Characterization of a U _L 49-Null Mutant: VP22 of Herpes Simplex Virus Type 1 Facilitates Viral Spread in Cultured Cells and the Mouse Cornea

Abstract

Herpes simplex virus type 1 (HSV-1) virions, like those of all herpesviruses, contain a proteinaceous layer termed the tegument that lies between the nucleocapsid and viral envelope. The HSV-1 tegument is composed of at least 20 different viral proteins of various stoichiometries. VP22, the product of the U L 49 gene, is one of the most abundant tegument proteins and is conserved among the alphaherpesviruses. Although a number of interesting biological properties have been attributed to VP22, its role in HSV-1 infection is not well understood. In the present study we have generated both a U L 49-null virus and its genetic repair and characterized their growth in both cultured cells and the mouse cornea. While single-step growth analyses indicated that VP22 is dispensable for virus replication at high multiplicities of infection (MOIs), analyses of plaque morphology and intra-and extracellular multistep growth identified a role for VP22 in viral spread during HSV-1 infection at low MOIs. Specifically, VP22 was not required for either virion infectivity or cell-cell spread but was required for accumulation of extracellular virus to wild-type levels. We found that the absence of VP22 also affected virion composition. Intracellular virions generated by the U L 49-null virus contained reduced amounts of ICP0 and glycoproteins E and D compared to those generated by the wild-type and U L 49-repaired viruses. In addition, viral spread in the mouse cornea was significantly reduced upon infection with the U L 49-null virus compared to infection with the wild-type and U L 49-repaired viruses, identifying a role for VP22 in viral spread in vivo as well as in vitro.Herpes simplex virus type 1 (HSV-1) virions, like those of all herpesviruses, are composed of a nucleocapsid harboring the double-stranded linear DNA genome, a proteinaceous layer surrounding the nucleocapsid termed the tegument, and a host-derived lipid membrane envelope that contains viral glycoproteins. The tegument layer is a unique feature among herpesviruses and is composed of at least 20 different viral proteins of various stoichiometries. Tegument proteins have been shown to play a variety of roles in infection including the regulation of viral and host gene expression and the promotion of virus assembly and egress (6,19,34,39). Because tegument proteins enter the cell upon fusion of the viral envelope with the host cell membrane, they can potentially exert their activities prior to viral gene expression to provide herpesviruses an advantage early in infection.VP22, encoded by the U L 49 gene, is one of the most abundant HSV-1 tegument proteins, with an average of 2,000 copies present in each virion (18,22). VP22 is conserved among the alphaherpesviruses and has been studied in HSV-1, herpes simplex virus type 2 (HSV-2), bovine herpesvirus, and pseudorabies virus (PRV). Localization studies suggest that this protein is dynamically trafficked, as VP22 is present within both the cytoplasm and nucleus at different times in infection (16,36). Although VP22 is ...

The major tegument structural protein VP22 targets areas of dispersed nucleolin and marginalized chromatin during productive herpes simplex virus 1 infection

“…Unfortunately, this analysis has a major technical caveat. The Bac-ΔVP22 virus is exquisitely sensitive to the acquisition of second site mutations and absolutely must be grown on complementing V49 cells { (Duffy et al, 2006) and data not shown}. Therefore, the Bac-ΔVP22 used in our studies possesses virion-derived VP22.…”

“…HSV-1(Bac-ΔVP22) virus is a VP22-null mutant and HSV-1 (Bac-ΔVP22Repair) virus has the VP22 gene (U L 49) engineered back in (Duffy et al, 2006). All Bac-ΔVP22 stocks were grown on V49 cells, as described (Duffy et al, 2006). To obtain virus stocks, subconfluent monolayer Vero cells (~ 3 × 10 6 cells) were inoculated at a multiplicity of infection (MOI) of 0.01 for 2 hours at 37°C in 199V (Life Technologies) supplemented with 1% newborn calf serum or 2% newborn calf serum.…”

“…HSV-1(Bac-F) virus represents HSV-1(F) virus (Duffy et al, 2006) generated from a bacterial artificial chromosome (Bac) construct (Tanaka et al, 2003). HSV-1(Bac-ΔVP22) virus is a VP22-null mutant and HSV-1 (Bac-ΔVP22Repair) virus has the VP22 gene (U L 49) engineered back in (Duffy et al, 2006). All Bac-ΔVP22 stocks were grown on V49 cells, as described (Duffy et al, 2006).…”

“…However, the role of VP22 during HSV-1 infection remains unclear. Full-length VP22 is required for efficient viral cell-to-cell spread during infection in animals and cultured cells (Duffy et al, 2006;Pomeranz and Blaho, 2000). VP22 of HSV-1 possesses two nuclear localization signals (NLS) (Kotsakis et al, 2001) and the protein accumulates to high levels in the nuclei of either infected cells (Pomeranz and Blaho, 1999) or VP22-expressing stable cell lines (Pomeranz and Blaho, 2000).…”

Deletion of the Herpes Simplex Virus 1 U L 49 Gene Results in mRNA and Protein Translation Defects That Are Complemented by Secondary Mutations in U L 41

“…Previous studies with a U L 49-null virus showed that the absence of VP22 results in reduced plaque size and a nearly global shutoff protein synthesis at late times in infection (7,8). Interestingly, passage of the U L 49-null virus on noncomplementing cells led to a rescue of the small plaque phenotype thought to be the result of a secondary, compensatory mutation (7). Further work by other investigators showed that U L 49 mutants propagated on noncomplementing cells frequently acquire secondary mutations in the U L 41 gene.…”