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The human antiviral factor TRIM11 is under the regulation of HIV-1 Vpr.

Yuan T, Yao W, Huang F, Sun B, Yang R - PLoS ONE (2014)

Bottom Line: In this study, we demonstrated that TRIM11 mainly suppressed the early steps of HIV-1 transduction, resulting in decreased reverse transcripts.Additionally, we found that TRIM11 could inhibit HIV-1 long terminal repeat (LTR) activity, which may be related to its inhibitory effects on NF-κB.These results suggest that the antiviral factor TRIM11 is indirectly regulated by HIV-1 Vpr through unknown mechanisms and that the concentration of Vpr is essential to these processes.

View Article: PubMed Central - PubMed

Affiliation: Research Group of HIV Molecular Epidemiology and Virology, Center for Emerging Infectious Diseases, The State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, The People's Republic of China.

ABSTRACT
TRIM11 has been reported to be able to restrict HIV-1 replication, but the detailed aspects of the interfering mechanisms remain unclear. In this study, we demonstrated that TRIM11 mainly suppressed the early steps of HIV-1 transduction, resulting in decreased reverse transcripts. Additionally, we found that TRIM11 could inhibit HIV-1 long terminal repeat (LTR) activity, which may be related to its inhibitory effects on NF-κB. Deletion mutant experiments showed that the RING domain of TRIM11 was indispensable in inhibiting the early steps of HIV-1 transduction but was dispensable in decreasing NF-κB and LTR activities. Moreover, we found that low levels of Vpr decreased TRIM11 protein levels, while high levels increased them, and these regulations were independent of the VprBP-associated proteasome machinery. These results suggest that the antiviral factor TRIM11 is indirectly regulated by HIV-1 Vpr through unknown mechanisms and that the concentration of Vpr is essential to these processes. Thus, our work confirms TRIM11 as a host cellular factor that interferes with the early steps of HIV-1 replication and provides a connection between viral protein and host antiviral factors.

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Effects of TRIM11 on HIV-1 transduction.A. Lysates from the HEK293 cells that were stably transduced with either pCDH or pCDH-TRIM11 were subjected to a western blot with the indicated antibodies. The numbers under each line display the relative ratios between the TRIM11 signals and actin signals. B. HEK293 cells stably expressing TRIM11 or a control pCDH vector were inoculated with various amounts of HIV-1 Vpr− viruses. Luciferase assays were performed at 24 h post-infection (hpi). C. Lysates from HEK293 cells that were stably transduced with shRNA targeting TRIM11 or GFP were subjected to a western blot with the indicated antibodies. The numbers under each line display the relative ratios between the TRIM11 signals and actin signals. D. TRIM11 knock-down and control cell lines were inoculated with various amounts of HIV-1 Vpr− viruses. Luciferase assays were performed at 24 hpi. E. Western blot analysis of TRIM11 expression in HEK293 cells transfected with control siRNA or TRIM11 siRNA#1, siRNA#2 and siRNA#3 for 24 h. The number under each line displays the relative ratios between the TRIM11 signals and actin signals. F. HEK293 cells transfected with control siRNA or TRIM11 siRNA were inoculation with 50 ng/ml p24gag HIV-1 Vpr− virus. Luciferase activity assays were performed at 24 hpi. Error bars represent the standard deviations from three independent replicates of the same experiment.
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pone-0104269-g001: Effects of TRIM11 on HIV-1 transduction.A. Lysates from the HEK293 cells that were stably transduced with either pCDH or pCDH-TRIM11 were subjected to a western blot with the indicated antibodies. The numbers under each line display the relative ratios between the TRIM11 signals and actin signals. B. HEK293 cells stably expressing TRIM11 or a control pCDH vector were inoculated with various amounts of HIV-1 Vpr− viruses. Luciferase assays were performed at 24 h post-infection (hpi). C. Lysates from HEK293 cells that were stably transduced with shRNA targeting TRIM11 or GFP were subjected to a western blot with the indicated antibodies. The numbers under each line display the relative ratios between the TRIM11 signals and actin signals. D. TRIM11 knock-down and control cell lines were inoculated with various amounts of HIV-1 Vpr− viruses. Luciferase assays were performed at 24 hpi. E. Western blot analysis of TRIM11 expression in HEK293 cells transfected with control siRNA or TRIM11 siRNA#1, siRNA#2 and siRNA#3 for 24 h. The number under each line displays the relative ratios between the TRIM11 signals and actin signals. F. HEK293 cells transfected with control siRNA or TRIM11 siRNA were inoculation with 50 ng/ml p24gag HIV-1 Vpr− virus. Luciferase activity assays were performed at 24 hpi. Error bars represent the standard deviations from three independent replicates of the same experiment.

Mentions: TRIM11 has been identified to potently interfere with HIV-1 replication in a screening of TRIM proteins that were transiently expressed in HEK293 cells [9]. To confirm these results, we constructed two HEK293 cell lines that stably expressed HA-TRIM11 or a control vector pCDH (Figure 1A). We first performed cell proliferation assay by counting cell numbers and found that cells stably expressing TRIM11 had minimal effect on cell proliferation (Figure S1A). The two cell lines were then inoculated with different amounts of HIV-1 Vpr− viruses, and the luciferase activities of the infected cells w examined to confirm the infection levels. In contrast with the control vector, the overexpression of TRIM11 resulted in a marked inhibition of HIV-1 transduction (Figure 1B). To assess whether TRIM11 expression at physiological levels possesses antiviral activity, we also constructed a knockdown cell line that stably expressing short hairpin ribonucleic acid (shRNA) that is directed against TRIM11. Because GFP is not expressed in mammalian cells, the cell line stably expressing shRNA that targets GFP served as the non-targeting control. As shown in Figure 1C, compared with the control cells, TRIM11 protein levels were significantly reduced in the knockdown cell line. Luciferase activity was monitored at 24 h after the inoculation of different amounts of HIV-1 Vpr− viruses in these two cell lines. The results showed that the TRIM11 knockdown cell line facilitated HIV-1 transduction by an average of approximately seven-fold (Figure 1D). To confirm the effect of endogenous TRIM11 on HIV-1 transduction, we knocked down TRIM11 expression by siRNAs. Three specific siRNAs for TRIM11 and a control siRNA were transfected into HEK293 cells. Western blot analysis showed that only siRNA#2 and siRNA#3 have apparent effects on reducing TRIM11 compared with the control siRNA. Single-round infectivity assay revealed that knockdown of TRIM11 by specific siRNA also facilitated HIV-1 transduction (Figure 1F). Taken together these results suggest that TRIM11 exhibits an inhibitory activity on HIV-1 transduction.


The human antiviral factor TRIM11 is under the regulation of HIV-1 Vpr.

Yuan T, Yao W, Huang F, Sun B, Yang R - PLoS ONE (2014)

Effects of TRIM11 on HIV-1 transduction.A. Lysates from the HEK293 cells that were stably transduced with either pCDH or pCDH-TRIM11 were subjected to a western blot with the indicated antibodies. The numbers under each line display the relative ratios between the TRIM11 signals and actin signals. B. HEK293 cells stably expressing TRIM11 or a control pCDH vector were inoculated with various amounts of HIV-1 Vpr− viruses. Luciferase assays were performed at 24 h post-infection (hpi). C. Lysates from HEK293 cells that were stably transduced with shRNA targeting TRIM11 or GFP were subjected to a western blot with the indicated antibodies. The numbers under each line display the relative ratios between the TRIM11 signals and actin signals. D. TRIM11 knock-down and control cell lines were inoculated with various amounts of HIV-1 Vpr− viruses. Luciferase assays were performed at 24 hpi. E. Western blot analysis of TRIM11 expression in HEK293 cells transfected with control siRNA or TRIM11 siRNA#1, siRNA#2 and siRNA#3 for 24 h. The number under each line displays the relative ratios between the TRIM11 signals and actin signals. F. HEK293 cells transfected with control siRNA or TRIM11 siRNA were inoculation with 50 ng/ml p24gag HIV-1 Vpr− virus. Luciferase activity assays were performed at 24 hpi. Error bars represent the standard deviations from three independent replicates of the same experiment.
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Related In: Results  -  Collection

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pone-0104269-g001: Effects of TRIM11 on HIV-1 transduction.A. Lysates from the HEK293 cells that were stably transduced with either pCDH or pCDH-TRIM11 were subjected to a western blot with the indicated antibodies. The numbers under each line display the relative ratios between the TRIM11 signals and actin signals. B. HEK293 cells stably expressing TRIM11 or a control pCDH vector were inoculated with various amounts of HIV-1 Vpr− viruses. Luciferase assays were performed at 24 h post-infection (hpi). C. Lysates from HEK293 cells that were stably transduced with shRNA targeting TRIM11 or GFP were subjected to a western blot with the indicated antibodies. The numbers under each line display the relative ratios between the TRIM11 signals and actin signals. D. TRIM11 knock-down and control cell lines were inoculated with various amounts of HIV-1 Vpr− viruses. Luciferase assays were performed at 24 hpi. E. Western blot analysis of TRIM11 expression in HEK293 cells transfected with control siRNA or TRIM11 siRNA#1, siRNA#2 and siRNA#3 for 24 h. The number under each line displays the relative ratios between the TRIM11 signals and actin signals. F. HEK293 cells transfected with control siRNA or TRIM11 siRNA were inoculation with 50 ng/ml p24gag HIV-1 Vpr− virus. Luciferase activity assays were performed at 24 hpi. Error bars represent the standard deviations from three independent replicates of the same experiment.
Mentions: TRIM11 has been identified to potently interfere with HIV-1 replication in a screening of TRIM proteins that were transiently expressed in HEK293 cells [9]. To confirm these results, we constructed two HEK293 cell lines that stably expressed HA-TRIM11 or a control vector pCDH (Figure 1A). We first performed cell proliferation assay by counting cell numbers and found that cells stably expressing TRIM11 had minimal effect on cell proliferation (Figure S1A). The two cell lines were then inoculated with different amounts of HIV-1 Vpr− viruses, and the luciferase activities of the infected cells w examined to confirm the infection levels. In contrast with the control vector, the overexpression of TRIM11 resulted in a marked inhibition of HIV-1 transduction (Figure 1B). To assess whether TRIM11 expression at physiological levels possesses antiviral activity, we also constructed a knockdown cell line that stably expressing short hairpin ribonucleic acid (shRNA) that is directed against TRIM11. Because GFP is not expressed in mammalian cells, the cell line stably expressing shRNA that targets GFP served as the non-targeting control. As shown in Figure 1C, compared with the control cells, TRIM11 protein levels were significantly reduced in the knockdown cell line. Luciferase activity was monitored at 24 h after the inoculation of different amounts of HIV-1 Vpr− viruses in these two cell lines. The results showed that the TRIM11 knockdown cell line facilitated HIV-1 transduction by an average of approximately seven-fold (Figure 1D). To confirm the effect of endogenous TRIM11 on HIV-1 transduction, we knocked down TRIM11 expression by siRNAs. Three specific siRNAs for TRIM11 and a control siRNA were transfected into HEK293 cells. Western blot analysis showed that only siRNA#2 and siRNA#3 have apparent effects on reducing TRIM11 compared with the control siRNA. Single-round infectivity assay revealed that knockdown of TRIM11 by specific siRNA also facilitated HIV-1 transduction (Figure 1F). Taken together these results suggest that TRIM11 exhibits an inhibitory activity on HIV-1 transduction.

Bottom Line: In this study, we demonstrated that TRIM11 mainly suppressed the early steps of HIV-1 transduction, resulting in decreased reverse transcripts.Additionally, we found that TRIM11 could inhibit HIV-1 long terminal repeat (LTR) activity, which may be related to its inhibitory effects on NF-κB.These results suggest that the antiviral factor TRIM11 is indirectly regulated by HIV-1 Vpr through unknown mechanisms and that the concentration of Vpr is essential to these processes.

View Article: PubMed Central - PubMed

Affiliation: Research Group of HIV Molecular Epidemiology and Virology, Center for Emerging Infectious Diseases, The State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, The People's Republic of China.

ABSTRACT
TRIM11 has been reported to be able to restrict HIV-1 replication, but the detailed aspects of the interfering mechanisms remain unclear. In this study, we demonstrated that TRIM11 mainly suppressed the early steps of HIV-1 transduction, resulting in decreased reverse transcripts. Additionally, we found that TRIM11 could inhibit HIV-1 long terminal repeat (LTR) activity, which may be related to its inhibitory effects on NF-κB. Deletion mutant experiments showed that the RING domain of TRIM11 was indispensable in inhibiting the early steps of HIV-1 transduction but was dispensable in decreasing NF-κB and LTR activities. Moreover, we found that low levels of Vpr decreased TRIM11 protein levels, while high levels increased them, and these regulations were independent of the VprBP-associated proteasome machinery. These results suggest that the antiviral factor TRIM11 is indirectly regulated by HIV-1 Vpr through unknown mechanisms and that the concentration of Vpr is essential to these processes. Thus, our work confirms TRIM11 as a host cellular factor that interferes with the early steps of HIV-1 replication and provides a connection between viral protein and host antiviral factors.

Show MeSH
Related in: MedlinePlus