Objective: The HIV-1 nucleocapsid protein (NC) is involved in transfer RNALys3 annealing to the primer binding site of viral genomic RNA by means of two basic regions that are similar to the N-terminal portion of the arginine-rich motif (ARM) of Tat. As Tat is known to be asymmetrically arginine dimethylated by protein arginine methyltransferase 6 (PRMT6) in its ARM, we investigated whether NC could also act as a substrate for this enzyme.
Methods: Arginine methylation of NC was demonstrated in vitro and in vivo, and sites of methylation were determined by mutational analysis. The impact of the arginine methylation of NC was measured in RNA annealing and reverse transcription initiation assays. An arginine methyltransferase inhibitor (AMI)3.4 was tested for its effects on viral infectivity and replication in vivo.
Results: NC is a substrate for PRMT6 both in vitro and in vivo. NC possesses arginine dimethylation sites in each of its two basic regions at positions R10 and R32, and methylated NC was less able than wild-type to promote RNA annealing and participate in the initiation of reverse transcription. Exposure of HIV-1-infected MT2 and primary cord blood mononuclear cells to AMI3.4 led to increased viral replication, whereas viral infectivity was not significantly affected in multinuclear-activation galactosidase indicator assays.
Conclusion: NC is an in-vivo target of PRMT6, and arginine methylation of NC reduces RNA annealing and the initiation of reverse transcription. These findings may lead to ways of driving HIV-infected cells out of latency with drugs that inhibit PRMT6.
From the aMcGill University AIDS Centre, Canada
bTerry Fox Molecular Oncology Group, Canada
cBloomfield Centre for Research on Aging at the Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Canada
dDepartments of Medicine, Canada
eOncology, McGill University, Montréal, Québec, Canada.
Received 14 July, 2006
Revised 1 November, 2006
Accepted 7 December, 2006
Correspondence to Mark A. Wainberg, McGill AIDS Centre, Lady Davis Institute, Jewish General Hospital, 3755 Côte-Ste-Catherine Road, Montréal, Québec H3T 1E2, Canada. Tel: +1 514 340 8260; fax: +1 514 340 7537; e-mail: firstname.lastname@example.org