Characterization of the Alphaherpesvirus Tegument Protein US2
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Members of the Herpesviridae are large enveloped, double-stranded DNA viruses, whose virions are comprised of a viral genome-containing icosahedral capsid, a layer of tegument and a glycoprotein-embedded envelope. The tegument contains numerous viral proteins and cellular proteins. Most of the tegument proteins are poorly understood and require further investigation. This study focuses on one of the tegument proteins, Us2, and utilizes two model alphaherpesviruses: pseudorabies virus (PRV) and herpes simplex virus (HSV). Us2 is conserved among all alphaherpesvirus with the exception of varicella-zoster virus (VZV). The amino acid sequence of all Us2 orthologs share three N-terminal conserved regions whereas the C-terminal sequences are highly variable. PRV Us2 contains a C-terminal prenylation motif that targets Us2 to the plasma membrane. Although it is indispensable for virus growth in cell culture, deletion of Us2 gene in PRV caused an accumulation of virions in the cytoplasm of infected primary cells. Furthermore, PRV Us2 spatially regulates MAPK ERK activity by sequestering it to the plasma membrane. Inhibition of ERK kinase activity caused a delay in the release of extracellular viruses and the defect was more profound in PRV Us2-null virus infected cells. Altogether, these data suggest a requirement for ERK activity and significance of PRV Us2-ERK interaction in virus egress. To understand the mechanism of Us2-ERK interaction, PRV Us2 determinants for ERK interaction were mapped. Our data revealed that the N-terminal 214 residues are the minimal sequence of Us2 required for interaction with ERK. In addition, PRV Us2 oligomerizes and forms complexes with ERK via the ERK common docking (CD) domain that facilitates the interaction of ERK with many of its substrates. Unlike PRV Us2, HSV-2 Us2 does not have any putative membrane targeting signals. However, our data revealed that HSV-2 Us2 localizes to the plasma membrane and is lipid raft associated. In addition, HSV-2 Us2 interacts directly with ubiquitin. As ubiquitination is responsible for proteasomal degradation and is involved in endocytosis and lysosomal degradation, these findings suggest that Us2 may be involved in proteasomal degradation pathways that counteract host defenses, or participate in final envelopment in endocytic compartments by facilitating the endocytosis of viral envelope proteins.