The herpes simplex virus tegument protein pUL21 is required for viral genome retention within capsids

Abstract

During virion morphogenesis herpes simplex virus nucleocapsids transit from the nucleoplasm to the cytoplasm, through a process called nuclear egress, where the final stages of virion assembly occur. Coupled to nuclear egress is a poorly understood quality-control mechanism that preferentially selects genome-containing C-capsids, rather than A- and B-capsids that lack genomes, for transit to the cytoplasm. We and others have reported that cells infected with HSV strains deleted for the tegument protein pUL21 accumulate both empty A-capsids and C-capsids in the cytoplasm of infected cells. Quantitative microscopy experiments indicated that C-capsids were preferentially selected for envelopment at the inner nuclear membrane and that nuclear integrity remained intact in cells infected with pUL21 mutants, prompting alternative explanations for the accumulation of A-capsids in the cytoplasm. More A-capsids were also found in the nuclei of cells infected with pUL21 mutants compared to their wild type (WT) counterparts, suggesting pUL21 might be required for optimal genome packaging or genome retention within capsids. In support of this, more viral genomes were prematurely released into the cytoplasm during pUL21 mutant infection compared to WT infection and led to enhanced activation of cellular cytoplasmic DNA sensors. Mass spectrometry and western blot analysis of WT and pUL21 mutant capsids revealed an increased association of the known pUL21 binding protein, pUL16, with pUL21 mutant capsids, suggesting that premature and/or enhanced association of pUL16 with capsids might result in capsid destabilization. Further supporting this idea, deletion of pUL16 from a pUL21 mutant strain rescued genome retention within capsids. Taken together, these findings suggest that pUL21 regulates pUL16 addition to nuclear capsids and that premature, and/or, over-addition of pUL16 impairs HSV genome retention within capsids.

Applying 3D correlative structured illumination microscopy and X-ray tomography to characterise herpes simplex virus-1 morphogenesis

“…This suggests that, if anything, our cryoSIM and CLXT data may underestimate the importance of pUL21 in this process. In addition, we note that our CLXT studies cannot differentiate mature genome-containing ‘C’ capsids from the genome-free ‘A’ and ‘B’ capsids that are known to accumulate in the absence of HSV-1 pUL16 77 and pUL21 19,78 . The reduced extent of nuclear egress at 16 hpi for the ΔVP16 virus ( Fig.…”

The Herpes Simplex Virus pUL16 and pUL21 Proteins Prevent Capsids from Docking at Nuclear Pore Complexes

“…However, the NPC dilation hypothesis is difficult to reconcile with the observations that NPC gating function appears unperturbed in HSV infected cells [ 69 ]. Moreover, NPC dilation as a means of trafficking capsids from the nucleus to the cytoplasm is incompatible with the observation that DNA-containing capsids, rather than abundant nuclear capsids that lack genomes, are preferentially translocated from the nucleus to the cytoplasm [ 64 , 72 ]. We did not notice any differences in nuclear pore dimensions between WT, Δ16, Δ21, or Δ21/Δ16 infected cells that could explain the differential recruitment of cytoplasmic nucleocapsids to NPCs ( Fig 3D ).…”

“…African green monkey kidney cells (Vero), porcine kidney cells (PK15), HeLa cells, life-extended human foreskin fibroblasts (T12) human keratinocytes (HaCaT) and HaCaT cells stably expressing pUL21 or pUL16 (HaCaT21 and HaCaT16, respectively) [ 38 , 44 ] and life-extended primary human fibroblasts (T12), a gift from Dr. W. A. Bresnahan (University of Minnesota) [ 73 ], were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a 5% CO 2 environment. HSV-1 KOS mutants deficient in pUL21 (Δ21), pUL16 (Δ16), or pUL21 and pUL16 (Δ21/Δ16) have been described previously [ 38 , 42 , 64 ]. HSV-2 186 mutants deficient in pUL21 (Δ21), pUL16 (Δ16) have been described previously [ 34 , 43 ].…”

“…All pUL21 deficient and pUL16 deficient viruses were propagated in HaCaT21 and HaCaT16 cells, respectively. The HSV-1 KOS Δ21/Δ16 virus was propagated on a 1:1 mixture of HaCaT21 and HaCaT16 cell monolayers as previously described [ 64 ]. 5-ethynyl-2’-deoxycytidine (EdC) was incorporated into the genome of HSV-2 186 WT virus as previously described [ 64 ] to produce HSV-2 186 WT EdC virus stocks.…”

Disruption of Herpes Simplex Virus Type 2 pUL21 Phosphorylation Impairs Secondary Envelopment of Cytoplasmic Nucleocapsids

“…The capsid-associated tegument component pUL21 is a multifunctional protein unique to the alphaherpesvirus subfamily of the Herpesviridae . Known functions of pUL21 include facilitating transport of capsids along microtubules (1–3), promoting secondary envelopment of capsids (4), regulating nuclear egress of capsids (5, 6), directing protein phosphatase 1 alpha (PP1α) to specific substrates for dephosphorylation (7, 8), triggering autophagic degradation of cyclic GMP-AMP synthase (cGAS) (9), enabling retention of viral genomes within capsids (10) and, most recently, preventing capsids from docking to nuclear pores after nuclear egress (11). With so many important roles played by pUL21 during viral infection, it is not surprising that viruses deficient in pUL21 display a small plaque phenotype and reduced viral replication in comparison to wild type (WT) virus (1, 4, 8, 12–15).…”