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Protein Arginine N-methyltransferases 5 and 7 Promote HIV-1 Production - PubMed

  • ️Wed Jan 01 2020

Protein Arginine N-methyltransferases 5 and 7 Promote HIV-1 Production

Hironobu Murakami et al. Viruses. 2020.

Abstract

Current therapies for human immunodeficiency virus type 1 (HIV-1) do not completely eliminate viral reservoirs in cells, such as macrophages. The HIV-1 accessory protein viral protein R (Vpr) promotes virus production in macrophages, and the maintenance of Vpr is essential for HIV-1 replication in these reservoir cells. We identified two novel Vpr-binding proteins, i.e., protein arginine N-methyltransferases (PRMTs) 5 and 7, using human monocyte-derived macrophages (MDMs). Both proteins found to be important for prevention of Vpr degradation by the proteasome; in the context of PRMT5 and PRMT7 knockdowns, degradation of Vpr could be prevented using a proteasome inhibitor. In MDMs infected with a wild-type strain, knockdown of PRMT5/PRMT7 and low expression of PRMT5 resulted in inefficient virus production like Vpr-deficient strain infections. Thus, our findings suggest that PRMT5 and PRMT7 support HIV-1 replication via maintenance of Vpr protein stability.

Keywords: HIV-1; PRMT5; PRMT7; Vpr; Vpr-binding protein; macrophage; pathogenesis; stability; virus production.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1

Identification of the Vpr-binding protein PRMT5. (a) Separation of Vpr-binding proteins by SDS-PAGE. The red arrow indicates the binding protein identified in this study. The blue arrows indicate FLAG-tagged-mRFP-FLAG-tagged-Vpr (mRFP-Vpr lane) and FLAG-tagged mRFP (mRFP lane). The black arrows indicate the heavy and light chains of the anti-FLAG IgG monoclonal antibody. (b) Peaks from MALDI-TOF MS analysis of the Vpr-binding protein after band excision and trypsin digestion. For peptide mass fingerprinting, the peak list was compared against the NCBI nr database using the MASCOT program. (c) The amino acid sequence of PRMT5 with matched peptide fragments identified by MALDI-TOF MS shown in red.

Figure 2
Figure 2

Binding of Vpr by PRMT family members. (a) GST pull-down assay using recombinant PRMT5. Purified GST, GST-Vpr, and PRMT5 expressed by E. coli strain BL21 cells were resolved by SDS-PAGE on 10% gels and stained with Coomassie brilliant blue (CBB; left panel). Recombinant PRMT5 after GST removal was incubated with GST-Vpr or GST as a control (immobilized on GST beads) and detected by immunoblotting with an anti-PRMT5 MAbs (right panel). (b) GST pull-down assays with other PRMT family members and GST-Vpr. Arrowheads indicate Vpr-binding PRMT proteins (PRMT2, PRMT7, and PRMT9). (c) Co-immunoprecipitation using HA-tagged-Vpr (HA-Vpr)-expressing cells. HA-Vpr and HA tag alone were expressed in HeLa cells and co-immunoprecipitated using anti-HA MAbs. PRMT2, PRMT5, PRMT7, and PRMT9 were detected by immunoblotting with antibodies specific to each protein.

Figure 3
Figure 3

Downregulation of HA-Vpr after siRNA knockdown of PRMTs and rescue by inhibitors. (a) HA-Vpr expression using the bicistronic vector HA-Vpr-IRES-ZsGreen1-pCAGGS with siRNA against PRMT2, PRMT5, PRMT7, or PRMT9. Proteins were visualized by immunoblotting at 48 h post-transfection. Lanes marked “0” indicate transfection with siRandom as a negative control. (b) HeLa cells were co-transfected with HA-Vpr-IRES-ZsGreen1-pCAGGS together with siPRMT5 (50 nM), siPRMT7 (50 nM), or siRandom (50 nM) as indicated. Lanes marked “−” indicate transfection of siRandom. Proteins were visualized by immunoblotting at 48 h post-transfection. HeLa cells were cotransfected with HA-Vpr-IRES-ZsGreen1-pCAGGS and siRNAs against PRMT5 and/or PRMT7, and then treated with 50 µM lactacystin (LC) (c) or 50 µM E-64d (d) for 6 h starting at 48 h post-transfection. Proteins were visualized by immunoblotting.

Figure 4
Figure 4

Knockdown of PRMT5 and PRMT7 in MDMs affects virus production. (a) Expression of PRMT5 and PRMT7 at 12 days post-infection was measured by qPCR. The experiments were independently conducted in triplicate. (b) MDMs were transfected with siPRMT5 and siPRMT7, and then infected with NF462 WT or NF462 ΔR strains of HIV-1. Virus production was measured using p24 ELISA at 0, 4, 8, and 12 days post-infection. Statistically significant differences are indicated by single asterisks (* p < 0.05).

Figure 5
Figure 5

Differential expression of PRMT 5 in MDMs affected virus production. Expression of PRMT5 (a) and PRMT7 (b) mRNA in MDMs from six healthy donors. (c) MDMs from each donor were infected with the NF462 WT or NF462 ΔR strain, and virus production was measured using p24 ELISA at 0, 4, 8, and 12 days post-infection. Values shown are mean values, with error bars indicating standard deviations. Experiments were independently conducted in triplicate. Statistically significant differences are indicated by single and double asterisks (* p < 0.05 and ** p < 0.01, respectively).

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