The small basic HIV-1 nucleocapsid protein (NC) consists of 55 amino acids and is synthesized as part of the Gag precursor polyprotein, which is cleaved into smaller components within the virion by the viral protease. NC binds to the stem loop (SL) 1 through the SL4 regions of viral RNA to specifically package HIV-1 genomic RNA into virions, and mediates strand transfer during reverse transcription [1–4]. Both of two zinc finger motifs of NC are required for these activities . NC is also involved in transfer RNALys3 annealing to the primer binding site (PBS) of viral genomic RNA by means of two basic regions that flank the first zinc finger motif [6–8]. Furthermore, these basic regions are involved in Gag–Gag interactions and facilitate efficient capsid assembly, which leads to proper particle formation . The only posttranslational modification of NC described to date is low-level ubiquitination at still undefined sites, which may be important in the context of Gag and HIV-1 release .
Arginine methylation is a posttranslational modification that involves the addition of one or two methyl groups to the nitrogen atoms of the guanidino group of arginine . These S-adenosyl-L-methionine-dependent (AdoMet) methylations are carried out by protein arginine methyltransferases (PRMT), a series of enzymes found only in eukaryotes . Arginine methylation has been implicated in RNA processing, transcriptional regulation, signal transduction, DNA repair, and contributes to the ‘histone code’ [11,13–19]. Two major types of arginine methylation have been described: type I methyltransferases catalyze the formation of ω-NG-monomethylarginine and ω-NG,NG-dimethylarginine (asymmetric); type II enzymes produce ω-NG-monomethylarginine and ω-NG,N′G-dimethylarginine (symmetric) [11,13,20,21]. In humans, nine different PRMT have been described : PRMT1 [22,23], PRMT3 [24,25], PRMT4 , PRMT6  and PRMT8  are all type I enzymes (Fig. 1a), whereas PRMT5 [28,29], PRMT7 [21,30] and PRMT9  are type II enzymes. The classification and activity of PRMT2 [23,32] has not yet been established.
The 41900Mr PRMT6 is located in the nucleus and is the only methyltransferase shown thus far to possess automethylation activity . The non-histone chromatin protein HMGA1a and the DNA polymerase β are the only host substrates that have been proposed to be methylated by PRMT6 [33–35]. Furthermore, PRMT6 recognizes sequences that are different from the glycine and arginine-rich (GAR) motifs, which are the targets for most other PRMT [11,15]. A peptidyl arginine deiminase was recently shown to have limited arginine demethylating activity, i.e. it recognizes only monomethylarginine-containing proteins as substrates [11,36–38]. Whether the demethylation of PRMT6 substrates might also be catalyzed by peptidyl arginine deiminase remains to be determined.
Some AdoMet analogues were shown to inhibit methyltransferases directly . More recently, a series of small molecules termed arginine methyltransferase inhibitors (AMI) were shown to act specifically against PRMT and not to act as competitors of AdoMet. AMI3.4 (Fig. 1b) is a second-generation compound that showed specific inhibition only of PRMT3 and PRMT6 but not of other PRMT (M.T. Bedford, personal communication).
Diseases such as multiple sclerosis, spinal muscular atrophy, cardiovascular disease, as well as some types of cancer and viral pathogenesis have been related to methylation . For example, the methylation of hepatitis delta virus antigen by PRMT1 is essential for RNA replication , and hepatitis C virus downregulates PRMT1 methylation of the helicase of non-structural protein 3 by increasing expression levels of protein phosphatase 2Ac . Our group demonstrated that HIV-1 Tat is methylated in its arginine rich motif (ARM) by PRMT6, and that this negatively regulates transactivation activity . These findings are also consistent with data on HIV-1 regulation by the transcription elongation factor originally named ‘suppressor of Ty 5’, which is methylated by both PRMT1 and PRMT5, showing that an increase in methylation can have a negative impact on viral replication . It is unknown, however, whether NC may be a substrate for PRMT6. Close analysis of the two basic regions of NC shows that both have similarity to the N-terminal portion of the ARM of HIV-1 Tat.
Here, we report the in-vitro and in-vivo methylation of NC by PRMT6. Methylation of the two basic regions of NC resulted in decreased tRNALys3 annealing efficiency to the PBS and diminished initiation of reverse transcription. Consistent with these findings, viral replication of different wild-type HIV-1 strains in MT2 cells or in cord blood mononuclear cells (CBMC) was increased when cells were treated with the PRMT6 inhibitor AMI3.4. The same compound had no significant effect on early viral replication events in multinuclear-activation galactosidase indicator (MAGI) cells, suggesting that arginine methylation of the basic regions of NC must have occurred before the initiation of new infection.
Materials and methods
Glutathione-S-transferase (GST)-tagged PRMT6 was recloned from pGEX-6P1  into pGEX-4T1 with restriction enzymes EcoRI and BamHI, then transformed into BL21(Lys3) for isopropyl-β-D-thiogalactopyranoside-induced expression.
Histidine-tagged Tat72 and Tat86 were prepared as previously described . Histidine-tagged NC, minimal Gag (mGag), Gag without C-terminal p6 (GagΔp6), reverse transcriptase (RT) and myc-epitope-tagged PRMT6 were described earlier [8,39,44]. Histidine-tagged mutant NC proteins were generated using the QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, California, USA) and transformed into BL21 (Invitrogen, Carlsbad, California, USA) for isopropyl-β-D-thiogalactopyranoside-induced expression. The following primers were purchased in cartridge purity (Invitrogen): R7A: 5′–G CAG AGA GGC AAT TTT GCG AAC CAA AGA AAG ATT GTT AAG–3′ and 5′–CTT AAC AAT CTT TCT TTG GTT CGC AAA ATT GCC TCT CTG C–3′, R10A: 5′–GGC AAT TTT AGG AAC CAA GCA AAG ATT GTT AAG TGT TTC–3′ and 5′–GAA ACA CTT AAC AAT CTT TGC TTG GTT CCT AAA ATT GCC–3′, R29A: 5′–C ACA GCC AGA AAT TGC GCG GCC CCT AGG AAA AAG GGC–3′ and 5′–GCC CTT TTT CCT AGG GGC CGC GCA ATT TCT GGC TGT G–3′, and R32A: 5′–GA AAT TGC AGG GCC CCT GCG AAA AAG GGC TGT TGG AAA TG–3′ and 5′–CA TTT CCA ACA GCC CTT TTT CGC AGG GGC CCT GCA ATT TC–3′ (introduced mutations underlined).
Human placental tRNALys3 was purchased from Bio S&T (Montréal, Québec, Canada). HIV-1 DNA (pHIV-PBS) containing the region spanning the 5’ untranslated region to the PBS was in vitro transcribed and purified as previously described .
Anti-HisG IgG (Mo) and anti-Mo IgG (Sh, horseradish peroxidase coupled) antibodies were purchased from Invitrogen and United States Biological (Swampscott, Massachusetts, USA), respectively.
AMI3.4 was solubilized in 100% dimethyl sulphoxide (DMSO) at a concentration of 10 mmol/l (Fig. 1b).
In-vitro methylation assays
Aliquots of 1–2 μg recombinant histidine-tagged NC (wild-type or mutated), Tat86, Tat72, mGag, GagΔp6, RT, or bovine serum albumin (New England Biolabs, Pickering, Ontario, Canada) were incubated with 3–4 μg GST-tagged PRMT6 in the presence of 0.55 μCi [methyl-3H]-S-adenosyl-L-methionine (Perkin Elmer Life Sciences, Boston, Massachusetts, USA) and TE buffer (1.67 mmol/l Tris, 0.33 mmol/l ethylenediamine tetraacetic acid, pH 7.4) for 3 h at 37°C in a final volume of 10 μl. Reactions were stopped by adding 10 μl 2× Lämmli buffer (Bio-Rad Laboratories, Hercules, California, USA), followed by boiling for 5 min and centrifugation at 16 000g for 2 min. Samples were loaded on 15% polyacrylamide gels containing sodium dodecyl sulphate (SDS) and a high level of N,N,N′,N′-tetramethylethylenediamine. Gels were stained with Coomassie brilliant blue R-250 solution (Bio-Rad) and, after destaining, soaked in Amplify (Amersham Biosciences, Little Chalfont, Bucks, UK) for 30 min. Gels were dried and exposed for fluorography on Hyperfilm MP (Amersham) for 1–3 days. Gels and films were quantified using Molecular Analyst (Bio-Rad).
Aliquots of 15 μg recombinant histidine-tagged wild-type NC were incubated with 10 μg GST-tagged PRMT6 (wild-type or mutated) or no enzyme in the presence of 10 μmol/l S-adenosyl-L-methionine (Sigma Chemical Co., St Louis, Missouri, USA) and TE buffer for 3 h at 37°C in a final volume of 16 μl. Reactions were used in a series of dilutions of NC (final concentration in initiation reaction: 3, 6, 9, 12 and 15 μmol/l) with 1× RT buffer (50 mmol/l Tris-HCl pH 7.8, 50 mmol/l sodium chloride) to a final volume of 10 μl. These dilutions were mixed with 3 μl PBS-RNA (0.4 μmol/l) and 1.5 μl tRNALys3 (0.4 μmol/l) in 1× RT buffer in a final volume of 15 μl, then incubated for 15 min at 37°C. Alternatively, a heat-annealing control containing 1× RT buffer instead of NC was incubated at 70°C, 50°C then 37°C for 10 min each. RT initiation reactions were set up by mixing the annealing samples with dithiothreitol (10 mmol/l), magnesium chloride (6 mmol/l), 0.5 μl RNase inhibitor (Invitrogen), RT (0.5 μg), deoxyguanidine triphosphate (10 μmol/l), 2′-deoxythymidine 5′-triphosphate (10 μmol/l), dideoxyadenosine triphosphate (10 μmol/l) and [32P]-deoxycytidine 5’-triphosphate (10 μmol/l) all in 1× RT buffer to a final volume of 30 μl and incubated for 30 min at 37°C. Reactions were stopped with 2× loading dye, boiled for 3 min, separated by urea-polyacrylamide gel electrophoresis (PAGE; 5%) and exposed on Kodak BioMax MR film (Amersham).
In-vivo methylation assays
Briefly, five or 20 plates (10 cm) of HeLa cells were transfected with pT-REx-DEST30-HNC or pVAX-mycPRMT6 (wild type) in a manner similar to that previously described . At 48 h post-transfection, the cells were pulse-labelled with 50 μCi/5 ml L-[methyl-3H]-methionine (Amersham) for 3 h in the presence of cycloheximide (100 μg/ml) and chloramphenicol (40 μg/ml) (both Sigma) in Dulbecco's modified essential medium lacking the amino acids methionine, cysteine and glutamine to prevent protein synthesis. The cells were lysed in radio immunoprecipitation assay buffer (150 mmol/l sodium chloride, 50 mmol/l Tris, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, pH 8) containing complete mini ethylenediamine tetraacetic acid-free protease inhibitor (Roche Diagnostic Systems, Inc., Branchburg, New Jersey, USA), partly purified with Ni-NTA (Qiagen, Valencia, California, USA) and processed as described for the in-vitro methylation assays. Exposure times on Hyperfilm MP were 2 weeks. NC was confirmed by Western blot with anti-HisG antibody that was detected with an anti-Mo secondary antibody conjugated to horseradish peroxidase using the ECL Plus Western blotting detection system on Hyperfilm ECL (Amersham).
MT2 cells and cord blood mononuclear cell assays
MT2 cells were pre-incubated for 2 h in RPMI medium (Gibco BRL, Gaithersburg, Maryland, USA) supplemented with different concentrations of AMI3.4. All samples were supplemented with DMSO up to the same concentration as in the assay containing 3 μmol/l AMI3.4 (solubilized in DMSO). After centrifugation, the cells were resuspended in fresh RPMI medium containing DMSO and AMI3.4, and then infected with different wild-type HIV-1 laboratory strain or clinical isolates. At 2 h post-infection, the cells were centrifuged and resuspended in RPMI medium supplemented with DMSO and AMI3.4. The infected cells were then transferred into 96-well plates in triplicate at a concentration of 5 × 104 cells per well, and grown for 3–4 days at 37°C with 5% carbon dioxide. Supernatants were harvested and assayed for p24 by enzyme-linked immunosorbent assay with the Vironostika HIV-1 antigen (Biomérieux, Marcy L'Etoile, France). Mock-infected cells were counted. The values of p24 levels were divided by the number of counted cells to provide normalization. Cultures not exposed to AMI3.4 were assigned a value of 100% and P values were determined by an unpaired t-test with Prism 4 (GraphPad Software Inc., San Diego, California, USA).
CBMC obtained from the Department of Obstetrics, Jewish General Hospital, Montreal, were treated in a similar way. The medium used was, however, additionally complemented with IL-2 as described . The same HIV-1 strains were used for infection and 5 × 105 cells were transferred to each well. Cells were refed after 3 days and supernatants collected after 7 days.
Multinuclear-activation galactosidase indicator assay
HeLa-CD4-LTR-β-gal (MAGI) cells were infected with 10 ng BH10 virus as described . The medium was supplemented with different concentrations of AMI3.4. All wells, except for one of the two controls, were supplemented with DMSO up to the same concentration as in the assay with 3 μmol/l AMI3.4 (solubilized in DMSO). The number of blue colonies counted in wells containing neither AMI3.4 nor DMSO was defined as 100%. Results were normalized with cell counts as described above.
HIV-1 nucleocapsid protein is methylated by PRMT6 in vitro
The HIV-1 Tat protein contains an ARM that was shown to be a substrate for PRMT6 . This means that PRMT6 recognizes sequences different from the GAR motif, and this prompted us to ask whether any other HIV-1 proteins might also be substrates of PRMT6. Accordingly, we tested four purified recombinant histidine-tagged HIV-1 proteins for potential in-vitro methylation by PRMT6: RT, NC, Gag that was deleted of the C-terminal p6 peptide (GagΔp6), and a minimal Gag protein consisting only of the C-terminal part of the capsid protein followed by the spacer peptide SP1 and NC (mGag). After completion of the methylation reaction, the samples were separated by SDS–PAGE and exposed for fluorography (Fig. 1c). The results show that NC, mGag and GagΔp6 were all methylated by PRMT6. No signals were detected for either of the two subunits of RT, i.e. p51 and p66. The positive controls Tat72 and Tat86 were detected as intense bands. The negative control bovine serum albumin did not produce any signal. In some reactions, a weak band could be detected at the level of PRMT6 as a result of the previously reported automethylation activity of this methyltransferase .
As NC is part of both mGag and GagΔp6, it is not evident whether other portions of Gag besides NC may also be methylated by PRMT6. The results of Figure 1d show that the process of in-vitro methylation for NC is not linear; thus, reliable quantification to compare the methylation of NC with that of mGag and GagΔp6 may not be possible. Therefore, increasing the substrate concentration by a factor of two does not necessarily mean that signal intensity will double. Our findings thus demonstrate that NC is an in-vitro substrate for PRMT6.
Nucleocapsid protein has one methylation site in each of its two basic regions
To characterize the site(s) of arginine methylation within NC further, we carried out mutational analyses. Our recombinant NC protein is derived from HXB2 (Fig. 2a). At first glance, sequence comparisons of Tat and NC reveal little similarity. Closer inspection, however, shows that the two basic regions of NC, containing RXXRK sequences, resemble the N-terminal portion of the ARM of Tat, which contains RXXRR.
To map the methylation sites of NC, four mutants were cloned, each carrying a single R to A substitution. These four mutants, as well as wild-type NC, were subjected to PRMT6 methylation. After separation by SDS–PAGE and Coomassie blue staining (Fig. 2b, lower left panel), the gel was exposed for fluorography as described (Fig. 2b, upper left panel). We quantified the bands, taking into account the amount of NC that had been loaded, with wild-type NC set at 100% (Fig. 2b, right panel). The band intensities of NC proteins containing R7A or R29A substitutions were 100 and 92%, respectively, compared with wild type, ruling these residues out as methyl acceptors for PRMT6. In contrast, R10A or R32A substitutions in NC led to reduced band intensities of 67 and 37%, respectively, suggesting that both residues are probably substrates for PRMT6. Residue R32 may be a more efficient substrate for methylation than R10.
To confirm this interpretation of the results, we constructed and expressed a doubly mutated NC with substitutions at both R10A and R32A. The methylation band intensity of the double mutant was reduced to 11% compared with wild-type NC set at 100% (Fig. 2b, right panel), suggesting that residues R10 and R32 are the only two efficient methyl acceptors in NC.
Methylation of nucleocapsid protein reduces the efficiency of tRNALys3 annealing to primer binding site
We next wished to monitor the effect of PRMT6 arginine methylation on NC-mediated annealing of tRNALys3 to the PBS. Accordingly, wild-type NC was exposed to either wild-type PRMT6 or to a mutant PRMT6 that contains a mutation that abolishes methylation at its active site . Reactions were then mixed with tRNALys3 and PBS-RNA for NC-mediated annealing, followed by the initiation of RT, separation by urea–PAGE and quantification of [32P]- deoxycytidine 5′-triphosphate incorporation on film (Fig. 3). The highest efficiency of RNA annealing was set at 100%, and an optimum NC concentration of approximately 9–10 μmol/l was based on previous results . Non-methylated NC, when incubated in the buffer without any PRMT6, attained the highest levels of annealing, i.e. 100%. The percentage of annealing dropped at higher NC concentrations, because efficiency is highest at a ratio of six nucleotide residues of template RNA per NC molecule . Non-methylated NC, subjected to mutated PRMT6, was somewhat less efficient, with the highest levels of annealing reached at 12 μmol/l NC, suggesting that PRMT6 might itself exert a modest negative effect on annealing. In contrast, annealing was markedly impaired when using wild-type PRMT6, showing that RNA annealing was inhibited when arginine residues of NC are methylated.
NC is an in-vivo target of PRMT6
To prove in-vivo relevance, we wished to visualize the extent of NC methylation by PRMT6 in cell-based assays. Accordingly, we transfected 20 tissue culture plates of HeLa cells with both NC and PRMT6 or NC alone. As a control, we also transfected five plates with either NC or PRMT6. Cells were pulse labelled with L-[methyl-3H]-methionine, and partly purified lysates were separated by SDS–PAGE for subsequent Coomassie staining and fluorography. As expected, the Coomassie stained gels revealed similar overall background protein levels as well as NC protein levels, with the exception of the PRMT6 only transfection (Fig. 4a, left panel).
The fluorograph shows that NC methylation was detected and that this signal was stronger in the aftermath of co-transfection with PRMT6 (Fig. 4a, middle panel). The control did not reveal NC methylation, but there was higher overall signal intensity in the case of PRMT6 transfection, with signals approximately twice as strong as for the NC sole transfection. This was also observed in the NC/PRMT6 co-transfection. Calculations of band levels show that the percentage of methylated NC was not significantly increased when PRMT6 was overexpressed. The latter finding corresponds well with our observation that NC methylation is not linear.
The other bands in the fluorograph were not caused by the incorporation of labelled methionine during protein synthesis, because relevant amino acids were omitted from the medium and the drugs cycloheximide and chloramphenicol were present. Rather, these signals probably originated from co-purified methylated proteins that were modified by PRMT or other enzymes that may methylate unrelated proteins.
To confirm that the relevant signals indeed related to NC, we carried out Western blots of partly purified lysates using anti-HisG antibody (Fig. 4a, right panel). The presence of NC was readily visualized. Together, these results show that NC is an in-vivo target for PRMT6 arginine methylation.
AMI3.4 increases viral replication
We next tested the PRMT6 inhibitor AMI3.4 in both MT2 cells and CBMC to determine its effect on HIV replication. MT2 cells were pre-treated with either AMI3.4 or with the non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine as a positive control, and then infected with HIV-1 wild-type viruses (laboratory strain or clinical isolates). After 3–4 days, we measured viral p24 levels in culture supernatants. In the case of nevirapine-treated cells, all wild-type strains showed a drop in p24 levels (data not shown). With AMI3.4-treated MT2 cells, however, an increase in p24 values was observed at drug concentrations of 1 and 3 μmol/l (Fig. 4b). Depending on the HIV-1 strain, viral replication was increased up to 5.6-fold (Table 1). In some cases, we also measured RT activity, which showed increases of a range similar to the p24 measurements (data not shown).
We also tested HIV-1 wild-type clinical isolates in CBMC. Nevirapine, as expected, led to a significant decrease in p24 levels for all strains (data not shown). In the case of AMI3.4, increased p24 values were observed, but to a lesser extent than in MT2 cells (Fig. 4c). Viral replication of the clinical isolates was also increased approximately 40–80% in the presence of AMI3.4 (Table 1).
To test the effects of AMI3.4 on viral infectiousness, we also performed infections of MAGI cells with HIV-1 BH10 in the presence of this compound (Fig. 4d). AMI3.4 did not possess any direct effect on viral infectiousness.
We have shown that the HIV-1 NC protein is both an in-vitro and in-vivo substrate for PRMT6, making NC, after Tat, the second HIV-1 protein that is known to be methylated by this enzyme. Neither of these substrates contains a GAR motif, which is the common recognition motif for PRMT. In the case of Tat, arginine methylation occurs within the ARM .
Using a mutational approach, we have shown that amino acid residues R10 and R32 are responsible for approximately one-third and two-thirds of the methylation signal, respectively. We also investigated the effects of arginine methylation on NC-mediated annealing of tRNALys3 to the PBS, a process in which the two basic regions that flank the first zinc finger of NC are involved . The efficiency of annealing of tRNALys3 by methylated NC was reduced by approximately threefold compared with wild-type NC. These in-vitro annealing results suggest that the methylation of NC may slow down HIV-1 reverse transcription.
The in-vivo NC methylation signals were weak, even with PRMT6 co-transfection. Our data are consistant with recent findings on a different PRMT6 substrate termed HMGA1a, which yielded only weak methylation signals after 2 months of exposure . It is thus difficult to demonstrate in-vivo methylation of PRMT6 substrates, even with PRMT6 overexpression and large amounts of sample.
Our in-vivo data in both MT2 cells and CBMC show that the AMI3.4-mediated inhibition of PRMT6, which recognizes NC and Tat as substrates, leads to increased viral replication of all virus strains tested. These findings are consistent with previous observations that broadly active methylase inhibitors can upregulate HIV gene transcription . One of the HIV-1 elements contributing to such increased viral replication is Tat, which was shown to be less efficient in its transactivation ability when methylated , i.e. transcription of viral RNA was accelerated when Tat methylation by PRMT6 was blocked by small interfering RNA. AMI3.4 also blocked the methylation of NC, a finding that may explain the increase in viral replication. Although our viral replication data demonstrate that methylation of both NC and Tat can compromise viral replication, it is not possible to calculate individual contributions of the two proteins in this process. It is significant, however, that a single drug can simultaneously affect the function of two distinct HIV-1 proteins.
In contrast, MAGI assays did not reveal any effect of AMI3.4. This suggests that arginine methylation must have occurred during the previous round of infection, possibly in the context of the Gag polyprotein, which was also identified as a target of PRMT6, at late stages of the viral life cycle.
Does this mean that arginine methylation of HIV-1 proteins by PRMT6 is required by the virus to fine-tune different stages of the viral life cycle? May methylation represent a type of host defence mechanism that regulates viral replication? The fact that the inhibition of arginine methylation causes increased viral replication may mean that HIV regulates its own replication capacity by allowing arginine methylation to maintain viral replication at an optimal level. In contrast, arginine methylation of HIV-1 proteins might serve as a host cell defence mechanism by lowering overall levels of viral replication. This may also be of benefit to the virus in establishing latency in resting CD4 T cells. Current research in our laboratory will attempt to determine whether the use of PRMT6 inhibitors may represent a strategy to drive such cells out of latency.
The authors would like to thank Dr Mark T. Bedford for providing AMI3.4, Maureen Oliveira for p24 and RT assays, Dr Héctor A. Roldán for purified Gag proteins and NC plasmids, Daniela Moisi and Michel Ntemgwa for DNA sequencing.
Sponsorship: This work was supported by a grant to M.A.W. from the Canadian Institutes of Health Research (CIHR). C.F.I. was partly supported by a Swiss National Science Foundation fellowship award. F.A.F. is the recipient of a CIHR doctoral fellowship award.
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