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

After entry into cells, herpes simplex virus (HSV) nucleocapsids dock at nuclear pore complexes (NPCs) through which viral genomes are released into the nucleoplasm where viral gene expression, genome replication, and early steps in virion assembly take place. After their assembly, nucleocapsids are translocated to the cytoplasm for final virion maturation. Nascent cytoplasmic nucleocapsids are prevented from binding to NPCs and delivering their genomes to the nucleus from which they emerged, but how this is accomplished is not understood. Here we report that HSV pUL16 and pUL21 deletion mutants accumulate empty capsids at the cytoplasmic face of NPCs late in infection. Additionally, prior expression of pUL16 and pUL21 prevented incoming nucleocapsids from docking at NPCs, delivering their genomes to the nucleus and initiating viral gene expression. Both pUL16 and pUL21 localized to the nuclear envelope, placing them in an appropriate location to interfere with nucleocapsid/NPC interactions.

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

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.