The HBx protein has been described as a promiscuous transactivator of viral and cellular genes, acting from both cytoplasmic and nuclear locations (2
). Recent studies have provided clues for HBx transcriptional activity by showing that the viral protein is recruited to the promoter of cellular and HBV genes and interferes with epigenetic regulation (6
). Here, using affinity purification, we found that HBx interacts with PRMT1, a protein of the arginine methyltransferase family involved in multiple cellular functions, including transcriptional regulation. We showed that HBx inhibits PRMT1 methyltransferase activity. Thus, what might be the benefit of such inhibition for HBV? We demonstrated first that PRMT1 overexpression results in the downregulation of HBV transcription and, on the contrary, that depletion of PRMT1 correlates with an increase in HBV transcription. Using an enzymatically inactive PRMT1, we showed that transcriptional repression is mediated by the methyltransferase activity of PRMT1. Finally, using ChIP experiments, we confirmed that PRMT1 is recruited to the HBV cccDNA in the context of infection. Our data suggest that PRMT1 is recruited to the cccDNA and inhibits its transcription through the methylation of target proteins. HBx in turn might counteract the repressive effect of PRMT1 on HBV transcription by inhibiting its catalytic activity.
PRMT1 has been shown to methylate a large number of proteins, including proteins involved in transcriptional regulation and chromatin remodeling. Interestingly, HIV long terminal repeat (LTR) transcription has been shown to be repressed by PRMT1 together with PRMT5. Both PRMT1 and PRMT5 act through the methylation of SPT5, which decreases its binding to RNA polymerase II and, subsequently, transcriptional elongation (24
). SPT5 and its partner SPT4 are general transcription elongation factors that can regulate transcription elongation of a large number of cellular and viral genes in both positive and negative manners (52
). It will thus be interesting to test whether HBV transcription/elongation is also regulated by the SPT5 and SPT4 complex and whether SPT5 recruitment is enhanced during transcriptional activation.
Alternatively, the repressive activity of PRMT1 on HBV transcription could be dependent on HBx expression. One hypothesis is that PRMT1 could methylate HBx. Using an in vitro
methylation assay, we could not, however, observe the methylation of HBx by PRMT1 under our experimental conditions, but we cannot completely exclude that methylation occurs in vivo
, and further work is needed to determine if HBx activity is modulated by PRMT1. Since methylation modulates protein-protein or protein-nucleic acid interactions (49
), PRMT1 could also target one of the HBx-interacting partners involved in HBx transcriptional activity. Further studies will be necessary in order to understand the mechanism of PRMT1 repression.
In response to PRMT1 inhibition, the virus seems to have developed a strategy to evade this repression. Indeed, we observed that HBx inhibits the methyltransferase activity of PRMT1 toward substrates such as the polypeptide containing the N-terminal portion of human fibrillarin (GST-GAR), H4, as well as whole cellular proteins (). Interestingly, while we observed that in the presence of HBx, the methyltransferase activity of PRMT1 seems to be largely decreased, a protein with a molecular mass of approximately 40 kDa appears to have increased methylation (). Little is known regarding the regulation of PRMT1 activity; however, it has been shown that its activity can be regulated in a substrate-dependent manner through its interaction with cellular partners such as the immediate-early gene TIS21 and the leukemia-associated protein BTG1 or CCR4-associated factor 1 (hCAF1) (56
). HBx might thus behave as such a regulator.
PRMT1 participates in broad cellular processes, including signal transduction, cell proliferation, transcriptional regulation, chromatin structure regulation, RNA metabolism, and DNA repair (58
). Yu and collaborators demonstrated that a total loss of PRMT1 in mouse embryonic fibroblasts (MEFs) leads to DNA damage, cell cycle progression delay, checkpoint defects, as well as cell division aberration (59
). PRMT1 controls the DNA damage response pathway in part through the methylation of MRE11 and 53BP1 (60
). Interestingly, HBx contributes to HBV-induced hepatocarcinogenesis, and it is now well established that HBx expression correlates with mitotic aberrations such as chromosome segregation defects and polyploidy and impairs DNA damage checkpoint control (2
). Thus, deregulation of PRMT1 activity could contribute to the oncogenic activity of HBx.
Finally, PRMT1 might play an important role in the JAK-STAT pathway. Silencing of PRMT1 expression reduces the growth-inhibitory effect of beta interferon (IFN-β) (65
). However, the mechanism is not fully understood. PRMT1 has been shown to interact with the cytoplasmic domain of the interferon receptor IFNAR1 (65
). PRMT1 is also believed to methylate STAT1 and thus decrease its interaction with the protein inhibitor of activated STAT1 (PIAIS1) (49
). However, this mechanism is a subject of controversy and awaits further analysis, since others have reported that PIAIS1 could be a substrate of PRMT1 (66
). Our present data showing that HBx inhibits PRMT1 activity are in line with a previous report demonstrating that HBV interferes with interferon signaling through the inhibition of PRMT1 activity (67
). Thus, PRMT1 inhibition by HBx could participate in the resistance of some chronic HBV carriers to IFN-α treatment (68
Given that arginine methylation is an important and prevalent mechanism for protein function regulation, it is not surprising that viral proteins not only are arginine methyltransferase substrates but also are able to target arginine methyltransferase activity. PRMT6 has been shown to inhibit HIV-1 transcription through the methylation of Tat, Rev, and the nucleocapsid proteins (69
). PRMT1 has been shown to methylate and inhibit hepatitis C virus (HCV) NS3 protein, but in turn, HCV counteracts PRMT1 repressive activity by activating protein phosphatase 2, which inhibits PRMT1 (72
). Interestingly, by inhibiting PRMT1, HCV modulates the expression of cellular genes involved in hepatocarcinogenesis and inhibits DNA repair damage, suggesting a role of PRMT1 inhibition in HCV-mediated HCC (73
). Similarly, the E6 oncoproteins of low-risk and high-risk human papillomavirus interact with histone methyltransferases CARM1, PRMT1, and SET7, inhibiting their activities, which leads to the suppression of p53 activity (74
). Together with our current study, these results emphasize the important role of arginine methyltransferases in virus replication and associated pathogenesis.