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

Valas S, Rolland M, Perrin C, Perrin G, Mamoun RZ - Retrovirology (2008)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: AFSSA-Niort, Laboratoire d'Etudes et de Recherches Caprines, 79012 Niort, France. s.valas@niort.afssa.fr

ABSTRACT

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.

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Genetic homologies of the internal exon sequence carrying the SDrev and SDrtm sites among SRLV strains. All available sequences belonging to the three phylogenetic groups (A, B and C) are aligned. Dashes indicate deletions. A consensus sequence is presented. The conserved SD motifs are boxed.
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Figure 8: Genetic homologies of the internal exon sequence carrying the SDrev and SDrtm sites among SRLV strains. All available sequences belonging to the three phylogenetic groups (A, B and C) are aligned. Dashes indicate deletions. A consensus sequence is presented. The conserved SD motifs are boxed.

Mentions: To assess the biological importance of the SD6140 site and of the Rtm protein for SRLVs, we assumed that it should be conserved in all SRLV genomes, as the rev SD6123 site is. To look for the conservation of the SD6140 site among SRLV strains, previously described env sequences representative of highly divergent phylogenetic clusters were aligned (Fig. 8). This alignment confirmed that the 5' region of the SRLV env gene was extremely variable, except two quasi perfect repeat sequences (GGTAAG) corresponding to the SD6123 and SD6140 sites of Cork genome. Remarkably, the 17 nt distance between the two SD sites was also perfectly conserved among all SRLV sequences. Moreover, the downstream SD site was even better conserved than the SD site used for the rev mRNA synthesis. The high conservation of all these genetic features (sequence, frame, and nt distance) within a highly variable region strongly suggested that the Rtm protein, like the rev protein, is very important for SRLVs.


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

Valas S, Rolland M, Perrin C, Perrin G, Mamoun RZ - Retrovirology (2008)

Genetic homologies of the internal exon sequence carrying the SDrev and SDrtm sites among SRLV strains. All available sequences belonging to the three phylogenetic groups (A, B and C) are aligned. Dashes indicate deletions. A consensus sequence is presented. The conserved SD motifs are boxed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2291067&req=5

Figure 8: Genetic homologies of the internal exon sequence carrying the SDrev and SDrtm sites among SRLV strains. All available sequences belonging to the three phylogenetic groups (A, B and C) are aligned. Dashes indicate deletions. A consensus sequence is presented. The conserved SD motifs are boxed.
Mentions: To assess the biological importance of the SD6140 site and of the Rtm protein for SRLVs, we assumed that it should be conserved in all SRLV genomes, as the rev SD6123 site is. To look for the conservation of the SD6140 site among SRLV strains, previously described env sequences representative of highly divergent phylogenetic clusters were aligned (Fig. 8). This alignment confirmed that the 5' region of the SRLV env gene was extremely variable, except two quasi perfect repeat sequences (GGTAAG) corresponding to the SD6123 and SD6140 sites of Cork genome. Remarkably, the 17 nt distance between the two SD sites was also perfectly conserved among all SRLV sequences. Moreover, the downstream SD site was even better conserved than the SD site used for the rev mRNA synthesis. The high conservation of all these genetic features (sequence, frame, and nt distance) within a highly variable region strongly suggested that the Rtm protein, like the rev protein, is very important for SRLVs.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: AFSSA-Niort, Laboratoire d'Etudes et de Recherches Caprines, 79012 Niort, France. s.valas@niort.afssa.fr

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus