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Egress of HSV-1 capsid requires the interaction of VP26 and a cellular tetraspanin membrane protein.

Wang L, Liu L, Che Y, Wang L, Jiang L, Dong C, Zhang Y, Li Q - Virol. J. (2010)

Bottom Line: In this study, a member of the tetraspanin superfamily, CTMP-7, was shown to physically interact with HSV-1 protein VP26, and the VP26-CTMP-7 complex was detected both in vivo and in vitro.The interaction of VP26 with CTMP-7 plays an essential role in normal HSV-1 replication.Together, our data support the notion that biological events mediated by a VP26 - CTMP-7 interaction aid in viral capsid enveloping and egress from the cell during the HSV-1 infectious process.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute Of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical College, Kunming 650118, P. R. China.

ABSTRACT
HSV-1 viral capsid maturation and egress from the nucleus constitutes a self-controlled process of interactions between host cytoplasmic membrane proteins and viral capsid proteins. In this study, a member of the tetraspanin superfamily, CTMP-7, was shown to physically interact with HSV-1 protein VP26, and the VP26-CTMP-7 complex was detected both in vivo and in vitro. The interaction of VP26 with CTMP-7 plays an essential role in normal HSV-1 replication. Additionally, analysis of a recombinant virus HSV-1-UG showed that mutating VP26 resulted in a decreased viral replication rate and in aggregation of viral mutant capsids in the nucleus. Together, our data support the notion that biological events mediated by a VP26 - CTMP-7 interaction aid in viral capsid enveloping and egress from the cell during the HSV-1 infectious process.

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CTMP-7 is a VP26 interacting protein. a. Binding assay of the proteins and VP26. The positive control was the fusion yeast containing pGBK-p53 and pACT-LT. The negative control was the fusion yeast containing pGBK-Lam and pACT-LT. b. Amino acid sequence features of CTMP-7. The amino acid sequence of CTMP-7 contains the typical tetraspanin-enriched domain with four transmembrane regions (shown in red) and a topological domain (shown in black). c. Co-immunoprecipitation of the VP26 and CTMP-7 interaction complex and immunoblot by anti-CTMP-7 antibody. Vero cells transfected by VP26 and CTMP gene and labeled with 35S-methionine were lysed in RIPA buffer and interacted with anti-VP26 or anti-CTMP antibodies. Lane 1: The immunoprecipitated complexes of cells co-transfected with VP26 and CTMP genes by anti-VP26 antibody; Lane 2: The immunoprecipitated complexes of cells co-transfected with VP26 and CTMP genes by anti-CTMP-7 antibody. Lane 3: The immunoprecipitated complexes of cells transfected with pcDNA mock by anti-VP26 antibody; and Lane 4: The immunoprecipitated complexes of cells transfected with pcDNA mock by anti-CTMP-7 antibody. Lane 5: Control cells lysate; Lane 6: Negative control with normal mouse IgG. d. Mapping the region of CTMP-7 interaction with VP26. The plasmids encoding CTMP-7 amino acid residues 1-61, 61-150 and 150-249 were constructed and transfected into yeast Y187. These transfected Y187 clones were fused with AH109 transfected with pGBK-VP26. These fused clones were identified on QDO plates and their β-galactosidase activity was analyzed.
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Figure 1: CTMP-7 is a VP26 interacting protein. a. Binding assay of the proteins and VP26. The positive control was the fusion yeast containing pGBK-p53 and pACT-LT. The negative control was the fusion yeast containing pGBK-Lam and pACT-LT. b. Amino acid sequence features of CTMP-7. The amino acid sequence of CTMP-7 contains the typical tetraspanin-enriched domain with four transmembrane regions (shown in red) and a topological domain (shown in black). c. Co-immunoprecipitation of the VP26 and CTMP-7 interaction complex and immunoblot by anti-CTMP-7 antibody. Vero cells transfected by VP26 and CTMP gene and labeled with 35S-methionine were lysed in RIPA buffer and interacted with anti-VP26 or anti-CTMP antibodies. Lane 1: The immunoprecipitated complexes of cells co-transfected with VP26 and CTMP genes by anti-VP26 antibody; Lane 2: The immunoprecipitated complexes of cells co-transfected with VP26 and CTMP genes by anti-CTMP-7 antibody. Lane 3: The immunoprecipitated complexes of cells transfected with pcDNA mock by anti-VP26 antibody; and Lane 4: The immunoprecipitated complexes of cells transfected with pcDNA mock by anti-CTMP-7 antibody. Lane 5: Control cells lysate; Lane 6: Negative control with normal mouse IgG. d. Mapping the region of CTMP-7 interaction with VP26. The plasmids encoding CTMP-7 amino acid residues 1-61, 61-150 and 150-249 were constructed and transfected into yeast Y187. These transfected Y187 clones were fused with AH109 transfected with pGBK-VP26. These fused clones were identified on QDO plates and their β-galactosidase activity was analyzed.

Mentions: Five proteins interacting with VP26 were identified by yeast two-hybrid analysis using VP26 as the bait protein and a human embryonic lung mRNA library as the target (Supplementary Table 1). These interactions were further confirmed by a β-gal activity assay (Fig.1a). One of the VP26 interacting proteins is a cellular membrane protein type III and is a homologue of tetraspanin family member cellular tetraspanin membrane protein 7 (CTMP-7, GenBank accession number is NP_004606)). CTMP-7 is composed of 249 amino acid residues (28 kDa) and includes a typical tetraspanin-enriched domain (TEM) (Fig. 1b). We further confirmed binding of CTMP-7 to VP26 in HSV-1 infected human embryonic lung fibroblasts by co-immunoprecipitation with anti-CTMP-7 antibody and anti-VP26 antibody (Fig. 1c). Analysis of the CTMP-7 - VP26 interaction by β-gal activity assay suggested that the interaction involved the C-terminal portion of CTMP-7 (Fig.1d).


Egress of HSV-1 capsid requires the interaction of VP26 and a cellular tetraspanin membrane protein.

Wang L, Liu L, Che Y, Wang L, Jiang L, Dong C, Zhang Y, Li Q - Virol. J. (2010)

CTMP-7 is a VP26 interacting protein. a. Binding assay of the proteins and VP26. The positive control was the fusion yeast containing pGBK-p53 and pACT-LT. The negative control was the fusion yeast containing pGBK-Lam and pACT-LT. b. Amino acid sequence features of CTMP-7. The amino acid sequence of CTMP-7 contains the typical tetraspanin-enriched domain with four transmembrane regions (shown in red) and a topological domain (shown in black). c. Co-immunoprecipitation of the VP26 and CTMP-7 interaction complex and immunoblot by anti-CTMP-7 antibody. Vero cells transfected by VP26 and CTMP gene and labeled with 35S-methionine were lysed in RIPA buffer and interacted with anti-VP26 or anti-CTMP antibodies. Lane 1: The immunoprecipitated complexes of cells co-transfected with VP26 and CTMP genes by anti-VP26 antibody; Lane 2: The immunoprecipitated complexes of cells co-transfected with VP26 and CTMP genes by anti-CTMP-7 antibody. Lane 3: The immunoprecipitated complexes of cells transfected with pcDNA mock by anti-VP26 antibody; and Lane 4: The immunoprecipitated complexes of cells transfected with pcDNA mock by anti-CTMP-7 antibody. Lane 5: Control cells lysate; Lane 6: Negative control with normal mouse IgG. d. Mapping the region of CTMP-7 interaction with VP26. The plasmids encoding CTMP-7 amino acid residues 1-61, 61-150 and 150-249 were constructed and transfected into yeast Y187. These transfected Y187 clones were fused with AH109 transfected with pGBK-VP26. These fused clones were identified on QDO plates and their β-galactosidase activity was analyzed.
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Figure 1: CTMP-7 is a VP26 interacting protein. a. Binding assay of the proteins and VP26. The positive control was the fusion yeast containing pGBK-p53 and pACT-LT. The negative control was the fusion yeast containing pGBK-Lam and pACT-LT. b. Amino acid sequence features of CTMP-7. The amino acid sequence of CTMP-7 contains the typical tetraspanin-enriched domain with four transmembrane regions (shown in red) and a topological domain (shown in black). c. Co-immunoprecipitation of the VP26 and CTMP-7 interaction complex and immunoblot by anti-CTMP-7 antibody. Vero cells transfected by VP26 and CTMP gene and labeled with 35S-methionine were lysed in RIPA buffer and interacted with anti-VP26 or anti-CTMP antibodies. Lane 1: The immunoprecipitated complexes of cells co-transfected with VP26 and CTMP genes by anti-VP26 antibody; Lane 2: The immunoprecipitated complexes of cells co-transfected with VP26 and CTMP genes by anti-CTMP-7 antibody. Lane 3: The immunoprecipitated complexes of cells transfected with pcDNA mock by anti-VP26 antibody; and Lane 4: The immunoprecipitated complexes of cells transfected with pcDNA mock by anti-CTMP-7 antibody. Lane 5: Control cells lysate; Lane 6: Negative control with normal mouse IgG. d. Mapping the region of CTMP-7 interaction with VP26. The plasmids encoding CTMP-7 amino acid residues 1-61, 61-150 and 150-249 were constructed and transfected into yeast Y187. These transfected Y187 clones were fused with AH109 transfected with pGBK-VP26. These fused clones were identified on QDO plates and their β-galactosidase activity was analyzed.
Mentions: Five proteins interacting with VP26 were identified by yeast two-hybrid analysis using VP26 as the bait protein and a human embryonic lung mRNA library as the target (Supplementary Table 1). These interactions were further confirmed by a β-gal activity assay (Fig.1a). One of the VP26 interacting proteins is a cellular membrane protein type III and is a homologue of tetraspanin family member cellular tetraspanin membrane protein 7 (CTMP-7, GenBank accession number is NP_004606)). CTMP-7 is composed of 249 amino acid residues (28 kDa) and includes a typical tetraspanin-enriched domain (TEM) (Fig. 1b). We further confirmed binding of CTMP-7 to VP26 in HSV-1 infected human embryonic lung fibroblasts by co-immunoprecipitation with anti-CTMP-7 antibody and anti-VP26 antibody (Fig. 1c). Analysis of the CTMP-7 - VP26 interaction by β-gal activity assay suggested that the interaction involved the C-terminal portion of CTMP-7 (Fig.1d).

Bottom Line: In this study, a member of the tetraspanin superfamily, CTMP-7, was shown to physically interact with HSV-1 protein VP26, and the VP26-CTMP-7 complex was detected both in vivo and in vitro.The interaction of VP26 with CTMP-7 plays an essential role in normal HSV-1 replication.Together, our data support the notion that biological events mediated by a VP26 - CTMP-7 interaction aid in viral capsid enveloping and egress from the cell during the HSV-1 infectious process.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute Of Medical Biology, Chinese Academy of Medicine Science, Peking Union Medical College, Kunming 650118, P. R. China.

ABSTRACT
HSV-1 viral capsid maturation and egress from the nucleus constitutes a self-controlled process of interactions between host cytoplasmic membrane proteins and viral capsid proteins. In this study, a member of the tetraspanin superfamily, CTMP-7, was shown to physically interact with HSV-1 protein VP26, and the VP26-CTMP-7 complex was detected both in vivo and in vitro. The interaction of VP26 with CTMP-7 plays an essential role in normal HSV-1 replication. Additionally, analysis of a recombinant virus HSV-1-UG showed that mutating VP26 resulted in a decreased viral replication rate and in aggregation of viral mutant capsids in the nucleus. Together, our data support the notion that biological events mediated by a VP26 - CTMP-7 interaction aid in viral capsid enveloping and egress from the cell during the HSV-1 infectious process.

Show MeSH
Related in: MedlinePlus