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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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Rtm binds to the cytoplasmic domain of Env protein. Purified recombinant GST-CD™ or GST alone were incubated with in vitro-translated Rtm protein labeled with [35S]methionine. The beads were washed six times in the presence of different ionic strengths of KCl, as indicated, and the bound proteins were subjected to 15% SDS-PAGE analysis and autoradiography.
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Figure 7: Rtm binds to the cytoplasmic domain of Env protein. Purified recombinant GST-CD™ or GST alone were incubated with in vitro-translated Rtm protein labeled with [35S]methionine. The beads were washed six times in the presence of different ionic strengths of KCl, as indicated, and the bound proteins were subjected to 15% SDS-PAGE analysis and autoradiography.

Mentions: Considering that the major part of the Rtm sequence corresponded to the cytoplasmic domain of TM and that the homologue domain of HIV TM was reported to self-assemble as an oligomer [26], we looked for an interaction between the Rtm and the cytoplasmic domain of TM. In this attempt, a GST pull-down assay was performed to identify potential interaction of Rtm with Env protein. In vitro-translated, radiolabeled Rtm protein was incubated with either a GST fusion protein containing the entire cytoplasmic domain of TM (GST-CD™) or with GST alone coupled to glutathione-Sepharose beads. Equal amounts of protein were used in all binding experiments, as verified by SDS-PAGE and Coomassie blue staining (data not shown). After extensive washing of the bead-bound complexes in different stringent conditions, the bound proteins were analysed by SDS-PAGE and autoradiography. As shown in Fig. 7, the Rtm protein interacted with the GST-CD™, and this interaction was resistant to high ionic strength washes. In contrast, no significant interaction was observed in association with the GST protein alone.


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)

Rtm binds to the cytoplasmic domain of Env protein. Purified recombinant GST-CD™ or GST alone were incubated with in vitro-translated Rtm protein labeled with [35S]methionine. The beads were washed six times in the presence of different ionic strengths of KCl, as indicated, and the bound proteins were subjected to 15% SDS-PAGE analysis and autoradiography.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Rtm binds to the cytoplasmic domain of Env protein. Purified recombinant GST-CD™ or GST alone were incubated with in vitro-translated Rtm protein labeled with [35S]methionine. The beads were washed six times in the presence of different ionic strengths of KCl, as indicated, and the bound proteins were subjected to 15% SDS-PAGE analysis and autoradiography.
Mentions: Considering that the major part of the Rtm sequence corresponded to the cytoplasmic domain of TM and that the homologue domain of HIV TM was reported to self-assemble as an oligomer [26], we looked for an interaction between the Rtm and the cytoplasmic domain of TM. In this attempt, a GST pull-down assay was performed to identify potential interaction of Rtm with Env protein. In vitro-translated, radiolabeled Rtm protein was incubated with either a GST fusion protein containing the entire cytoplasmic domain of TM (GST-CD™) or with GST alone coupled to glutathione-Sepharose beads. Equal amounts of protein were used in all binding experiments, as verified by SDS-PAGE and Coomassie blue staining (data not shown). After extensive washing of the bead-bound complexes in different stringent conditions, the bound proteins were analysed by SDS-PAGE and autoradiography. As shown in Fig. 7, the Rtm protein interacted with the GST-CD™, and this interaction was resistant to high ionic strength washes. In contrast, no significant interaction was observed in association with the GST protein alone.

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