pUL21 regulation of pUs3 kinase activity influences the nature of nuclear envelope deformation by the HSV-2 nuclear egress complex

Abstract

It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.

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

“…The capsid-associated tegument protein pUL21 is one of several viral proteins that function in nuclear egress and is conserved amongst the alphaherpesvirus subfamily [29][30][31][32]. pUL21 forms a tripartite complex in the mature HSV virion, interacting with tegument protein pUL16, which in turn interacts with pUL11 [33][34][35][36][37].…”

“…During the early stages of infection, pUL21 has been implicated in the trafficking of capsids to nuclei in HSV-1, HSV-2, and pseudorabies virus (PRV) infected cells as well as being required for optimal viral gene expression [31,[38][39][40]. Late in infection, pUL21 regulates nuclear egress, tegument formation, cell-to-cell spread of infection and actives the ceramide transport protein, CERT [29,32,33,35,[41][42][43][44]. Despite significant research into several pUL21 activities, the function pUL21 performs on capsids has yet to be thoroughly investigated.…”

Herpes simplex virus 1 protein pUL21 stimulates cellular ceramide transport by activating CERT

“…It is tempting to speculate that the accelerated replication kinetics of pUL21 V382E HSV-1 arises, at least in part, from an increased rate of nuclear capsid egress owing to increased Cer abundance. This effect would be distinct from the previously demonstrated role of pUL21 in promoting nuclear egress via regulating the phosphorylation of NEC components [18, 20]. Increased Cer abundance could also stimulate secondary envelopment of virus particles at post-Golgi membranes.…”

“…Mutant HSV-1 lacking pUL21 expression exhibits a 10- to 100-fold replication defect and severely impaired cell-to-cell spread, both of which can be at least partially ascribed to the phosphomodulatory role of pUL21 as a protein phosphatase 1 (PP1) adaptor [18]. PP1 is a highly active and abundant cellular phosphatase [19] and pUL21 recruits PP1 to promote dephosphorylation of multiple substrates, including the viral protein pUL31 that is implicated in viral nuclear egress [18, 20] and the cellular protein CERT that regulates sphingomyelin metabolism [18].…”

The Oligomeric Assemblies of Cytomegalovirus Core Nuclear Egress Proteins Are Associated with Host Kinases and Show Sensitivity to Antiviral Kinase Inhibitors

“…Data of the present study indicate that formation of dimers, hexamers and higher oligomers may represent a strong intrinsic assembly property of the viral core NEC that may be significant for later steps of binding activities and regulation. Specifically, the NEC association of protein kinases, such as vCDK/pUL97, CDK1, CDK2, protein kinase C (PKC) and possibly even more, is another important characteristic shared between herpesviral NECs that leads to the phosphorylation-driven regulatory activities of the multicomponent extensions of these complexes [ 8 , 9 , 10 , 12 , 13 , 19 , 36 , 49 ]. Some of these events of site-specific phosphorylation of NEC-associated proteins have been functionally described, such as lamin A/C pSer22 phosphorylation [ 7 , 20 , 38 ], while others remain to be further investigated, such as the phosphorylation of core NEC proteins themselves, p32/gC1qR phosphorylation and the association of a variety of further phosphoproteins [ 4 ].…”