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Automethylation of protein arginine methyltransferase 6 (PRMT6) regulates its stability and its anti-HIV-1 activity.

Singhroy DN, Mesplède T, Sabbah A, Quashie PK, Falgueyret JP, Wainberg MA - Retrovirology (2013)

Bottom Line: In addition, PRMT6 inhibits HIV-1 replication in cell culture by directly methylating and interfering with the functions of several HIV-1 proteins, i.e. Tat, Rev and nucleocapsid (NC).PRMT6 also displays automethylation capacity but the role of this post-translational modification in its antiretroviral activity remains unknown.These results show that PRMT6 automethylation plays a role in the stability of this protein and that this event is indispensible for its anti-HIV-1 activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: McGill University AIDS Centre, Lady Davis for Medical Research, Jewish General Hospital, 3755 Cote Sainte Catherine, Montreal, QC, H3T 1E2, Canada.

ABSTRACT

Background: Protein arginine methyltransferase 6 (PRMT6) is a nuclear enzyme that methylates arginine residues on histones and transcription factors. In addition, PRMT6 inhibits HIV-1 replication in cell culture by directly methylating and interfering with the functions of several HIV-1 proteins, i.e. Tat, Rev and nucleocapsid (NC). PRMT6 also displays automethylation capacity but the role of this post-translational modification in its antiretroviral activity remains unknown.

Results: Here we report the identification by liquid chromatography-mass spectrometry of R35 within PRMT6 as the target residue for automethylation and have confirmed this by site-directed mutagenesis and in vitro and in vivo methylation assays. We further show that automethylation at position 35 greatly affects PRMT6 stability and is indispensable for its antiretroviral activity, as demonstrated in HIV-1 single-cycle TZM-bl infectivity assays.

Conclusion: These results show that PRMT6 automethylation plays a role in the stability of this protein and that this event is indispensible for its anti-HIV-1 activity.

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Related in: MedlinePlus

PRMT6 automethylation is necessary for its HIV-1 restriction activity. The indicated forms of PRMT6 plasmid were co-transfected with HIV-1 pNL4.3 proviral DNA into 293T cells. At 48 hours after transfection, cell culture fluids containing virus were collected, quantified by QPCR (A) or by HIV RT activity assay (B), and titrated onto TZM-bl cells (C, D). The amount of transfected PRMT6 expression, i.e. not protein stability, was quantified by Western blots with anti-Myc and anti-actin antibodies followed by densitometric analysis normalized against actin (E). Endogenous levels of PRMT6 in HeLa cells (lane 2), TZM-bl cells (lane 3) and 293T cells (lane 4) were compared to transfected PRMT6 in HeLa cells (lane 1) (F). 15 μg of protein from whole cell extracts were loaded into each well used for analysis in the Western blots in (E) and (F). Actin was used as a loading control. Infectivity was measured by luciferase assay at 48 hours after infection. The control experiment was transfection of pNL4.3 with an empty plasmid (referred to on the figure as pNL4.3 only). Each experiment was performed in triplicate on three separate occasions, with similar results being obtained each time. T-tests were performed for both (A) and (B), showing that only PRMT6-WT was significantly different than the control *: p < 0.05. Two-way ANOVA was performed for (C) and (D), showing that only PRMT6-WT statistically inhibited HIV replication *: p < 0.001.
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Figure 6: PRMT6 automethylation is necessary for its HIV-1 restriction activity. The indicated forms of PRMT6 plasmid were co-transfected with HIV-1 pNL4.3 proviral DNA into 293T cells. At 48 hours after transfection, cell culture fluids containing virus were collected, quantified by QPCR (A) or by HIV RT activity assay (B), and titrated onto TZM-bl cells (C, D). The amount of transfected PRMT6 expression, i.e. not protein stability, was quantified by Western blots with anti-Myc and anti-actin antibodies followed by densitometric analysis normalized against actin (E). Endogenous levels of PRMT6 in HeLa cells (lane 2), TZM-bl cells (lane 3) and 293T cells (lane 4) were compared to transfected PRMT6 in HeLa cells (lane 1) (F). 15 μg of protein from whole cell extracts were loaded into each well used for analysis in the Western blots in (E) and (F). Actin was used as a loading control. Infectivity was measured by luciferase assay at 48 hours after infection. The control experiment was transfection of pNL4.3 with an empty plasmid (referred to on the figure as pNL4.3 only). Each experiment was performed in triplicate on three separate occasions, with similar results being obtained each time. T-tests were performed for both (A) and (B), showing that only PRMT6-WT was significantly different than the control *: p < 0.05. Two-way ANOVA was performed for (C) and (D), showing that only PRMT6-WT statistically inhibited HIV replication *: p < 0.001.

Mentions: We have shown that the expression level of PRMT6 is important for inhibition of HIV-1 replication, and now wished to assess the impact of the R35A mutation on this activity (Figure 6). Proviral DNA coding for pNL4-3 virus was co-transfected into 293T cells together with either a control empty plasmid or plasmids coding for PRMT6-WT or R35A mutant proteins. The resulting viruses were quantified by RT activity assay and by QPCR (Figure 6A and B) and their infectiousness was tested in TZM-bl reporter cells by normalizing the amount of infecting virus for either RT activity or viral RNA (Figure 6C and D). Since PRMT6-WT and R35A display differences in their stability (Figure 5), we verified the proper expression of these proteins in these experiments by measuring PRMT6 protein expression at 24 hours post transfection by Western blot (Figure 6E). Densitometric quantification indicated that PRMT6-WT expression levels were 20% lower than those of PRMT6-R35A in these experiments, measures that are independent from differences in stability. The data show that expression of PRMT6-WT decreased HIV-1 infectivity, but that the R35A mutation restored viral infectivity to levels similar to those of virus grown in the absence of PRMT6. This indicates that prevention of PRMT6 automethylation results in a significant decrease in PRMT6 anti-HIV activity of approximately 90% (Figure 6).


Automethylation of protein arginine methyltransferase 6 (PRMT6) regulates its stability and its anti-HIV-1 activity.

Singhroy DN, Mesplède T, Sabbah A, Quashie PK, Falgueyret JP, Wainberg MA - Retrovirology (2013)

PRMT6 automethylation is necessary for its HIV-1 restriction activity. The indicated forms of PRMT6 plasmid were co-transfected with HIV-1 pNL4.3 proviral DNA into 293T cells. At 48 hours after transfection, cell culture fluids containing virus were collected, quantified by QPCR (A) or by HIV RT activity assay (B), and titrated onto TZM-bl cells (C, D). The amount of transfected PRMT6 expression, i.e. not protein stability, was quantified by Western blots with anti-Myc and anti-actin antibodies followed by densitometric analysis normalized against actin (E). Endogenous levels of PRMT6 in HeLa cells (lane 2), TZM-bl cells (lane 3) and 293T cells (lane 4) were compared to transfected PRMT6 in HeLa cells (lane 1) (F). 15 μg of protein from whole cell extracts were loaded into each well used for analysis in the Western blots in (E) and (F). Actin was used as a loading control. Infectivity was measured by luciferase assay at 48 hours after infection. The control experiment was transfection of pNL4.3 with an empty plasmid (referred to on the figure as pNL4.3 only). Each experiment was performed in triplicate on three separate occasions, with similar results being obtained each time. T-tests were performed for both (A) and (B), showing that only PRMT6-WT was significantly different than the control *: p < 0.05. Two-way ANOVA was performed for (C) and (D), showing that only PRMT6-WT statistically inhibited HIV replication *: p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: PRMT6 automethylation is necessary for its HIV-1 restriction activity. The indicated forms of PRMT6 plasmid were co-transfected with HIV-1 pNL4.3 proviral DNA into 293T cells. At 48 hours after transfection, cell culture fluids containing virus were collected, quantified by QPCR (A) or by HIV RT activity assay (B), and titrated onto TZM-bl cells (C, D). The amount of transfected PRMT6 expression, i.e. not protein stability, was quantified by Western blots with anti-Myc and anti-actin antibodies followed by densitometric analysis normalized against actin (E). Endogenous levels of PRMT6 in HeLa cells (lane 2), TZM-bl cells (lane 3) and 293T cells (lane 4) were compared to transfected PRMT6 in HeLa cells (lane 1) (F). 15 μg of protein from whole cell extracts were loaded into each well used for analysis in the Western blots in (E) and (F). Actin was used as a loading control. Infectivity was measured by luciferase assay at 48 hours after infection. The control experiment was transfection of pNL4.3 with an empty plasmid (referred to on the figure as pNL4.3 only). Each experiment was performed in triplicate on three separate occasions, with similar results being obtained each time. T-tests were performed for both (A) and (B), showing that only PRMT6-WT was significantly different than the control *: p < 0.05. Two-way ANOVA was performed for (C) and (D), showing that only PRMT6-WT statistically inhibited HIV replication *: p < 0.001.
Mentions: We have shown that the expression level of PRMT6 is important for inhibition of HIV-1 replication, and now wished to assess the impact of the R35A mutation on this activity (Figure 6). Proviral DNA coding for pNL4-3 virus was co-transfected into 293T cells together with either a control empty plasmid or plasmids coding for PRMT6-WT or R35A mutant proteins. The resulting viruses were quantified by RT activity assay and by QPCR (Figure 6A and B) and their infectiousness was tested in TZM-bl reporter cells by normalizing the amount of infecting virus for either RT activity or viral RNA (Figure 6C and D). Since PRMT6-WT and R35A display differences in their stability (Figure 5), we verified the proper expression of these proteins in these experiments by measuring PRMT6 protein expression at 24 hours post transfection by Western blot (Figure 6E). Densitometric quantification indicated that PRMT6-WT expression levels were 20% lower than those of PRMT6-R35A in these experiments, measures that are independent from differences in stability. The data show that expression of PRMT6-WT decreased HIV-1 infectivity, but that the R35A mutation restored viral infectivity to levels similar to those of virus grown in the absence of PRMT6. This indicates that prevention of PRMT6 automethylation results in a significant decrease in PRMT6 anti-HIV activity of approximately 90% (Figure 6).

Bottom Line: In addition, PRMT6 inhibits HIV-1 replication in cell culture by directly methylating and interfering with the functions of several HIV-1 proteins, i.e. Tat, Rev and nucleocapsid (NC).PRMT6 also displays automethylation capacity but the role of this post-translational modification in its antiretroviral activity remains unknown.These results show that PRMT6 automethylation plays a role in the stability of this protein and that this event is indispensible for its anti-HIV-1 activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: McGill University AIDS Centre, Lady Davis for Medical Research, Jewish General Hospital, 3755 Cote Sainte Catherine, Montreal, QC, H3T 1E2, Canada.

ABSTRACT

Background: Protein arginine methyltransferase 6 (PRMT6) is a nuclear enzyme that methylates arginine residues on histones and transcription factors. In addition, PRMT6 inhibits HIV-1 replication in cell culture by directly methylating and interfering with the functions of several HIV-1 proteins, i.e. Tat, Rev and nucleocapsid (NC). PRMT6 also displays automethylation capacity but the role of this post-translational modification in its antiretroviral activity remains unknown.

Results: Here we report the identification by liquid chromatography-mass spectrometry of R35 within PRMT6 as the target residue for automethylation and have confirmed this by site-directed mutagenesis and in vitro and in vivo methylation assays. We further show that automethylation at position 35 greatly affects PRMT6 stability and is indispensable for its antiretroviral activity, as demonstrated in HIV-1 single-cycle TZM-bl infectivity assays.

Conclusion: These results show that PRMT6 automethylation plays a role in the stability of this protein and that this event is indispensible for its anti-HIV-1 activity.

Show MeSH
Related in: MedlinePlus