A Role for the HSV Tegument Protein pUL21 in Retention of Viral Genomes Within Capsids

Abstract

Herpes simplex virus (HSV) virions are comprised of a double-stranded DNA genome packaged at high pressure within an icosahedral capsid, surrounded by tegument proteins and a lipid envelope studded with viral glycoproteins. Viral DNA (vDNA) synthesis and packaging into newly assembled capsids occur in the cell nucleus, whereas final envelopment of capsids occurs at cytoplasmic membranes. Thus, large 125nm diameter vDNA-containing C-capsids must transit to the cytoplasm through a process called nuclear egress (NE). Coupled to NE is a quality-control mechanism that preferentially selects C-capsids rather than empty A-capsids for this translocation. However, cells infected with HSV strains deleted for viral protein pUL21 have both A- and C- capsids in the cytoplasm of infected cells. Two possible explanations for this observation are: 1) NE quality-control is impaired in the absence of pUL21; or 2) unstable vDNA packaging in the absence of pUL21 leads to premature ejection of vDNA. Transmission electron microscopy was used to quantify A- and C-capsids that had underwent primary envelopment into the perinuclear space. In general, fewer perinuclear virions containing A-capsids were observed for pUL21 mutants compared to WT strains, suggesting that preferential selection of C-capsids in NE was functional in the absence of pUL21. Similar to what was observed in the cytoplasm, significantly more nuclear A-capsids were found in cells infected with pUL21 mutants compared to their WT counterparts, suggesting that vDNA retention was impaired in the absence of pUL21. In support of this view, during pUL21 mutant infection more fluorescent EdC-labeled vDNA was present in the cytoplasm compared to WT. In response to cytoplasmic vDNA, interferon regulatory factor 3 translocation to the nucleus was significantly higher in cells infected with pUL21 mutants compared WT viruses. Collectively, these results indicate more vDNA is prematurely ejected into the cytoplasm and favours the hypothesis that pUL21 facilitates vDNA retention within HSV capsids. Considering this, WT and pUL21 mutant nuclear capsid composition was examined and capsids in the absence of pUL21 had increased association of the viral protein pUL16. Our findings suggest that pUL21 regulates pUL16 addition to nuclear capsids and that over-addition of pUL16 may impair vDNA retention.