perspective of Rev activity may enhance understanding of an essential lentiviral protein and stimulate new strategies for treatment and prevention of lentivirus infections in vivo.

Structural Model of the Rev Regulatory Protein from Equine Infectious Anemia Virus

Rev is an essential regulatory protein in the equine infectious anemia virus (EIAV) and other lentiviruses, including HIV-1. It binds incompletely spliced viral mRNAs and shuttles them from the nucleus to the cytoplasm, a critical prerequisite for the production of viral structural proteins and genomic RNA. Despite its important role in production of infectious virus, the development of antiviral therapies directed against Rev has been hampered by the lack of an experimentally-determined structure of the full length protein. We have used a combined computational and biochemical approach to generate and evaluate a structural model of the Rev protein. The modeled EIAV Rev (ERev) structure includes a total of 6 helices, four of which form an anti-parallel four-helix bundle. The first helix contains the leucine-rich nuclear export signal (NES). An arginine-rich RNA binding motif, RRDRW, is located in a solvent-exposed loop region. An ERLE motif required for Rev activity is predicted to be buried in the core of modeled structure where it plays an essential role in stabilization of the Rev fold. This structural model is supported by existing genetic and functional data as well as by targeted mutagenesis of residues predicted to be essential for overall structural integrity. Our predicted structure should increase understanding of structure-function relationships in Rev and may provide a basis for the design of new therapies for lentiviral diseases.

Naturally arising point mutations in non-essential domains of equine infectious anemia virus Rev alter Rev-dependent nuclear-export activity

Equine infectious anemia virus (EIAV) exhibits a high rate of genetic variation in vivo, and results in a clinically variable disease in infected horses. In vivo populations of EIAV have been characterized by the presence of distinct, genetic subpopulations of Rev that differ in phenotype and fluctuate in dominance in a manner coincident with each clinical stage of disease. This study examined the specific mutations that arose in vivo and altered the phenotype. The Rev protein was found to be highly conserved, and only 10 aa mutations were observed at a frequency greater than 10 % in the sample population. Nine of these mutations were capable of significantly altering Rev activity, either as single mutations in the context of the founder variant, or in the context of cumulatively fixed mutations. The results indicated that limited genetic variation outside the essential functional domains of Rev can alter the phenotype and may confer a selective advantage in vivo.

Characterization of Functional Domains of Equine Infectious Anemia Virus Rev Suggests a Bipartite RNA-Binding Domain

Equine infectious anemia virus (EIAV) Rev is an essential regulatory protein that facilitates expression of viral mRNAs encoding structural proteins and genomic RNA and regulates alternative splicing of the bicistronic tat/rev mRNA. EIAV Rev is characterized by a high rate of genetic variation in vivo, and changes in Rev genotype and phenotype have been shown to coincide with changes in clinical disease. To better understand how genetic variation alters Rev phenotype, we undertook deletion and mutational analyses to map functional domains and to identify specific motifs that are essential for EIAV Rev activity. All functional domains are contained within the second exon of EIAV Rev. The overall organization of domains within Rev exon 2 includes a nuclear export signal, a large central region required for RNA binding, a nonessential region, and a C-terminal region required for both nuclear localization and RNA binding. Subcellular localization of green fluorescent protein-Rev mutants indicated that basic residues within the KRRRK motif in the C-terminal region of Rev are necessary for targeting of Rev to the nucleus. Two separate regions of Rev were necessary for RNA binding: a central region encompassing residues 57 to 130 and a C-terminal region spanning residues 144 to 165. Within these regions were two distinct, short arginine-rich motifs essential for RNA binding, including an RRDRW motif in the central region and the KRRRK motif near the C terminus. These findings suggest that EIAV Rev utilizes a bipartite RNA-binding domain.

828. Optimizing the Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element (WPRE) for Safety and Function in Lentiviral Vectors

We have been developing a gene transfer system based on the non-primate lentivirus equine infectious anemia virus (EIAV) for human gene transfer. Previously, we reported that recombinant EIAV vectors pseudotyped with the G-glycoprotein of the vesicular stomatitis virus (VSV) are able to efficiently transduce muscle, hematopoietic, and neuronal cells. The vector has been optimized for vector titers and trans-gene expression in transduced cells by incorporating various cis-acting elements (EIAV Rev responsive element (RRE), Mason Pfizer monkey transport element (CTE), post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE)) into the vector. The optimal vector configuration resulted in placement of the RRE upstream of the internal promoter and the WPRE into the 3-prime untranslated region downstream of the reporter gene. Although this configuration of vector yields the highest viral titers and transgene protein expression in target cells, safety concerns have been raised regarding the potential oncogenic activity of the truncated WHV X protein encoded in this element (Kingsman et al, Gene Therapy 12, 3, 2005). The WPRE used in our vectors is a 900 bp tripartite element described by Donello et al (J Virol, 78, 5085, 1998). The gamma and the alpha sub-elements provide the majority of the post-transcriptional effects on localization of viral RNA with some contribution from the beta sub-element which also encodes the WHV X protein, the function of which is still not clearly understood. To test whether the X protein is being expressed from our constructs, we fused a luciferase reporter gene in frame with the X protein. Luciferase activity was observed in transduced cells, which clearly suggests that the truncated X protein is being expressed. We are determining the levels of expression in primary human hepatocytes and in transduced mouse liver. We have constructed a minimal WPRE sequence of 513 bp that retains its complete cis-acting function. This element contains a 32 amino acid portion of protein X. We are currently making mutations in the protein X promoter which is a 21 nucleotide domain immediately upstream of the ATG codon of protein X and mutating the start codon and X ORF to prevent any aberrant X protein expression in target cells.

Subpopulations of equine infectious anemia virus Rev coexist in vivo and differ in phenotype.

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.

A nuclear kinesin-like protein interacts with and stimulates the activity of the leucine-rich nuclear export signal of the human immunodeficiency virus type 1 rev protein.

The Rev protein of human immunodeficiency virus type 1 (HIV-1) is essential for the nucleocytoplasmic transport of unspliced and partially spliced HIV mRNAs containing the Rev response element (RRE). In a yeast two-hybrid screen of a HeLa cell-derived cDNA expression library for human factors interacting with the Rev leucine-rich nuclear export sequence (NES), we identified a kinesin-like protein, REBP (Rev/Rex effector binding protein), highly homologous to Kid, the carboxy-terminal 75-residue region of which interacts specifically with the NESs of HIV-1 Rev, human T-cell leukemia virus type 1 Rex, and equine infectious anemia virus Rev but not with functionally inactive mutants thereof. REBP is a nuclear protein that colocalizes with Rev in the nucleoplasm and nuclear periphery of transfected cells. Specific, albeit weak, interaction between REBP and Rev could be demonstrated in coimmunoprecipitation assays in BSC-40 cells. REBP can modestly enhance Rev-dependent RRE-linked reporter gene expression both independently and in cooperation with the nucleoporin cofactor Rab/hRIP. Thus, REBP displays the characteristics expected of an authentic mediator of Rev NES function and may play a role in RRE RNA transport during HIV infection.

Genetic and Biological Variation in Equine Infectious Anemia Virus Rev Correlates with Variable Stages of Clinical Disease in an Experimentally Infected Pony

Genetic and biological variation in the regulatory protein Rev of equine infectious anemia virus (EIAV) were examined throughout a clinically dynamic disease course of an experimentally infected pony. Following infection with the virulent EIAVWyo, the pony underwent a variable disease course, including an acute fever episode at 12 days postinfection (DPI), multiple recurrent fever episodes until 135 DPI, a prolonged subclinical period, and two late fever episodes. Viral RNA was isolated from the inoculum and sequential sera samples, and the rev exon 2/gp45 overlapping ORFs were amplified, cloned, and sequenced. Novel variants were found throughout infection, and genetic analyses indicated that both the Rev and gp45 ORFs were under selective pressure. The Rev variant predominant in the inoculum, R1, remained predominant during the early periods following infection (until 35 DPI); however, R1 was replaced by new predominant variants during the recurrent fever period (67–135 DPI). R1 reemerged as the predominant variant during the afebrile period, but a new predominant variant, R93, was associated with the late fever episodes. Rev variants predominant during recurrent febrile and late-febrile periods had significantly higher Rev-mediated nuclear export activity than the variants predominant during the acute and afebrile periods. Statistical correlation was found between Rev activity and different stages of clinical disease. Together, these results suggest that genetic and biological variation in rev may be a contributing factor in EIAV disease progression.

PAQ: Partition Analysis of Quasispecies.

The complexities of genetic data may not be accurately described by any single analytical tool. Phylogenetic analysis is often used to study the genetic relationship among different sequences. Evolutionary models and assumptions are invoked to reconstruct trees that describe the phylogenetic relationship among sequences. Genetic databases are rapidly accumulating large amounts of sequences. Newly acquired sequences, which have not yet been characterized, may require preliminary genetic exploration in order to build models describing the evolutionary relationship among sequences. There are clustering techniques that rely less on models of evolution, and thus may provide nice exploratory tools for identifying genetic similarities. Some of the more commonly used clustering methods perform better when data can be grouped into mutually exclusive groups. Genetic data from viral quasispecies, which consist of closely related variants that differ by small changes, however, may best be partitioned by overlapping groups. We have developed an intuitive exploratory program, Partition Analysis of Quasispecies (PAQ), which utilizes a non-hierarchical technique to partition sequences that are genetically similar. PAQ was used to analyze a data set of human immunodeficiency virus type 1 (HIV-1) envelope sequences isolated from different regions of the brain and another data set consisting of the equine infectious anemia virus (EIAV) regulatory gene rev. Analysis of the HIV-1 data set by PAQ was consistent with phylogenetic analysis of the same data, and the EIAV rev variants were partitioned into two overlapping groups. PAQ provides an additional tool which can be used to glean information from genetic data and can be used in conjunction with other tools to study genetic similarities and genetic evolution of viral quasispecies.

Binding of equine infectious anemia virus rev to an exon splicing enhancer mediates alternative splicing and nuclear export of viral mRNAs.

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs.

Binding of Equine Infectious Anemia Virus Rev to an Exon Splicing Enhancer Mediates Alternative Splicing and Nuclear Export of Viral mRNAs

Binding of Equine Infectious Anemia Virus Rev to an Exon Splicing Enhancer Mediates Alternative Splicing and Nuclear Export of Viral mRNAs

Leptomycin B inhibits equine infectious anemia virus Rev and feline immunodeficiency virus rev function but not the function of the hepatitis B virus posttranscriptional regulatory element.

Human immunodeficiency virus type 1 Rev export depends upon the presence of the nuclear export signal (NES), a leucine-rich stretch of hydrophobic amino acids. Recently, the nuclear NES-binding receptor has been identified as CRM1 or exportin 1. Rev export has been shown to be CRM1 dependent. The function of the atypical NES-containing Rev-like proteins of equine infectious anemia virus (EIAV) and feline immunodeficiency virus (FIV) is inhibited by leptomycin B, a drug that specifically blocks NES-CRM1 interactions. These data suggest that the function of atypical NES-containing proteins is CRM1 dependent. In contrast to the inhibition of EIAV Rev and FIV Rev, the cytoplasmic accumulation of hepatitis B virus (HBV) posttranscriptional regulatory element (PRE)-containing and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-containing RNAs is not inhibited by leptomycin B treatment. We conclude that the HBV PRE, like the MPMV CTE, functions independently of an NES receptor-exportin 1 interaction.

Differential requirements for alternative splicing and nuclear export functions of equine infectious anemia virus Rev protein.

The Rev protein of equine infectious anemia virus (ERev) exports unspliced and partially spliced viral RNAs from the nucleus. Like several cellular proteins, ERev regulates its own mRNA by mediating an alternative splicing event. To determine the requirements for these functions, we have identified ERev mutants that affect RNA export or both export and alternative splicing. Mutants were further characterized for subcellular localization, nuclear-cytoplasmic shuttling, and multimerization. None of the nuclear export signal (NES) mutants are defective for alternative splicing. Furthermore, the NES of ERev is similar in composition but distinct in spacing from other leucine-rich NESs. Basic residues at the C terminus of ERev are involved in nuclear localization, and disruption of the C-terminal residues affects both functions of ERev. ERev forms multimers, and no mutation disrupts this activity. In two mutants with substitutions of charged residues in the middle of ERev, RNA export is affected. One of these mutants is also defective for ERev-mediated alternative splicing but is identical to wild-type ERev in its localization, shuttling, and multimerization. Together, these results demonstrate that the two functions of ERev both require nuclear import and at least one other common activity, but RNA export can be separated from alternative splicing based on its requirement for a functional NES.

Biological characterization of Rev variation in equine infectious anemia virus.

Sequence analysis identified significant variation in the second exon of equine infectious anemia virus (EIAV) rev. Functional analysis indicated that limited amino acid variation in Rev significantly altered the export activity of the protein but did not affect Rev-dependent alternative splicing. EIAV Rev can mediate export through two independent cis-acting Rev-responsive elements (RREs), and differences among Rev variants were more pronounced when both RREs were present. Variation in Rev may be an important mechanism for regulation of virus replication in vivo and may contribute to changes in clinical disease.

Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.

We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.

Nuclear transport of human immunodeficiency virus type 1, visna virus, and equine infectious anemia virus Rev proteins: identification of a family of transferable nuclear export signals.

The human immunodeficiency virus type 1 Rev trans activator binds directly to unspliced viral mRNA in the nucleus and activates its transport to the cytoplasm. In additon to the sequences that confer RNA binding and nuclear localization, Rev has a carboxy-terminal region, the activation domain, whose integrity is essential for biological activity. Because it has been established that Rev constitutively exits and reenters the nucleus and that the activation domain is required for nuclear exit, it has been proposed that Rev's activation domain is a nuclear export signal (NES). Here, we used microinjection-based assays to demonstrate that the activation domain of human immunodeficiency virus type 1 Rev imparts rapid nuclear export after its transfer to heterologous substrates. NES- mediated export is specific, as it is sensitive both to inactivation by missense mutation and to selective inhibition by an excess of the wild-type, but not mutant, activation domain peptide. Examination of the Rev trans activators of two nonprimate lentiviruses, visna virus and equine infectious anemia virus, revealed that their activation domains are also potent NESs. Taken together, these data demonstrate that nuclear export can be determined by positively acting peptide motifs, namely, NESs, and suggest that Rev proteins activate viral RNA transport by providing export ribonucleoproteins with specific information that targets them to the cytoplasm.

The mechanism of activity of the rev protein of EIAV differs from that of HIV

THE MECHANISM OF ACTIVITY OF THE REV PROTEIN OF EIAV DIFFERS FROM THAT OF HIV. A. Gazit, R. Rosin-Arbesfeld, M. Rivlin, P. Mashiah, S.R. Tronick and A. Yaniv. Two species of the Rev protein of equine infectious anemia virus (EIAV), are encoded by three alternatively spliced polycistronic transcripts. These two Rev proteins, of 18 and 16 kDa in size, differ in their N-termini, and thus, we presume, are distinct in their intracellular location and biological function. Interestingly, unlike the Rev protein of HIV, we found the EIAV 18 kDa Rev packaged in mature virions. The Rev protein of HIV is suggested to activate the cytoplasmic expression of the viral unspliced and partially spliced transcripts, through binding to a specific Rev responsive elment (RRE), located within the env region. In contrast, in EIAV, a similar secondary structure element which could serve as the RRE of EIAV Rev, could not be found. Extensive deletions within the 3' half of EIAV genome, established that the genomic region which spans the various splice sites of the EIAV middle-located exons, possesses two functions: a. CRS (cis-acting repressive sequences), responsible presumably for the nuclear retention of CRS-containing transcripts. b. RRE, responsible presumably for the direct or indirect binding of the 18 kDa Rev, thus overcoming the CRS-induced nuclear retention. In-vitro mutagenesis of the EIAV genome, coupled with complementation assays suggested that the 18 kDa Rev of EIAV functions via two mechanisms: a. when present at moderate levels, it facilitates the nuclear export of a tat-envtranscdpt, which serves as the major env transcript during in-vitro EIAV infection, b. when present at high levels, it acts as a positive splicing regulator which activates an alternate sub-optimal splice accepter site, thus giving rise to two distinct monocistronic env transcripts which enable potent expression of the viral Env glycoproteins. Thus, it is suggested that the EIAV 18 kDa Rev protein regulates EIAV replication and pathogenesis in a concentration-dependent manner. The biological function of the 16 kDa Rev is being investigated.

Identification of the activation domain of equine infectious anemia virus rev

Several members of the lentivirus family of complex retroviruses have been shown to encode proteins that are functionally equivalent to the Rev posttranscriptional regulatory protein of human immunodeficiency virus type 1 (HIV-1). Furthermore, the domain organization of HIV-1 Rev, featuring a highly basic N-terminal RNA binding domain and a leucin-rich C-terminal effector domain, has also been shown to be highly conserved among Rev proteins derived from not only the primate but also the ovine and caprine lentiviruses. Although it has therefore appeared highly probable that the lentivirus equine infectious anemia virus (EIAV) also encodes a Rev, the predicted amino acid sequence of this putative EIAV regulatory protein does not display any evident homology to the basic and leucine-rich motifs characteristic of other known Rev proteins. By fusion of different segments of the proposed EIAV Rev protein to the well-defined RNA binding domain of either HIV-1 or visna virus Rev, we have identified a segment of this EIAV protein that can efficiently substitute in cis for the otherwise essential activation motif. Interestingly, the minimal EIAV Rev activation motif identified in this study comprises approximately 18 amino acids located toward the protein N terminus that lack any evident similarity to the leucine-rich activation domains found in these other lentivirus Rev proteins. It therefore appears that the Rev protein of EIAV, while analogous in function to Rev proteins defined in lentiviruses of primate, ovine, and caprine origin, is nevertheless distinguished by an entirely novel domain organization.

The open reading frame ORF S3 of equine infectious anemia virus is expressed during the viral life cycle

The genome of equine infectious anemia virus (EIAV) contains several small open reading frames (ORFs), the importance of which in the development of the virus is not clear. We investigated the possibility that the largest of these ORFs (ORF S3) is expressed during the course of the viral infection. The ORF S3 information was expressed in Escherichia coli, and the antigen was used to raise monospecific antiserum. A 20-kDa protein expressed in cells producing EIAV was identified as the gene product of ORF S3. Furthermore, sera from EIAV-infected animals specifically recognized this protein, indicating that the ORF S3 antigen is expressed in vivo as well. A model for the expression of this new viral antigen is presented. The proposed splicing pattern is similar to that of the VEP-1 protein of maedi-visna-virus, which tempts us to speculate that ORF S3 defines the second exon of the EIAV Rev protein.