Temporal Regulation of Herpes Simplex Virus Type 2 VP22 Expression and Phosphorylation

Abstract

The VP22 protein of herpes simplex virus type 2 (HSV-2) is a major component of the virion tegument. Previous work with HSV-1 indicated that VP22 is phosphorylated during infection, and phosphorylation may play a role in modulating VP22 localization in infected cells. It is not clear, however, when phosphorylation occurs in infected cells or how it is regulated. Less is known about the synthesis and phosphorylation of HSV-2 VP22. To study the complete biosynthetic history of HSV-2 VP22, we generated a monoclonal antibody to the carboxy terminus of VP22. Using immunoprecipitation and Western blot analyses, we show that HSV-2 VP22 can be found in three distinct isoforms in infected cells, two of which are phosphorylated. Like HSV-1 VP22, HSV-2 VP22 is synthesized ca. 4 h after infection, and the isoform later incorporated into virions is hypophosphorylated. In addition, we demonstrate for the first time (i) that newly synthesized VP22 is phosphorylated rapidly after synthesis, (ii) that this phosphorylation occurs in a virus-dependent manner, (iii) that the HSV-2 kinase UL13 is capable of inducing phosphorylation of VP22 in the absence of other viral proteins, (iv) that phosphorylated VP22 is very stable in infected cells, (v) that phosphorylated isoforms of VP22 are gradually dephosphorylated late in infection to produce the virion tegument form, and (vi) that this dephosphorylation occurs independently of viral DNA replication or virion assembly. These results indicate that HSV-2 VP22 is a stable protein that undergoes highly regulated, virus-dependent phosphorylation events in infected cells.The tegument of the herpesvirus virion is an amorphous structure that lies between the capsid and the envelope. The function of this subvirion structure appears to be twofold: to link the capsid of the newly forming viral particle with the envelope and to introduce regulatory proteins into a newly infected cell. The capacity of herpes simplex virus type 1 (HSV-1) and HSV-2 tegument proteins to regulate viral gene expression and to subvert cellular functions has been the focus of considerable study. For example, it is known that the tegument proteins VP16 and vhs transactivate viral ␣ genes and degrade host and viral mRNAs, respectively (14,15,22). These and other tegument proteins help to create a cellular environment that is advantageous to virus replication. VP22 is an abundant HSV tegument protein that possesses the remarkable property of intercellular spread (5). VP22 also can associate with and stabilize microtubules (7), interact with the transactivating protein VP16 (6), and associate with chromatin (13, 23). Although the function(s) of VP22 has not been established, its properties suggest that VP22 must play an important role in the HSV life cycle.Posttranslational modification is a common mechanism to regulate protein localization and function. VP22 is a 300-amino-acid protein that has been shown in both HSV-1 and HSV-2 to be posttranslationally modified by phosphorylation (1,8,13,23). Phosphorylation of HSV-1 VP22...

Viral Serine/Threonine Protein Kinases

“…In addition, the CHPK of HSV phosphorylates the tegument proteins VP13/14 and VP22, which may facilitate their release from the capsid during entry (45,70,152). Alphaherpesvirus CHPK-mediated phosphorylation of tegument and/or cellular proteins in combination with phosphorylation by the alphaherpesvirus kinase US3 (see below) may regulate the directionality of microtubule-based virus transport in neuronal axons (39).…”

Bovine herpesvirus-1 US3 protein kinase: critical residues and involvement in the phosphorylation of VP22

“…By examining the phosphorylation of BoHV-1 VP22 from a qualitative perspective we noticed that VP22 was not only phosphorylated by CK2, but also to a lower extent by US3. In contrast, it has been noted that, in HSV-2, US3 does not appear to be responsible for VP22 phosphorylation in Vero cells expressing US3 and VP22 transiently, although this was not confirmed in kinase assays (Geiss et al, 2001). Although according to the rules outlined above there are potential recognition sites for US3 in VP22 from HSV-1 (ACM62272: RRSSSR).…”

“…Recently, UL13 was confirmed to directly phosphorylate VP22 in HSV-1 (Asai et al, 2007). However, in co-transfected Vero cells, US3 from HSV-2 did not appear to be responsible for phosphorylating VP22 (Geiss et al, 2001). This, however, was not confirmed with kinase assays.…”

“…Initial experiments involving the UL13 protein kinase of HSV-1 implicated it as a putative viral factor involved in the phosphorylation of VP22 (Coulter et al, 1993). Subsequent studies demonstrated that cellular casein kinase 2 (CK2) is the major kinase responsible for VP22 phosphorylation (Elliott et al, 1996, but that UL13 is also involved, both in HSV-1 and HSV-2 (Geiss et al, 2001;Morrison et al, 1998). Recently, UL13 was confirmed to directly phosphorylate VP22 in HSV-1 (Asai et al, 2007).…”

Molecular cloning and characterization of the pseudorabies virus US1 gene

“…Phosphorylation site prediction revealed 22 potential phosphorylation sites in PICP22, including 2 potential tyrosine phosphorylation sites. Tyrosine phosphorylation is involved in the replication of several herpesviruses (Geiss et al, 2001;Ren et al, 2001) and in shifting protein translocation from the cytoplasm to the nucleus during productive virus infection (Pomeranz and Blaho, 1999). Phosphorylation of VZV ORF63 is associated with its subcellular localization and transcriptional regulatory properties (Habran et al, 2005;Mueller et al, 2010), and phosphorylation of ICP22 is involved in HSV-1 virulence (Brandt and Kolb, 2003).…”