Characterization of a Herpes Simplex Virus 1 (HSV-1) Chimera in Which the Us3 Protein Kinase Gene Is Replaced with the HSV-2 Us3 Gene

Abstract

Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) play important roles in viral replication and pathogenicity. To investigate type-specific differences between HSV-1 Us3 and HSV-2 Us3 in cells infected by viruses with all the same viral gene products except for their Us3 kinases, we constructed and characterized a recombinant HSV-1 in which its Us3 gene was replaced with the HSV-2 Us3 gene. Replacement of HSV-1 Us3 with HSV-2 Us3 had no apparent effect on viral growth in cell cultures or on the range of proteins phosphorylated by Us3. HSV-2 Us3 efficiently compensated for HSV-1 Us3 functions, including blocking apoptosis, controlling infected cell morphology, and downregulating cell surface expression of viral envelope glycoprotein B. In contrast, replacement of HSV-1 Us3 by HSV-2 Us3 changed the phosphorylation status of UL31 and UL34, which are critical viral regulators of nuclear egress. It also caused aberrant localization of these viral proteins and aberrant accumulation of primary enveloped virions in membranous vesicle structures adjacent to the nuclear membrane, and it reduced viral cell-cell spread in cell cultures and pathogenesis in mice. These results clearly demonstrated biological differences between HSV-1 Us3 and HSV-2 Us3, especially in regulation of viral nuclear egress and phosphorylation of viral regulators critical for this process. Our study also suggested that the regulatory role(s) of HSV-1 Us3, which was not carried out by HSV-2 Us3, was important for HSV-1 cell-cell spread and pathogenesis in vivo. IMPORTANCEA previous study comparing the phenotypes of HSV-1 and HSV-2 suggested that the HSV-2 Us3 kinase lacked some of the functions of HSV-1 Us3 kinase. The difference between HSV-1 and HSV-2 Us3 kinases appeared to be due to the fact that some Us3 phosphorylation sites in HSV-1 proteins are not conserved in the corresponding HSV-2 proteins. Therefore, we generated recombinant HSV-1 strains YK781 (Us3-chimera) with HSV-2 Us3 and its repaired virus YK783 (Us3-repair) with HSV-1 Us3, to compare the activities of HSV-1 Us3 and HSV-2 Us3 in cells infected by viruses with the same HSV-1 gene products except for their Us3 kinases. We report here that some processes in viral nuclear egress and pathogenesis in vivo that have been attributed to HSV-1 Us3 could not be carried out by HSV-2 Us3. Therefore, our study clarified the biological differences between HSV-1 Us3 and HSV-2 Us3, which may be relevant to viral pathogenesis in vivo.

Roles of the Phosphorylation of Herpes Simplex Virus 1 UL51 at a Specific Site in Viral Replication and Pathogenicity

“…These membranous invagination structures were present at a lower but consistent frequency in HaCaT cells infected with wild-type HSV-1(F), YK633 (UL51-S184A/D-repair), or YK639 (ΔUL51-repair) ( Fig. 7), as reported previously (45). The quantification of virus particles at different morphogenetic stages showed that 24.4% and 27.8% of enveloped virions in HaCaT cells infected with YK631 (UL51-S184A) or YK638 (ΔUL51), respectively, were present in the perinuclear space and invagination structures.…”

“…Although the UL34 and UL31 proteins were colocalized along the nuclear rim in the majority of HaCaT cells infected with wild-type HSV-1(F), YK633 (UL51-S184A/D-repair), or YK639 (ΔUL51-repair) (69.9% to 77.2%), some UL34 and UL31 proteins were colocalized in punctate structures adjacent to the nuclear rim (22.8% to 30.1%) ( Fig. 9), as reported previously (45). In contrast, in HaCaT cells infected with YK631 (UL51-S184A) or YK638 (ΔUL51), UL31 and UL34 were colocalized in punctate structures adjacent to the nuclear rim in the majority of cells (76.2% or 80.5%, respectively) and were colocalized along the nuclear rim in a smaller percentage of cells (Fig.…”

“…Our data showed that null and S184A mutations in UL51 induced membranous invaginations containing primary enveloped virions adjacent to the nuclear membrane and in the perinuclear space. This led us to investigate the effects of these mutations on the intracellular localization of UL34 and UL31, which are critical for the nuclear egress of HSV-1 nucleocapsids (3-5, 46-49), because the phenotypes observed above were reported to be linked to the aberrant localization of UL34 and UL31 (38,45,47,48,50). To this end, HaCaT cells were infected with wild-type HSV-1(F), YK631 (UL51-S184A), YK632 (UL51-S184D), YK633 (UL51-S184A/D-repair), YK634 (UL51-T190A), YK636 (UL51-S226T228S235/AAA), YK638 (ΔUL51), or YK639 (ΔUL51-repair) at an MOI of 5 for 18 h, and the localization of UL34 and UL31 in these infected cells was analyzed by confocal microscopy.…”

Differentiating the Roles of UL16, UL21 and Us3 in the Nuclear Egress of Herpes Simplex Virus Capsids

“…By contrast, the strains utilized by Morimoto and colleagues express catalytically inactive forms of pUs3 raising the possibility that pUs3 is important for HSV-2 nuclear egress, but its kinase activity is not (29). Interestingly, replacement of HSV-1 Us3 with HSV-2 Us3 resulted in a chimeric strain that displayed nuclear egress deficiencies (32). These findings indicated that HSV-2 Us3 could not complement the loss of HSV-1 Us3 and further supported the idea that HSV-2 pUs3 does not function in nuclear egress.…”

Roles of Us8A and Its Phosphorylation Mediated by Us3 in Herpes Simplex Virus 1 Pathogenesis

“…In agreement with this, it has been reported that HSV-1 Us3 and HSV-2 Us3 differ in their contributions to viral neuroinvasiveness in mice (26,58). Recently, it has been reported that the replacement of HSV-1 Us3 with HSV-2 Us3 compensated for the phosphorylation of HSV-1 gB Thr-887, which is not conserved in HSV-2 gB, leading to the effect of its phosphorylation and the downregulation of the cell surface expression of gB in infected cells (59). These observations suggest that phosphorylation sites in viral proteins may have evolved to enable viral kinases to acquire new functions in viral protein regulation.…”