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M2BP inhibits HIV-1 virion production in a vimentin filaments-dependent manner

View Article: PubMed Central - PubMed

ABSTRACT

M2BP (also called 90K) is an interferon-stimulated gene product that is upregulated in HIV-1 infection. A recent study revealed that M2BP reduces the infectivity of HIV-1 by inhibiting the processing of the viral envelope protein. Here we report that in addition to reducing viral infectivity, M2BP inhibits HIV-1 virion production. We provide evidence showing that M2BP inhibits HIV-1 Gag trafficking to the plasma membrane in a vimentin-dependent manner. When vimentin filaments were collapsed by treating cells with acrylamide or by overexpression of a dominant-negative mutant of vimentin, M2BP inhibition of HIV-1 virion production was significantly relieved. We further show that M2BP interacts with both HIV-1 Gag and vimentin and thereby mediates their interactions. We propose that M2BP traps HIV-1 Gag to vimentin filaments to inhibit the transportation of HIV-1 Gag to the plasma membrane. These findings uncover a novel mechanism by which a host antiviral factor inhibits HIV-1 virion production.

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M2BP mediates the interaction between Gag and VIM.(A–C) HEK293T cells were transfected with plasmids expressing the proteins indicated. At 48 h posttransfection, cells were lysed and the cell lysates were immunoprecipitated with antibodies indicated, followed by Western blotting analyses. (D) Hela cells were transfected with plasmids indicated. At 48 h posttransfection, cells were lysed and the cell lysates were immunoprecipitated with anti-Flag antibody, followed by Western blotting analyses. (E) HeLa cells were transfected with pVIM-TagBFP, together with an empty vector or a plasmid expressing M2BP-mKO. At 36 h posttransfection, cells were transfected again with pHIV-1-iGFPΔEnv for 36 h. Cells were fixed in paraformaldehyde. Fluorescence images were obtained by confocal fluorescence microscopy.
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f8: M2BP mediates the interaction between Gag and VIM.(A–C) HEK293T cells were transfected with plasmids expressing the proteins indicated. At 48 h posttransfection, cells were lysed and the cell lysates were immunoprecipitated with antibodies indicated, followed by Western blotting analyses. (D) Hela cells were transfected with plasmids indicated. At 48 h posttransfection, cells were lysed and the cell lysates were immunoprecipitated with anti-Flag antibody, followed by Western blotting analyses. (E) HeLa cells were transfected with pVIM-TagBFP, together with an empty vector or a plasmid expressing M2BP-mKO. At 36 h posttransfection, cells were transfected again with pHIV-1-iGFPΔEnv for 36 h. Cells were fixed in paraformaldehyde. Fluorescence images were obtained by confocal fluorescence microscopy.

Mentions: To understand how vimentin filaments are involved in M2BP inhibition of HIV-1 localization on the plasma membrane, we analyzed the interactions between M2BP, Gag and VIM. Myc-tagged M2BP was transiently expressed in HEK293T cells together with Flag-tagged VIM or pHIV-1NL4-3ΔEnv-luc. M2BP was immunoprecipitated and its interactions with VIM and Gag were assayed by Western blotting. Data showed that M2BP interacted with both VIM (Fig. 8A) and HIV-1 Gag (Fig. 8B). Furthermore, in the absence of M2BP, Gag did not interact with VIM (Fig. 8C, lane 6). However, in the presence of M2BP, Gag interacted with VIM (Fig. 8C, lane 7). Notably, expression of Gag seemed to increase the interaction between M2BP and VIM (Fig. 8C, compare lanes 3 and 7), while overexpression of VIM or VIMmut did not affect the interaction between M2BP and Gag (Supplementary Fig. 5). To further analyze the interaction between Gag, endogenous M2BP and endogenous VIM, pGag-Flag was transfected into HeLa cells and cells were lysed for immunoprecipitation with anti-Flag antibody, followed by Western blotting analyses. The results showed that when Gag-Flag was immunoprecipitated, the endogenous M2BP and endogenous VIM co-immunoprecipitated (Fig. 8D). When the endogenous M2BP was knocked down, the levels of co-immunoprecipitated endogenous VIM were reduced (Fig. 8D). These results suggest that Gag, M2BP and VIM may form a complex coordinated by M2BP.


M2BP inhibits HIV-1 virion production in a vimentin filaments-dependent manner
M2BP mediates the interaction between Gag and VIM.(A–C) HEK293T cells were transfected with plasmids expressing the proteins indicated. At 48 h posttransfection, cells were lysed and the cell lysates were immunoprecipitated with antibodies indicated, followed by Western blotting analyses. (D) Hela cells were transfected with plasmids indicated. At 48 h posttransfection, cells were lysed and the cell lysates were immunoprecipitated with anti-Flag antibody, followed by Western blotting analyses. (E) HeLa cells were transfected with pVIM-TagBFP, together with an empty vector or a plasmid expressing M2BP-mKO. At 36 h posttransfection, cells were transfected again with pHIV-1-iGFPΔEnv for 36 h. Cells were fixed in paraformaldehyde. Fluorescence images were obtained by confocal fluorescence microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015019&req=5

f8: M2BP mediates the interaction between Gag and VIM.(A–C) HEK293T cells were transfected with plasmids expressing the proteins indicated. At 48 h posttransfection, cells were lysed and the cell lysates were immunoprecipitated with antibodies indicated, followed by Western blotting analyses. (D) Hela cells were transfected with plasmids indicated. At 48 h posttransfection, cells were lysed and the cell lysates were immunoprecipitated with anti-Flag antibody, followed by Western blotting analyses. (E) HeLa cells were transfected with pVIM-TagBFP, together with an empty vector or a plasmid expressing M2BP-mKO. At 36 h posttransfection, cells were transfected again with pHIV-1-iGFPΔEnv for 36 h. Cells were fixed in paraformaldehyde. Fluorescence images were obtained by confocal fluorescence microscopy.
Mentions: To understand how vimentin filaments are involved in M2BP inhibition of HIV-1 localization on the plasma membrane, we analyzed the interactions between M2BP, Gag and VIM. Myc-tagged M2BP was transiently expressed in HEK293T cells together with Flag-tagged VIM or pHIV-1NL4-3ΔEnv-luc. M2BP was immunoprecipitated and its interactions with VIM and Gag were assayed by Western blotting. Data showed that M2BP interacted with both VIM (Fig. 8A) and HIV-1 Gag (Fig. 8B). Furthermore, in the absence of M2BP, Gag did not interact with VIM (Fig. 8C, lane 6). However, in the presence of M2BP, Gag interacted with VIM (Fig. 8C, lane 7). Notably, expression of Gag seemed to increase the interaction between M2BP and VIM (Fig. 8C, compare lanes 3 and 7), while overexpression of VIM or VIMmut did not affect the interaction between M2BP and Gag (Supplementary Fig. 5). To further analyze the interaction between Gag, endogenous M2BP and endogenous VIM, pGag-Flag was transfected into HeLa cells and cells were lysed for immunoprecipitation with anti-Flag antibody, followed by Western blotting analyses. The results showed that when Gag-Flag was immunoprecipitated, the endogenous M2BP and endogenous VIM co-immunoprecipitated (Fig. 8D). When the endogenous M2BP was knocked down, the levels of co-immunoprecipitated endogenous VIM were reduced (Fig. 8D). These results suggest that Gag, M2BP and VIM may form a complex coordinated by M2BP.

View Article: PubMed Central - PubMed

ABSTRACT

M2BP (also called 90K) is an interferon-stimulated gene product that is upregulated in HIV-1 infection. A recent study revealed that M2BP reduces the infectivity of HIV-1 by inhibiting the processing of the viral envelope protein. Here we report that in addition to reducing viral infectivity, M2BP inhibits HIV-1 virion production. We provide evidence showing that M2BP inhibits HIV-1 Gag trafficking to the plasma membrane in a vimentin-dependent manner. When vimentin filaments were collapsed by treating cells with acrylamide or by overexpression of a dominant-negative mutant of vimentin, M2BP inhibition of HIV-1 virion production was significantly relieved. We further show that M2BP interacts with both HIV-1 Gag and vimentin and thereby mediates their interactions. We propose that M2BP traps HIV-1 Gag to vimentin filaments to inhibit the transportation of HIV-1 Gag to the plasma membrane. These findings uncover a novel mechanism by which a host antiviral factor inhibits HIV-1 virion production.

No MeSH data available.


Related in: MedlinePlus