Phosphoregulation of a Conserved Herpesvirus Tegument Protein by a Virally Encoded Protein Kinase in Viral Pathogenicity and Potential Linkage between Its Evolution and Viral Phylogeny

Abstract

Us3 proteins of herpes simplex virus 1 (HSV-1) and HSV-2 are multi-functional serine-threonine protein kinases. Here, we identified an HSV-2 tegument protein UL7 as a novel physiological substrate of HSV-2 Us3. Mutations in HSV-2 UL7, which precluded Us3 phosphorylation of the viral protein, significantly reduced mortality, viral replication in the vagina, and development of vaginal disease in mice following vaginal infection. These results indicated that Us3 phosphorylation of UL7 in HSV-2 was required for efficient viral replication and pathogenicity in vivo. Of note, this phosphorylation was conserved in UL7 of chimpanzee herpesvirus (ChHV), which phylogenically forms a monophyletic group with HSV-2 and the resurrected last common ancestral UL7 for HSV-2 and ChHV. In contrast, the phosphorylation was not conserved in UL7 of HSV-1, which belongs to a sister clade of the monophyletic group, the resurrected last common ancestral UL7 for HSV-1, HSV-2, and ChHV, and UL7s of other members of the genus Simplexvirus that are phylogenically close to these viruses. Thus, evolution of Us3 phosphorylation of UL7 coincided with the phylogeny of simplexviruses. Furthermore, artificially induced Us3 phosphorylation of UL7 in HSV-1 had no effect on viral replication and pathogenicity in mice in contrast to phosphorylation in HSV-2. Our results suggest that HSV-2 and ChHV have acquired and maintained Us3 phospho-regulation of UL7 during their evolution because the phospho-regulation had an impact on viral fitness in vivo, whereas most other simplexviruses have not because the phosphorylation was not necessary for efficient fitness of the viruses in vivo. IMPORTANCE It has been hypothesized that the evolution of protein phospho-regulation drives phenotypic diversity across species of organisms, which impacts fitness during their evolution. However, there is a lack of information regarding linkage between the evolution of viral phospho-regulation and phylogeny of virus species. In this study, we clarified the novel HSV-2 Us3 phospho-regulation of UL7 in infected cells, which is important for viral replication and pathogenicity in vivo. We also showed that evolution of Us3 phospho-regulation of UL7 was linked to phylogeny of viruses that are phylogenically close to HSV-2 and to phosphorylation requirements for the efficient in vivo viral fitness of HSV-2 and HSV-1, which are representative of viruses that have or have not evolved phospho-regulation, respectively. This study reports the first evidence showing that evolution of viral phospho-regulation coincides with phylogeny of virus species and supports the hypothesis regarding the evolution of viral phospho-regulation during viral evolution.

Effects of US3 protein kinase activity on localization of UL31/UL34 protein and nucleocapsids egress of duck plague virus

“…Interestingly, there were many viral proteins observed in our MS analysis ( Table 3 ). In addition to UL47 ( Kato et al, 2011 ), UL12 ( Daikoku et al, 1995 ), UL31 ( Mou et al, 2009 ), UL34 ( Ryckman and Roller, 2004 ), gB ( Kato et al, 2009 ), and UL7 ( Shibazaki et al, 2020 ) which had been found to be phosphorylated substrates of pUS3 in other alphaherpesviruses, other proteins including UL49, US10, UL37, UL54, LORF3, LORF4, and LORF5 have still not been reported to interact with pUS3. Among these proteins, LORF3, LORF4 and LORF5 are unique genes in avian herpesviruses.…”

Regulation of alphaherpesvirus protein via post-translational phosphorylation

“…In addition, some viral proteins, such as VP1/2, VP13/14, VP16 and VP22, are phosphorylated during virion assembly and dephosphorylated after virion maturation, suggesting that phosphorylation and dephosphorylation are mechanisms that regulate HSV-1 dissociation and assembly. In addition to regulating the performance and metabolism of the viruses themselves, the evolution of phosphorylation has driven phenotypic diversity among species, further influencing their adaptation during evolution [ 166 ]. From this perspective, the effect of viral protein phosphorylation on antiviral drugs effectivity is very helpful for treatment development, but not all of the viral protein phosphorylation events during physiological viral activities show a significant regulatory effect; therefore, further study of viruses in host cell protein phosphorylation and their corresponding regulatory functions will lead to a better understanding of the viral infection mechanisms and identification of effective targets for virus prevention and control.…”

“…For example, UL7 is phosphorylated by HSV-2 Us3. Although its phosphorylation exerted no effect on viral replication in cell culture, it was necessary for effective replication and pathogenicity of HSV-2 in vivo [ 166 ]; ICP1/2, also known as VP1/2, is a 270 kDa structural protein located in the HSV-1 tegument that is prone to serine phosphorylation, but the role played by phosphorylated VP1/2 has not been clarified [ 167 ]. The virion host shutoff (VHS) protein encoded by HSV UL41 specifically degrades mRNA, inducing the abrogation of host gene expression.…”

Establishment of a system to quantify wild‐type herpes simplex virus–induced cell–cell fusion reveals a role of N‐glycosylation of HSV‐1 envelope glycoprotein B in cell–cell fusion

“…9,10 Wild-type HSV-1(F), wild-type HSV-2(186), YK695(ΔgB), YK696(ΔgB-repair), YK683(gB-N141Q), and YK684(gB-N141Q-repair) (Figure 1) have also been previously described. 8,11,12…”