The Us2 gene product of herpes simplex virus 2 is a membrane-associated ubiquitin-interacting protein.

The Us2 gene encodes a tegument protein that is conserved in most members of the Alphaherpesvirinae. Previous studies on the pseudorabies virus (PRV) Us2 ortholog indicated that it is prenylated, associates with membranes, and spatially regulates the enzymatic activity of the MAP (mitogen-activated protein) kinase ERK (extracellular signal-related kinase) through direct binding and sequestration of ERK at the cytoplasmic face of the plasma membrane. Here we present an analysis of the herpes simplex virus 2 (HSV-2) Us2 ortholog and demonstrate that, like PRV Us2, HSV-2 Us2 is a virion component and that, unlike PRV Us2, it does not interact with ERK in yeast two-hybrid assays. HSV-2 Us2 lacks prenylation signals and other canonical membrane-targeting motifs yet is tightly associated with detergent-insoluble membranes and localizes predominantly to recycling endosomes. Experiments to identify cellular proteins that facilitate HSV-2 Us2 membrane association were inconclusive; however, these studies led to the identification of HSV-2 Us2 as a ubiquitin-interacting protein, providing new insight into the functions of HSV-2 Us2.

Analysis of filamentous process induction and nuclear localization properties of the HSV-2 serine/threonine kinase Us3.

The Us3 serine/threonine kinase encoded by all alphaherpesviruses performs several important functions during virus multiplication. For example, expression of pseudorabies virus (PRV) Us3 causes reorganization of the actin cytoskeleton into filamentous processes (FPs) that promote cell-to-cell spread of virus infection. PRV Us3-induced FP formation requires Us3 kinase activity. To determine whether these characteristics were shared by HSV-2 Us3, expression plasmids for wild type (WT) and kinase dead (KD) Us3 variants were constructed. Expression of WT Us3 resulted in robust FP formation whereas expression of the KD Us3 variants did not. In the course of these experiments we noted that KD/K220 mutant Us3s were excluded from the nucleus in comparison to WT or KD/D305A Us3, prompting us to investigate Us3 nuclear shuttling properties. Herein we describe determinants of HSV-2 Us3-induced FP formation and present evidence for the presence of a leucine-rich nuclear export signal within HSV-2 Us3.

Arginine methylation of the HIV-1 nucleocapsid protein results in its diminished function.

OBJECTIVE: The HIV-1 nucleocapsid protein (NC) is involved in transfer RNA3 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.