Characterization of Signal Sequences Determining the Nuclear/Nucleolar Import and Nuclear Export of the Caprine Arthritis-Encephalitis Virus Rev Protein.

Caprine arthritis-encephalitis virus (CAEV), a lentivirus, relies on the action of the Rev protein for its replication. The CAEV Rev fulfills its function by allowing the nuclear exportation of partially spliced or unspliced viral mRNAs. In this study, we characterized the nuclear and nucleolar localization signals (NLS and NoLS, respectively) and the nuclear export signal (NES) of the CAEV Rev protein. These signals are key actors in the nucleocytoplasmic shuttling of a lentiviral Rev protein. Several deletion and alanine substitution mutants were generated from a plasmid encoding the CAEV Rev wild-type protein that was fused to the enhanced green fluorescent protein (EGFP). Following cell transfection, images were captured by confocal microscopy and the fluorescence was quantified in the different cell compartments. The results showed that the NLS region is localized between amino acids (aa) 59 to 75, has a monopartite-like structure and is exclusively composed of arginine residues. The NoLS was found to be partially associated with the NLS. Finally, the CAEV Rev protein's NES mapped between aa 89 to 101, with an aa spacing between the hydrophobic residues that was found to be unconventional as compared to that of other retroviral Rev/Rev-like proteins.

Small ruminant lentiviral Vif proteins commonly utilize cyclophilin A, an evolutionarily and structurally conserved protein, to degrade ovine and caprine APOBEC3 proteins.

Mammals have co-evolved with retroviruses, including lentiviruses, over a long period. Evidence supporting this contention is that viral infectivity factor (Vif) encoded by lentiviruses antagonizes the anti-viral action of cellular apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) of the host. To orchestrate E3 ubiquitin ligase complex for APOBEC3 degradation, Vifs utilize mammalian proteins such as core-binding factor beta (CBFB; for primate lentiviruses) or cyclophilin A (CYPA; for Maedi-Visna virus [MVV]). However, the co-evolutionary relationship between lentiviral Vif and the mammalian proteins associated with Vif-mediated APOBEC3 degradation is poorly understood. Moreover, it is unclear whether Vif proteins of small ruminant lentiviruses (SRLVs), including MVV and caprine arthritis encephalitis virus (CAEV), commonly utilize CYPA to degrade the APOBEC3 of their hosts. In this study, molecular phylogenetic and protein homology modeling revealed that Vif co-factors are evolutionarily and structurally conserved. It was also found that not only MVV but also CAEV Vifs degrade APOBEC3 of both sheep and goats and that CAEV Vifs interact with CYPA. These findings suggest that lentiviral Vifs chose evolutionarily and structurally stable proteins as their partners (e.g., CBFB or CYPA) for APOBEC3 degradation and, particularly, that SRLV Vifs evolved to utilize CYPA as their co-factor in degradation of ovine and caprine APOBEC3.

Phage display identifies two Caprine Arthritis Encephalitis Virus env epitopes.

Using phage display and IgG of a goat infected with Caprine Arthritis Encephalitis Virus (CAEV) we obtained families of 7 mer constrained peptides with consensus motifs LxSDPF/Y and SWN/KHWSY and mapped the epitopes mimicked by them at the Env 6-LISDPY-11 and 67-WNTYHW-72 sites of the mature gp135 amino acid sequence. The first epitope fell into the N-terminal immunogenic aa1-EDYTLISDPYGFS- aa14 site identified previously with a synthetic peptide approach; the second epitope has not been described previously. The first epitope is mostly conserved across CAEV isolates whereas the second newly described epitope is extremely conserved in Small Ruminant Lentiviruses env sequences. As being immunodominant, the epitopes are candidate targets for mimotope-mediated diagnosis and/or neutralization.

Small-ruminant lentivirus enhances PrPSc accumulation in cultured sheep microglial cells.

Sheep scrapie is the prototypical transmissible spongiform encephalopathy (prion disease), which has a fundamental pathogenesis involving conversion of normal cellular prion protein (PrP(C) [C superscript stands for cellular]) to disease-associated prion protein (PrP(Sc) [Sc superscript stands for sheep scrapie]). Sheep microglial cell cultures, derived from a prnp 136VV/171QQ near-term fetal brain, were developed to study sheep scrapie in the natural host and to investigate potential cofactors in the prion conversion process. Two culture systems, a primary cell culture and a cell line transformed with the large T antigen of simian virus 40, were developed, and both were identified as microglial in origin as indicated by expression of several microglial phenotype markers. Following exposure to PrP(Sc), sheep microglial cells demonstrated relatively low levels (transformed cell line) to high levels (primary cell line) of PrP(Sc) accumulation over time. The accumulated PrP(Sc) demonstrated protease resistance, an inferred beta-sheet conformation (as determined by a commercial enzyme-linked immunosorbent assay), specific inhibition by anti-PrP antibodies, and was transmissible in a dose-dependent manner. Primary microglia coinfected with a small-ruminant lentivirus (caprine arthritis encephalitis virus-Cork strain) and PrP(Sc) demonstrated an approximately twofold increase in PrP(Sc) accumulation compared to that of primary microglia infected with PrP(Sc) alone. The results demonstrate the in vitro utility of PrP(Sc)-permissive sheep microglial cells in investigating the biology of natural prion diseases and show that small-ruminant lentiviruses enhance prion conversion in cultured sheep microglia.

TNFalpha and GM-CSF-induced activation of the CAEV promoter is independent of AP-1.

Caprine arthritis encephalitis virus transcription is under the control of the viral promoter within the long terminal repeat. Previous studies with the closely related maedi visna lentivirus have indicated that viral transcription is dependent upon the AP-1 transcription factor. Other studies have indicated a potential role for the cytokines TNFalpha and GM-CSF in CAEV pathogenesis by increasing viral loads in infected tissues. The hypotheses that AP-1 transcription factors are necessary for transcriptional activation of the CAEV promoter and that CAEV transcriptional activation results from treatment with the cytokines GM-CSF and TNFalpha were tested with a stably transduced U937 cell line. Here, we found that TNFalpha and GM-CSF activated CAEV transcription in U937 cells. However, this activation effect was not blocked by SP600125, an inhibitor of Jun N-terminal kinase. SP600125 effectively prevented Jun phosphorylation in cells subsequently treated with cytokines. The cytokines TNFalpha and GM-CSF therefore activate CAEV transcription, and this effect occurs independently of AP-1. A set of progressive deletion mutants was utilized to show that TNFalpha-induced expression depends on an element or elements within the U3 70-bp repeat.

Immunohistochemical detection of the p27 capsid protein of caprine arthritis-encephalitis virus (CAEV) in bone-marrow cells of seropositive goats.

Bone-marrow samples were collected from 48 CAEV-seropositive, symptomless goats (30 kids, 18 adults). The samples were formalin-fixed and processed for histological examination. In addition, all samples were examined immunohistochemically with a monoclonal antibody (1A7) against the p27 capsid protein of maedi-visna virus, an antibody which cross-reacts with the Ca-p27 of CAEV. Samples from 16 goats (10/30 kids, 6/18 adults) showed positive immunolabelling of bone-marrow stromal cells (fibrocytes, endothelial cells and adipocytes) and of scattered macrophages, whereas haematopoietic cells were negative. The detection of viral Ca-p27 protein in bone-marrow fibrocytes was consistent with previous in-vitro studies which indicated that such cells are semi-permissive for CAEV infection. It is speculated that bone-marrow stromal cells represent a viral reservoir in symptomless animals.

Cross-reactivity between caprine arthritis-encephalitis virus and type 1 human immunodeficiency virus.

BACKGROUND: Caprine arthritis encephalitis (CAE) is caused by the lentivirus caprine arthritis-encephalitis virus (CAEV), a member of the Retroviridae family that also includes the human immunodeficiency virus (HIV). Serum of CAEV-infected goats cross-reacts with HIV-1 antigens in enzyme-linked immunosorbent assay (ELISA) tests. We attempted to identify the proteins responsible for this cross-reactivity. METHODS: Fifty selected human sera (30 positive, 10 negative, and 10 indeterminate to HIV-1 by Western blot) and 50 selected goat sera (33 positive and 17 negative to CAEV by ELISA) were evaluated. Human and goat sera were tested by Western blot against HIV-1 and CAEV antigens. RESULTS: Cross-reactivity between surface glycoproteins gp120 (HIV-1) and gp135 (CAEV) was specific. Positive reaction of human sera to CAEV gp135 was more intense than that of goat sera to HIV-1 gp120. Surface glycoprotein sequences of the two viruses were compared by Lasergene software (Dynex Technologies, Inc., Chantilly, VA, USA). Three homologous regions were identified: the first in the internal domain of gp120; the second in the beta3 loop, and still another-with the greatest homology-in a short sequence of the proximal region of the external domain of gp120 between loops beta4 and beta8. CONCLUSIONS: Surface glycoproteins of HIV-1 and CAEV share structural regions essential for viral adsorption and for induction of neutralizing antibodies. Thus, human contact with CAEV eventually could be a possible source of HIV-1 false positive reactions and must be considered in the interpretation of HIV serologic results.

Caprine arthritis-encephalitis virus envelope surface glycoprotein regions interacting with the transmembrane glycoprotein: structural and functional parallels with human immunodeficiency virus type 1 gp120.

A sequence similarity between surface envelope glycoprotein (SU) gp135 of the lentiviruses maedi-visna virus and caprine arthritis-encephalitis virus (CAEV) and human immunodeficiency virus type 1 (HIV-1) gp120 has been described. The regions of sequence similarity are in the second and fifth conserved regions of gp120, and the similarity is highest in sequences coinciding with beta-strands 4 to 8 and 25, which are located in the most virion-proximal region of the gp120 inner domain. A subset of this structure, formed by gp120 beta-strands 4, 5, and 25, is conserved in most or all lentiviruses. Because of the orientation of gp120 on the virion, this highly conserved virion-proximal region of the gp120 core may interact with the transmembrane glycoprotein (TM) together with the amino and carboxy termini of full-length gp120. Therefore, interactions between SU and TM of lentiviruses may be structurally related. Here we tested whether the amino acid residues in the putative virion-proximal region of CAEV gp135 comprising putative beta-strands 4, 5, and 25, as well as its amino and carboxy termini, are important for stable interactions with TM. An amino acid change at gp135 position 119 or 521, located in the turn between putative beta-strands 4 and 5 and near beta-strand 25, respectively, specifically disrupted the epitope recognized by monoclonal antibody 29A. Thus, similar to the corresponding gp120 regions, these gp135 residues are located in close proximity to each other in the folded protein, supporting the hypothesis of a structural similarity between the gp120 virion-proximal inner domain and gp135. Amino acid changes in the amino- and carboxy-terminal and putative virion-proximal regions of gp135 increased gp135 shedding from the cell surface, indicating that these gp135 regions are involved in interactions with TM. Our results indicate structural and functional parallels between CAEV gp135 and HIV-1 gp120 that may be more broadly applicable to the SU of other lentiviruses.

The Tat protein of the caprine arthritis encephalitis virus interacts with the Notch2 EGF-like repeats and the epithelin/granulin precursor.

Using the yeast two-hybrid system, we screened a human placenta cDNA library and identified two proteins that interacted with the Tat protein of the caprine arthritis encephalitis virus (CAEV): the EGF-like repeats 1-6 of the extracellular domain of the human Notch2 receptor and the epithelin/granulin growth factor precursor. This interaction was also confirmed in mammalian cells. Using in vitro mutagenesis assays, we showed that each one of the three cysteine residues located within the cysteine-rich domain of the CAEV Tat protein is essential for the binding of Tat to both the Notch2 and the epithelin/granulin protein. It is thus suggested that the cysteine-rich domain of Tat plays a role in the interaction between the Tat and either Notch2 or the epithelin/granulin domains, both of which exhibit EGF-like-repeat-imposed spatial conformation. It is assumed that such interactions might modulate the physiological functions of Notch2 and epithelin/granulin, thereby affecting various pathologies associated with CAEV.

Characterization of the caprine arthritis encephalitis virus (CAEV) rev N-terminal elements required for efficient interaction with the RRE.

The Caprine Arthritis Encephalitis Virus (CAEV) genome encodes three structural (gag, pol, and env) and three accessory (rev, tat, and vif) genes. The Rev-C protein regulates Gag, Pol and Env expression by transporting their mRNAs to the cytoplasm. Rev trans-activation requires binding of Rev to an RNA structure called the Rev Response Element (RRE-C). Previous mutational analyses have shown that two domains of Rev are required for its function. The basic domain mediates RRE binding and multimer formation, and the nuclear export signal (NES) mediates trans-activation. Preliminary experiments demonstrate that Rev-C N-terminal deletion mutants bind the RRE less avidly than does wildtype Rev. As a result, it was hypothesized that an additional domain located in the N-terminal exon of Rev-C was required for optimal RRE binding. To test this hypothesis, Rev-C alanine scanning mutants were generated and in vitro RRE binding assays were performed. Alteration of Rev-C amino acids K13, E14, N15, V19, T20, M21 and R27 dramatically decreased affinity for RRE-C. These data demonstrate that Rev-C N-terminal amino acids are required for optimal RRE-C binding and suggest that a third functional domain exists within the N-terminus of Rev-C.

Analysis of caprine arthritis encephalitis virus (CAEV) temporal gene expression in infected cells.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus that is closely related to visna virus and more distantly related to the human lentivirus, Human Immunodeficiency Virus type 1 (HIV-1). The CAEV genome contains several small open reading frames (ORFs) that encode viral regulatory proteins. One of these non-structural proteins, Rev-C, is required for cytoplasmic transport of viral un/incompletely spliced mRNAs and efficient viral replication. In HIV-1 and visna virus, Rev is responsible for the temporal shift from non-structural protein synthesis to synthesis of structural proteins that is observed during the viral infectious cycle. Since it encodes a Rev protein, CAEV would be predicted to exhibit a similar temporal shift in gene expression during its replicative cycle. Immunoprecipitation analysis of 35S-pulse labeled, CAEV-infected goat synovial membrane (GSM) cells indicates that Rev-C is more abundant than is Gag at 12 h post-infection (PI); at later times PI Gag predominates. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) experiments using nuclear and cytoplasmic RNA from CAEV-infected GSM cells indicates that the viral unspliced gag mRNA accumulates significantly in the cytoplasm only after Rev is detected. These data indicate that a temporal shift from viral non-structural to structural gene expression occurs in CAEV infected GSM cells.

Viral and cellular specificities of caprine arthritis encephalitis virus Vif protein.

The caprine arthritis encephalitis virus (CAEV) Vif protein is necessary for a productive infection of susceptible goat cells. The vif gene is conserved among all primate and most nonprimate lentiviruses. However, previous reports demonstrated that, in their respective host cells, primate Vif deleted lentiviruses could not be complemented by nonprimate Vif proteins, suggesting that species-specific restrictions between Vif and the virus-producing cells may modulate the Vif function on viral infectivity. Here we bring further support to this hypothesis since we show that CAEV Vif, when expressed in goat cells, is able to increase the infectivity of Vif deleted human immunodeficiency type-1 virus and of murine leukemia virus. Moreover, we demonstrate in vitro interactions between different Vif proteins and NC domains of heterologous Gag precursors, supporting the notion that species specificity of lentiviral infection is not due to molecular interactions between Vif and viral components.

Efficient replication of caprine arthritis-encephalitis virus in goat granulosa cells.

Recent reports demonstrated the susceptibility of epithelial cells from different organs to caprine arthritis-encephalitis virus (CAEV) both in vitro and in vivo. Since granulosa cells (GC) are of epithelial origin and currently used for in vitro oocyte maturation, we addressed the question whether these cells are susceptible or resistant to CAEV infection. GC were isolated from goats from certified CAEV-free herds. PCR analysis on GC DNA using CAEV specific primers confirmed the absence of CAEV infection and immunocytochemistry using specific K813 anti-cytokeratin monoclonal antibodies confirmed the epithelial nature of GC. These cells were then inoculated with CAEV using two strains: the CAEV-pBSCA molecular clone and the CAEV-3112 French field isolate. Cytopathic effects (CPE) were observed on cell culture monolayers inoculated with both CAEV strains. Expression of CAEV proteins was shown both by immunocytochemistry using anti-p24 gag specific antibodies and by immunoprecipitation using an hyperimmune serum. Supernatant of infected cells were shown to contain high titers (ranging 10(5) tissue culture infectious doses 50 per ml: TCID(50) per ml) of infectious cytopathic viruses when assayed onto the indicator goat synovial membrane (GSM) cells. Our findings demonstrate the large cell tropism of CAEV and suggest that GC could serve as a reservoir for the virus during the sub-clinical phase of infection. Furthermore, given the high seroprevalence of CAEV in the all industrialised countries and the large number of ovaries derived from unknown serological status animals used for in vitro goat embryo production, one can conclude that these feeder cell cultures might be a potential source of early transmission of CAEV to goat embryos.

Tryptophan 95, an amino acid residue of the Caprine arthritis encephalitis virus vif protein which is essential for virus replication.

The Caprine arthritis encephalitis virus (CAEV) vif gene was demonstrated to be essential for efficient virus replication. CAEV Vif deletion mutants demonstrated an attenuated replication phenotype in primary goat cell cultures and resulted in abortive infection when inoculated into goats. In this study, we determined the in vitro replication phenotype of five CAEV Vif point mutant infectious molecular clones and the ability of the corresponding in vitro translated Vif proteins to interact with the CAEV Pr55(gag) in the glutathione S--transferase (GST) binding assay. Here we show that (i) three of the mutants (S170E, S170G, S197G) behaved as the wild-type CAEV according to virus replication and Vif--Gag interactions; (ii) one mutant (Vif 6mut) was replication incompetent and bound weakly to GST-Gag fusion proteins; and (iii) one mutant (Vif RG) was impaired for replication while retaining its interaction properties. This mutant points out the critical importance of the CAEV Vif tryptophan residue at position 95 for efficient virus replication, defining for this lentivirus a functional domain unrelated to the Gag binding region.

Definition of the RRE binding and activation domains of the caprine arthritis encephalitis virus Rev protein.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus which is closely related by nucleotide sequence and biological properties to visna virus and is more distantly related to the human AIDS virus, HIV-1. Previous studies indicated that the CAEV Rev protein (Rev-C) functions as a trans-activator of mRNA cytoplasmic transport and expression. The function of Rev-C is mediated through an RNA element (RRE-C) present between nucleotides (nt) 7906 and 8110 in the CAEV env gene. In this study, RNA/protein immunoprecipitation experiments were used to demonstrate that Rev-C binds directly to the 204-nt RRE-C in vitro. Competition assays illustrate that this interaction is specific for the positive sense RRE-C RNA. Glutaraldehyde crosslinking studies demonstrate that the wildtype Rev-C protein can also form multimeric complexes in vitro. Deletions or amino acid alterations within the basic domain of Rev-C reduce affinity for the RRE and disrupt assembly of Rev-C multimers in vitro, indicating that this domain is involved in RRE binding and Rev multimer formation. Mutations within the leucine-rich domain of Rev-C do not greatly effect RRE-C binding or self-assembly. However, previous results demonstrate that some leucine-rich domain mutants are unable to trans-activate. These data are consistent with the hypothesis that the leucine domain is the effector domain of Rev-C.

Restrictive type of replication of ovine/caprine lentiviruses in ovine fibroblast cell cultures.

Caprine arthritis-encephalitis virus (CAEV) is a natural lentivirus pathogen of goats. CAEV, like all members of the ovine/ caprine lentivirus family, has an in vivo tropism for cells of the monocyte/macrophage cell lineage and activation of viral gene expression is observed only following differentiation of monocytes to macrophages. In addition to cells of the monocyte/ macrophage lineage, CAEV and the closely related maedi visna virus of sheep (MVV) can also replicate productively in fibro-epithelial cells derived from synovial membrane of goats (GSM). However, these viruses varied greatly in their ability to replicate in fibroblasts. We studied the biological and biochemical properties of CAEV and maedi-visna virus (MVV) of sheep following inoculation into the three ovine/caprine cell types. Our data showed no substantial differences in virus titers, viral protein biosynthesis, or processing of the viral proteins between CAEV and MVV following inoculation into primary macrophages and GSM cells. However, unlike MVV, CAEV failed to replicate productively in ovine fibroblasts (sheep choroid plexus cells). This correlated with a specific but abnormal proteolytic cleavage of the envelope glycoprotein of the virus. This abnormal proteolytic cleavage represents a novel type of host cell restriction of lentivirus replication.

A Rev protein is expressed in caprine arthritis encephalitis virus (CAEV)-infected cells and is required for efficient viral replication.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus that is closely related to visna virus and more distantly related to the human lentivirus human immunodeficiency virus 1 (HIV-1). Like other lentiviruses, the genome of CAEV contains multiple small ORFs that encode viral regulatory proteins. Sequence analysis of the CAEV genome and cDNAs generated from mRNA in infected cells has suggested that one of these ORFs encodes a protein (Rev-C) that is analogous to Rev of visna virus and HIV. Antibodies generated to a carboxy-terminal peptide of the rev ORF immunoprecipitate an 18-kDa protein from cells transfected with the Rev cDNA clone. Immunoprecipitation and immunofluorescence analysis of CAEV-infected ovine primary cells show that the product of the rev ORF is expressed during infection and localizes to the nucleolus of infected cells. Also, sera from CAEV-infected goats specifically immunoprecipitates an in vitro-translated product from the full-length Rev cDNA clone as well as that from the unique second open reading frame of Rev-C which shows that the Rev-C protein is expressed during natural CAEV infection of animals. Insertion of either a mutation that creates two stop codons in the unique second open reading frame of Rev-C or a mutation in the basic domain of Rev-C into the CAEV infectious molecular clone renders the virus unable to replicate in primary goat synovial membrane cells. Analysis of the RNA and proteins produced from both Rev-deficient clones indicates that they are defective in the accumulation of structural gene mRNAs in the cytoplasm as well as in synthesis of structural proteins compared to the wild-type CAEV clone. These data indicate that CAEV encodes a Rev protein that is required for efficient viral replication in culture.

Caprine arthritis-encephalitis lentivirus SU is the ligand for infection of caprine synovial membrane cells.

Infection of goat synovial membrane (GSM) cells by caprine arthritis-encephalitis virus (CAEV) was inhibited by incubation of cells with CAEV gp 135 envelope glycoprotein (SU) expressed by recombinant vaccinia virus. Incubation of cells with protein expressed by a control recombinant vaccinia virus without the CAEV envelope gene did not inhibit CAEV infection. Removal of recombinant SU from blocking medium by adsorption with anti-SU IgG/protein G-Sepharose complexes resulted in loss of CAEV inhibition. Results support that CAEV infection of GSM cells is mediated by a specific interaction between SU and a cell surface receptor or receptor complex.