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

<p>Abstract</p> <p>Background</p> <p>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 <it>gag</it>, <it>pol</it>, and <it>env </it>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.</p> <p>Results</p> <p>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 <it>rev </it>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 <it>rev </it>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 <it>in vitro</it>, and its expression impairs the fusion activity of the Env protein.</p> <p>Conclusion</p> <p>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 <it>rtm </it>mRNA synthesis among CAEV and MVV strains strongly suggests that the Rtm protein plays a role in SRLV propagation <it>in vivo</it>, likely by competing with Env protein functions.</p

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Les infections à lentivirus chez la chèvre et le mouton (étude des interactions entre sous-type viral et espèce animale)

Les lentivirus de petits ruminants (SRLV) présentent une grande diversité génétique et sont classés en plusieurs groupes et sous-types. En France, la prévalence des infections à SRLV est élevée, mais peu de données sont disponibles sur la nature des souches virales qui y circulent. Dans ce travail, une méthode de génotypage (HMA) à grande échelle et basée sur les séquences des gènes gag et env a été adaptée à l étude des SRLV. Les enquêtes d épidémiologie moléculaire réalisées dans des élevages caprins et ovins du sud de la France ont révélé une forte prévalence des sous-types B1 et B2. Des transmissions inter-espèces des deux sous-types, ainsi que des phénomènes de co-infection et de recombinaison, ont été décrits dans des élevages mixtes. Cependant, la distribution inégale de ces sous-types viraux dans les deux espèces animales suggère des interactions virus/hôte spécifiques. Une analyse comparative des propriétés biologiques de deux virus appartenant à chaque sous-type a été réalisée in vitro sur différents types cellulaires et in vivo chez la chèvre et le mouton après inoculation par voie intra-trachéale. Les deux virus testés partagent le même phénotype mais diffèrent pour leur pouvoir. Le virus du sous-type B1 présente un pouvoir infectant plus élevé chez les chèvres que chez les moutons. Bien que la sensibilité des chèvres et des moutons à l infection par le virus du sous-type B2 soit identique, l expression virale est plus constante chez les moutons. Ces résultats expérimentaux corroborent les observations de terrain en démontrant une interaction spécifique entre sous-type viral et espèce animale.The small ruminant lentiviruses (SRLV) display a high genetic diversity and are currently classified into several groups, and an increasing number of subtypes. While a high prevalence of SRLV infection has been reported in sheep and goats from France, the prevalent SRLV strains circulating in this country are still poorly known. In this study, a large-scale screening method (HMA) based on the sequences of gag and env genes has been developed for monitoring the genetic evolution of SRLV. Molecular epidemiological surveys in sheep and goats flocks from southern of France revealed a high prevalence of subtypes B1 and B2. Interspecies transmission of both subtypes was reported in mixed flocks, as well as co-infection and recombination. However, subtypes B1 and B2 were found with different frequencies in sheep and goats, suggesting a differential adaptation of SRLV subtypes in these hosts. The biological properties of two field SRLV isolates belonging to subtypes B1 and B2 were compared in vitro using different cell types, and in vivo in sheep and goats experimentally infected by intratracheal inoculation. These strains share similar phenotype but exhibit different replicative properties in macrophages and synovial membrane cells. The subtype B1 virus has been shown to be particularly prone to infect goats, compared to sheep. While the subtype B2 virus infects sheep and goats with the same efficiency, viral expression is more frequently detected in sheep than in goats. These results confirm those obtained from field investigations showing specific interaction between SRLV subtypes and their natural hosts.POITIERS-BU Sciences (861942102) / SudocSudocFranceF

Epidemiological survey in single-species flocks from Poland reveals expanded genetic and antigenic diversity of small ruminant lentiviruses.

Small ruminant lentivirus (SRLV) infections are widespread in Poland and circulation of subtypes A1, A12, A13, B1 and B2 was detected. The present work aimed at extending previous study based on the analysis of a larger number of animals from single-species flocks. Animals were selected for genetic analysis based on serological reactivity towards a range of recombinant antigens derived from Gag and Env viral proteins. Phylogenetic analysis revealed the existence of subtypes B2 and A12 in both goats and sheep and subtypes A1 and B1 in goats only. In addition, two novel subtypes, A16 and A17, were found in goats. Co-infections with strains belonging to different subtypes within A and B groups were detected in 1 sheep and 4 goats originating from four flocks. Although the reactivity of serum samples towards the recombinant antigens confirmed immunological relatedness between Gag epitopes of different subtypes and the cross-reactive nature of Gag antibodies, eleven serum samples failed to react with antigens representing all subtypes detected up-to-date in Poland, highlighting the limitations of the serological diagnosis. These data showed the complex nature of SRLV subtypes circulating in sheep and goats in Poland and the need for improving SRLV-related diagnostic capacity

Bayesian phylogenetic tree based on MA fragment.

<p>Sequences from this study are shown in bold and their names are preceded by the flock origin No. and the animal species (s–sheep; g–goat). Sequences of co-infection variant strains are indicated by a (‘) and (“).</p

Origins and characteristics of the Polish SRLV strains selected for genetic characterization.

<p>Origins and characteristics of the Polish SRLV strains selected for genetic characterization.</p

Bayesian phylogenetic tree based on V1/V2 SU fragment.

<p>Sequences from this study are shown in bold and their names are preceded by the flock origin No. and the animal species (s–sheep; g–goat). Sequences of co-infection variant strains are indicated by a (‘) and (“).</p

Mean nucleotide distance in CA fragment of <i>gag</i> gene among (inter-genotype) genotypes A and B of SRLV.

<p>Mean nucleotide distance in CA fragment of <i>gag</i> gene among (inter-genotype) genotypes A and B of SRLV.</p

Bayesian phylogenetic tree based on CA fragment.

<p>Sequences from this study are shown in bold and their names are preceded by the flock origin No. and the animal species (s–sheep; g–goat). Sequences of co-infection variant strains are indicated by a (‘) and (“).</p