Mechanism of Nuclear Lamina Disruption and the Role of pUS3 in Herpes Simplex Virus 1 Nuclear Egress

Abstract

Herpes simplex virus capsid envelopment at the nuclear membrane is coordinated by nuclear egress complex (NEC) proteins, pUL34 and pUL31, and is accompanied by alteration in the nuclear architecture and local disruption of nuclear lamina. Here, we examined the role of capsid envelopment in the changes of the nuclear architecture by characterizing HSV-1 recombinants that do not form capsids. Typical changes in nuclear architecture and disruption of the lamina were observed in the absence of capsids, suggesting that disruption of the nuclear lamina occurs prior to capsid envelopment. Surprisingly, in the absence of capsid envelopment, lamin A/C becomes concentrated at the nuclear envelope in a pUL34-independent and cell type-specific manner, suggesting that ongoing nuclear egress may be required for the dispersal of lamins observed in wild-type infection. Mutation of virus-encoded protein kinase, pUS3, on a wild-type virus background has been shown to cause accumulation of perinuclear enveloped capsids, formation of NEC aggregates, and exacerbated lamina disruption. We observed that mutation of US3 in the absence of capsids results in identical NEC aggregation and lamina disruption phenotypes, suggesting that they do not result from accumulation of perinuclear virions. TEM analysis revealed that, in the absence of capsids, NEC aggregates correspond to multi-folded nuclear membrane structures, suggesting that pUS3 may control NEC self-association and membrane deformation. To determine the significance of the pUS3 nuclear egress function for virus growth, the replication of single and double UL34 and US3 mutants was measured, showing that the significance of pUS3 nuclear egress function is cell-type specific. Importance The nuclear lamina is an important player in infection by viruses that replicate in the nucleus. Herpesviruses alter the structure of the nuclear lamina to facilitate transport of capsids from the nucleus to the cytoplasm and use both viral and cellular effectors to disrupt the protein-protein interactions that maintain the lamina. Here we explore the role of capsid envelopment and the virus-encoded protein kinase, pUS3, in the disruption of lamina structure. We show that capsid envelopment is not necessary for the lamina disruption, or for US3 mutant phenotypes, including exaggerated lamina disruption, that accompany nuclear egress. These results clarify the mechanisms behind alteration of nuclear lamina structure and support a function for pUS3 in regulating the aggregation state of the nuclear egress machinery.

pUL21 is a viral phosphatase adaptor that promotes herpes simplex virus replication and spread

“…pUL21 may also provide spatial regulation, for example by associating with nuclear capsids [15,21] and thus promoting pUL31 dephosphorylation once these capsids have docked with the NEC to promote capsid primary envelopment [46]. This is consistent with recent data suggesting pUS3 activity controls NEC self-association and resulting nuclear membrane deformation [59]. Importantly, pUL21 also dephosphorylates distinct cellular targets such as CERT that are not phosphorylated by pUS3; as such pUL21 does not just counterbalance pUS3-driven protein phosphorylation but also actively remodels the phosphoproteomic landscape of infected cells.…”

pUL21 is a viral phosphatase adaptor that promotes herpes simplex virus replication and spread

“…pUL21 may also provide spatial regulation, for example by associating with nuclear capsids (de Wind et al, 1992;Takakuwa et al, 2001) and thus promoting pUL31 dephosphorylation once these capsids have docked with the NEC to promote capsid primary envelopment (Mou et al, 2007). This is consistent with recent data suggesting pUS3 activity controls NEC self-association and resulting nuclear membrane deformation (Bahnamiri and Roller, 2021). Importantly, pUL21 also dephosphorylates distinct cellular targets such as CERT that are not phosphorylated by pUS3; as such pUL21 does not just counterbalance pUS3-driven protein phosphorylation but also actively remodels the phosphoproteomic landscape of infected cells.…”

Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering

“…The role of UL31 phosphorylation in nuclear egress has not yet been elucidated fully. Whenever UL31 cannot be phosphorylated, either due to a missing or a catalytically inactive US3 kinase ( 33 , 39 , 41 , 42 ) or due to the substitution of these six serines for alanines, which mimics an unphosphorylated state, budded capsids accumulate in the perinuclear space and the viral titers are reduced ( Fig. 3 ) ( 33 ).…”