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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.

No MeSH data available.


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Vimentin filament collapse relieves M2BP inhibition of HIV-1 virion production.(A–D) HEK293T cells were transfected with the HIV-1 vector producing plasmids indicated, together with plasmids expressing M2BP and VIMmut. At 12 h posttransfection, chemicals indicated were added to the culture media. At 36 h posttransfection, culture supernatants were harvested to infect HeLa-CD4-CCR5 cells or analyzed for protein expressions. At 48 h postinfection, luciferase activity was measured. (A,C) The relative luciferase activity in the absence of M2BP was set as 100. Data presented are means ± SD of three independent experiments. (B,D) Protein expressions in the cell lysates and culture supernatants. The p24CA release level was calculated as described in the legend to Fig. 4D. Data presented are representative of three independent experiments. (E) An empty vector or a plasmid expressing VIMmut was nucleofected into MT4-M2BPi cells together with the HIV-1-producing plasmids indicated. A plasmid expressing firefly luciferase was included to serve as a control for transfection efficiency and sample handling. At 8 h posttransfection, IFNα-2b was added to the culture media. At 48 h posttransfection, p24CA levels in the culture supernatants were measured by ELISA (upper panel). Equal volumes of the culture supernatants were used to infect HEK293T cells. At 48 h postinfection, luciferase activity was measured in the recipient cells (lower panel). Relative CA levels and relative luc activity in the MT4-Ctrli cells without IFN treatment was set as 100. Data presented are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, n.s. p > 0.05
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f6: Vimentin filament collapse relieves M2BP inhibition of HIV-1 virion production.(A–D) HEK293T cells were transfected with the HIV-1 vector producing plasmids indicated, together with plasmids expressing M2BP and VIMmut. At 12 h posttransfection, chemicals indicated were added to the culture media. At 36 h posttransfection, culture supernatants were harvested to infect HeLa-CD4-CCR5 cells or analyzed for protein expressions. At 48 h postinfection, luciferase activity was measured. (A,C) The relative luciferase activity in the absence of M2BP was set as 100. Data presented are means ± SD of three independent experiments. (B,D) Protein expressions in the cell lysates and culture supernatants. The p24CA release level was calculated as described in the legend to Fig. 4D. Data presented are representative of three independent experiments. (E) An empty vector or a plasmid expressing VIMmut was nucleofected into MT4-M2BPi cells together with the HIV-1-producing plasmids indicated. A plasmid expressing firefly luciferase was included to serve as a control for transfection efficiency and sample handling. At 8 h posttransfection, IFNα-2b was added to the culture media. At 48 h posttransfection, p24CA levels in the culture supernatants were measured by ELISA (upper panel). Equal volumes of the culture supernatants were used to infect HEK293T cells. At 48 h postinfection, luciferase activity was measured in the recipient cells (lower panel). Relative CA levels and relative luc activity in the MT4-Ctrli cells without IFN treatment was set as 100. Data presented are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, n.s. p > 0.05

Mentions: It has been reported that M2BP regulates centriole biogenesis16, which participates in the regulation of the morphology of cytoskeletons40. This prompted us to ask whether cytoskeletons are involved in M2BP inhibition of HIV-1 virion production. We analyzed the antiviral activity of M2BP in the presence of nocodazole, cytochalasin D and acrylamide, which collapses microtubules, microfilaments and some intermediate filaments, respectively284142. HEK293T cells were transfected with pHIV-1NL4-3-luc and treated with the chemicals. The culture supernatants were used to infect recipient cells to evaluate the effects of the chemicals on M2BP inhibition of virion production. Treatment of the cells with acrylamide, but not nocodazole or cytochalasin D, partially relieved the inhibitory effect of M2BP on HIV-1 production (Fig. 6A). Further analyses revealed that treatment of the cells with acrylamide partially relieved M2BP inhibition of virion production without affecting Env processing (Fig. 6B), suggesting that intermediate filaments might be involved in M2BP inhibition of HIV-1 virion production. Notably, in the absence of exogenous M2BP, treatment of acrylamide increased the luciferase activity by about 2-fold (Supplementary Fig. 3), suggesting that collapsing intermediate filaments by acrylamide relieves the inhibitory effect of endogenous M2BP. Only a few types of intermediate filaments have been reported to be collapsed by acrylamide, which include vimentin filaments284344. We speculated that acrylamide might relieve M2BP inhibition of HIV-1 virion production by collapsing vimentin filaments. To test this idea, a vimentin mutant (VIMmut), whose overexpression collapses vimentin filaments (Supplementary Fig. 4)29, was analyzed for its ability to relieve M2BP inhibition of the production of VSV-G pseudotyped HIV-1NL4-3ΔEnv-luc. Indeed, overexpression of VIMmut relieved the inhibitory effect of M2BP on HIV-1 virion production (Fig. 6C,D). To determine whether vimentin filaments are involved in the antiviral function of endogenous M2BP in T cell lines, VIMmut were expressed in the MT4 cells expressing a control shRNA or the shRNA targeting M2BP. IFNα-2b treatment reduced the production of VSV-G-pseudotyped HIV-1 vector in the control cells (Fig. 6E). In comparison, in the MT4-M2BPi cells or in the control cells overexpressing VIMmut, IFN inhibition of viral production was significantly reduced (Fig. 6E). Collectively, these results indicate that M2BP inhibits HIV-1 virion production in a vimentin filaments-dependent manner.


M2BP inhibits HIV-1 virion production in a vimentin filaments-dependent manner
Vimentin filament collapse relieves M2BP inhibition of HIV-1 virion production.(A–D) HEK293T cells were transfected with the HIV-1 vector producing plasmids indicated, together with plasmids expressing M2BP and VIMmut. At 12 h posttransfection, chemicals indicated were added to the culture media. At 36 h posttransfection, culture supernatants were harvested to infect HeLa-CD4-CCR5 cells or analyzed for protein expressions. At 48 h postinfection, luciferase activity was measured. (A,C) The relative luciferase activity in the absence of M2BP was set as 100. Data presented are means ± SD of three independent experiments. (B,D) Protein expressions in the cell lysates and culture supernatants. The p24CA release level was calculated as described in the legend to Fig. 4D. Data presented are representative of three independent experiments. (E) An empty vector or a plasmid expressing VIMmut was nucleofected into MT4-M2BPi cells together with the HIV-1-producing plasmids indicated. A plasmid expressing firefly luciferase was included to serve as a control for transfection efficiency and sample handling. At 8 h posttransfection, IFNα-2b was added to the culture media. At 48 h posttransfection, p24CA levels in the culture supernatants were measured by ELISA (upper panel). Equal volumes of the culture supernatants were used to infect HEK293T cells. At 48 h postinfection, luciferase activity was measured in the recipient cells (lower panel). Relative CA levels and relative luc activity in the MT4-Ctrli cells without IFN treatment was set as 100. Data presented are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, n.s. p > 0.05
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f6: Vimentin filament collapse relieves M2BP inhibition of HIV-1 virion production.(A–D) HEK293T cells were transfected with the HIV-1 vector producing plasmids indicated, together with plasmids expressing M2BP and VIMmut. At 12 h posttransfection, chemicals indicated were added to the culture media. At 36 h posttransfection, culture supernatants were harvested to infect HeLa-CD4-CCR5 cells or analyzed for protein expressions. At 48 h postinfection, luciferase activity was measured. (A,C) The relative luciferase activity in the absence of M2BP was set as 100. Data presented are means ± SD of three independent experiments. (B,D) Protein expressions in the cell lysates and culture supernatants. The p24CA release level was calculated as described in the legend to Fig. 4D. Data presented are representative of three independent experiments. (E) An empty vector or a plasmid expressing VIMmut was nucleofected into MT4-M2BPi cells together with the HIV-1-producing plasmids indicated. A plasmid expressing firefly luciferase was included to serve as a control for transfection efficiency and sample handling. At 8 h posttransfection, IFNα-2b was added to the culture media. At 48 h posttransfection, p24CA levels in the culture supernatants were measured by ELISA (upper panel). Equal volumes of the culture supernatants were used to infect HEK293T cells. At 48 h postinfection, luciferase activity was measured in the recipient cells (lower panel). Relative CA levels and relative luc activity in the MT4-Ctrli cells without IFN treatment was set as 100. Data presented are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, n.s. p > 0.05
Mentions: It has been reported that M2BP regulates centriole biogenesis16, which participates in the regulation of the morphology of cytoskeletons40. This prompted us to ask whether cytoskeletons are involved in M2BP inhibition of HIV-1 virion production. We analyzed the antiviral activity of M2BP in the presence of nocodazole, cytochalasin D and acrylamide, which collapses microtubules, microfilaments and some intermediate filaments, respectively284142. HEK293T cells were transfected with pHIV-1NL4-3-luc and treated with the chemicals. The culture supernatants were used to infect recipient cells to evaluate the effects of the chemicals on M2BP inhibition of virion production. Treatment of the cells with acrylamide, but not nocodazole or cytochalasin D, partially relieved the inhibitory effect of M2BP on HIV-1 production (Fig. 6A). Further analyses revealed that treatment of the cells with acrylamide partially relieved M2BP inhibition of virion production without affecting Env processing (Fig. 6B), suggesting that intermediate filaments might be involved in M2BP inhibition of HIV-1 virion production. Notably, in the absence of exogenous M2BP, treatment of acrylamide increased the luciferase activity by about 2-fold (Supplementary Fig. 3), suggesting that collapsing intermediate filaments by acrylamide relieves the inhibitory effect of endogenous M2BP. Only a few types of intermediate filaments have been reported to be collapsed by acrylamide, which include vimentin filaments284344. We speculated that acrylamide might relieve M2BP inhibition of HIV-1 virion production by collapsing vimentin filaments. To test this idea, a vimentin mutant (VIMmut), whose overexpression collapses vimentin filaments (Supplementary Fig. 4)29, was analyzed for its ability to relieve M2BP inhibition of the production of VSV-G pseudotyped HIV-1NL4-3ΔEnv-luc. Indeed, overexpression of VIMmut relieved the inhibitory effect of M2BP on HIV-1 virion production (Fig. 6C,D). To determine whether vimentin filaments are involved in the antiviral function of endogenous M2BP in T cell lines, VIMmut were expressed in the MT4 cells expressing a control shRNA or the shRNA targeting M2BP. IFNα-2b treatment reduced the production of VSV-G-pseudotyped HIV-1 vector in the control cells (Fig. 6E). In comparison, in the MT4-M2BPi cells or in the control cells overexpressing VIMmut, IFN inhibition of viral production was significantly reduced (Fig. 6E). Collectively, these results indicate that 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.

No MeSH data available.


Related in: MedlinePlus