Characterization of a new 5' splice site within the caprine arthritis encephalitis virus genome: evidence for a novel auxiliary protein.

BACKGROUND: Lentiviral genomes encode multiple structural and regulatory proteins. Expression of the full complement of viral proteins is accomplished in part by alternative splicing of the genomic RNA. Caprine arthritis encephalitis virus (CAEV) and maedi-visna virus (MVV) are two highly related small-ruminant lentiviruses (SRLVs) that infect goats and sheep. Their genome seems to be less complex than those of primate lentiviruses since SRLVs encode only three auxiliary proteins, namely, Tat, Rev, and Vif, in addition to the products of gag, pol, and env genes common to all retroviruses. Here, we investigated the central part of the SRLV genome to identify new splice elements and their relevance in viral mRNA and protein expression. RESULTS: We demonstrated the existence of a new 5' splice (SD) site located within the central part of CAEV genome, 17 nucleotides downstream from the SD site used for the rev mRNA synthesis, and perfectly conserved among SRLV strains. This new SD site was found to be functional in both transfected and infected cells, leading to the production of a transcript containing an open reading frame generated by the splice junction with the 3' splice site used for the rev mRNA synthesis. This open reading frame encodes two major protein isoforms of 18- and 17-kDa, named Rtm, in which the N-terminal domain shared by the Env precursor and Rev proteins is fused to the entire cytoplasmic tail of the transmembrane glycoprotein. Immunoprecipitations using monospecific antibodies provided evidence for the expression of the Rtm isoforms in infected cells. The Rtm protein interacts specifically with the cytoplasmic domain of the transmembrane glycoprotein in vitro, and its expression impairs the fusion activity of the Env protein. CONCLUSION: The characterization of a novel CAEV protein, named Rtm, which is produced by an additional multiply-spliced mRNA, indicated that the splicing pattern of CAEV genome is more complex than previously reported, generating greater protein diversity. The high conservation of the SD site used for the rtm mRNA synthesis among CAEV and MVV strains strongly suggests that the Rtm protein plays a role in SRLV propagation in vivo, likely by competing with Env protein functions.

Establishment and characterization of a goat synovial membrane cell line susceptible to small ruminant lentivirus infection.

Primary goat synovial membrane (GSM) cells are widely used to study small ruminant lentiviruses (SRLV), i.e. maedi visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV), but their limited life-span of 15-20 passages in vitro is problematic. Here, we report that ectopic expression of the catalytic subunit of human telomerase (hTERT) was sufficient to immortalize primary GSM cells. Cultures of hTERT-transfected GSM cells have been passaged for 2 years without showing any phenotypic difference from the original primary GSM cells. The hTERT-transfected cells continued to grow beyond a population doubling number of 250, while no net telomere lengthening was observed for these cells. Moreover, the immortalized GSM cells were susceptible to infection by both CAEV and MVV and were able to propagate theses viruses. Such cell line provides a useful source of standard and robust cells for both research and veterinary purposes.

Characterisation of an Irish caprine lentivirus strain--SRLV phylogeny revisited.

Small ruminant lentiviruses (SRLV), i.e. caprine arthritis-encephalitis virus (CAEV) (which infects goats) and maedi-visna virus (MVV) (which infects sheep) are two closely related lentiviruses but the relationship between goat and sheep lentiviruses has not been clearly established. To better understand their genetic relationship, we reinvestigated the phylogeny of SRLV using new sequences from an Irish and a Norwegian strain together with sequences available from databases. The phylogenetic analyses were carried out on the gag, pol and env fragments using four methods: neighbor-joining (NJ), Fitch and Margoliash (Fitch), Fitch and Wagner parsimony (Pars) and maximum likelihood (ML). The tree topologies were consistent whether derived from any of the four methods or any of the gene fragments, but the phylogenetic analyses in the pol and env regions were more informative than in the gag region. The Tamura-Nei model with variable rates across sites (described by a gamma distribution) provides a more accurate description of SRLV evolution than simple methods. The newly described Irish lentivirus strain, which was isolated from a goat, was closely related to the lentivirus that infects sheep: MVV. The novel Norwegian CAEV strain belonged to a cluster specific to the CAEV strains from Norway. Together, both data confirm the previously reported subdivision of the different SRLV strains into six clades. The caprine and ovine lentivirus sequences are interspersed in phylogenetic trees, supporting the existence of cross-species transmission. Nevertheless, the transmission of an ovine lentivirus to a goat could trigger the emergence of some goat-adapted phylums. Our new sequences confirm the complex situation in SRLV phylogeny but more sequences are needed to elucidate more precisely the relationship between SRLV.