Phosphorylation of a Herpes Simplex Virus 1 dUTPase by a Viral Protein Kinase, Us3, Dictates Viral Pathogenicity in the Central Nervous System but Not at the Periphery

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We recently reported that herpes simplex virus 1 (HSV-1) protein kinase Us3 phosphorylated viral dUTPase (vdUTPase) at serine 187 (Ser-187) to upregulate its enzymatic activity, which promoted HSV-1 replication in human neuroblastoma SK-N-SH cells but not in human carcinoma HEp-2 cells. In the present study, we showed that endogenous cellular dUTPase activity in SK-N-SH cells was significantly lower than that in HEp-2 cells and that overexpression of cellular dUTPase in SK-N-SH cells increased the replication of an HSV-1 mutant with an alanine substitution for Ser-187 (S187A) in vdUTPase to the wild-type level. In addition, we showed that knockdown of cellular dUTPase in HEp-2 cells significantly reduced replication of the mutant vdUTPase (S187A) virus but not that of wild-type HSV-1. Furthermore, the replacement of Ser-187 in vdUTPase with aspartic acid, which mimics constitutive phosphorylation, and overexpression of cellular dUTPase restored viral replication to the wildtype level in cellular dUTPase knockdown HEp-2 cells. These results indicated that sufficient dUTPase activity was required for efficient HSV-1 replication and supported the hypothesis that Us3 phosphorylation of vdUTPase Ser-187 upregulated vdUTPase activity in host cells with low cellular dUTPase activity to produce efficient viral replication.virus. IMPORTANCEIt has long been assumed that dUTPase activity is important for replication of viruses encoding a dUTPase and that the viral dUTPase (vdUTPase) activity was needed if host cell dUTPase activity was not sufficient for efficient viral replication. In the present study, we showed that the S187A mutation in HSV-1 vdUTPase, which impaired its enzymatic activity, reduced viral replication in SK-N-SH cells, which have low endogenous cellular dUTPase activity, and that overexpression of cellular dUTPase restored viral replication to the wild-type level. We also showed that knockdown of cellular dUTPase in HEp-2 cells, which have higher dUTPase activity than do SK-N-SH cells, reduced replication of HSV-1 with the vdUTPase mutation but had no effect on wild-type virus replication. This is the first report, to our knowledge, directly showing that dUTPase activity is critical for efficient viral replication and that vdUTPase compensates for low host cell dUTPase activity to produce efficient viral replication.

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confidence: 88%

Section: Discussionmentioning

confidence: 99%

Phosphorylation of Herpes Simplex Virus 1 dUTPase Upregulated Viral dUTPase Activity To Compensate for Low Cellular dUTPase Activity for Efficient Viral Replication

Hirohata

Arii

Kawaguchi

2014

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“…We recently reported that Us3, an HSV-1-encoded serine/threonine protein kinase, phosphorylated vdUTPase at serine 187 (Ser-187), which upregulated its enzymatic activity in HSV-1-infected cells (15). We also showed that this phosphorylation promoted viral replication in human neuroblastoma SK-N-SH cells and viral replication and virulence in the central nervous system (CNS) of mice (15,16). Thus, regulation of vdUTPase activity by Us3 phosphorylation of vdUTPase Ser-187 appeared to be important for HSV-1 replication and pathogenicity.…”

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confidence: 99%

“…These results suggested that Us3 phosphorylation of vdUTPase Ser-187 upregulated its enzymatic activity and that this upregulation compensated for low cellular dUTPase activity for efficient HSV-1 replication, at least in cell cultures. This also appeared to be the case in vivo, based on our previous report that Us3 phosphorylation of vdUTPase Ser-187 promoted viral replication and virulence in the CNS of mice, in which most cells are not dividing, but played no obvious role in viral replication and pathogenicity in the eyes and vaginas of mice, in which the HSV-1 target epithelial cells are actively dividing (16,23,24). However, experimental data to directly prove that the dUTPase of HSV-1 and other viruses is needed to compensate for insufficient cellular dUTPase activity for viral replication and pathogenicity in some cells in vivo have not been reported thus far.…”

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confidence: 99%

Phosphorylation of Herpes Simplex Virus 1 dUTPase Regulates Viral Virulence and Genome Integrity by Compensating for Low Cellular dUTPase Activity in the Central Nervous System

Arii

Koyanagi

Kawaguchi

2015

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A mutation in herpes simplex virus 1 dUTPase (vdUTPase), which precluded its phosphorylation at Ser-187, decreased viral neurovirulence and increased mutation frequency in progeny virus genomes in the brains of mice where endogenous cellular dUTPase activity was relatively low, and overexpression of cellular dUTPase restored viral neurovirulence and mutation frequency altered by the mutation. Thus, phosphorylation of vdUTPase appeared to regulate viral virulence and genome integrity by compensating for low cellular dUTPase activity in vivo. IMPORTANCEMany DNA viruses encode a homolog of host cell dUTPases, which are known to function in accurate replication of cellular DNA genomes. The viral dUTPase activity has long been assumed to play a role in viral replication by preventing mutations in progeny virus genomes if cellular dUTPase activity was not sufficient. Here, we showed that a mutation in herpes simplex virus 1 dUTPase, which precluded its phosphorylation at Ser-187 and reduced its activity, decreased viral neurovirulence and increased mutation frequency in progeny virus genomes in the brains of mice where endogenous cellular dUTPase activity was relatively low. In contrast, overexpression of cellular dUTPase restored viral neurovirulence and mutation frequency altered by the mutation in the brains of mice. This is the first report, to our knowledge, directly showing that viral dUTPase activity regulates viral genome integrity and pathogenicity by compensating for insufficient cellular dUTPase activity in vivo. Preserving the integrity of their genetic information during genome replication is of vital importance for all organisms. Noncanonical nucleotides are constantly generated during nucleotide metabolism, and their misincorporation into the genome during replication may result in increased mutagenesis and overload of the DNA excision repair system, leading to multiple DNA strand breaks and cell death (1, 2). The most common noncanonical nucleoside triphosphate is dUTP, which is continuously produced in the pyrimidine biosynthesis pathway by phosphorylation of dUDP or deamination of dCTP (1, 3). dUTPase catalyzes dUTP cleavage to dUMP and pyrophosphate, thereby reducing misincorporation of dUTP into the genome (4, 5). In addition, dUTPase also plays a role in providing a substrate for thymidylate synthase, which converts dUMP to TMP, a major biosynthetic pathway for TTP (6-8). Interestingly, a number of viruses, including herpesviruses, poxviruses, adenoviruses, D-type retroviruses, and African swine fever virus (ASFV), encode their own dUTPases, suggesting the importance of dUTPases in the life cycles of these viruses (4, 9-11).In this study, we focused on the dUTPase encoded by herpes simplex virus 1 (HSV-1), one of the best-studied members of the Herpesviridae family, which causes a variety of diseases such as mucocutaneous diseases, keratitis, skin diseases, and encephalitis (12). HSV-1 dUTPase (vdUTPase) is encoded by the UL50 gene and is conserved throughout the Herpesviridae family (13,1...

“…1). Replacement of the phosphorylation site with an acidic amino acid, such as glutamic acid or aspartic acid, is a phosphomimetic mutation (a mutation that mimics the negative charges produced by phosphorylation) (31)(32)(33). We first inquired whether phosphorylation of pUL12 Tyr-371 had an effect on UL12 expression in HSV-1-infected cells.…”

Section: Identification Of Tyr-371 As a Phosphorylation Site In Pul12mentioning

confidence: 99%

The UL12 Protein of Herpes Simplex Virus 1 Is Regulated by Tyrosine Phosphorylation

Fujii

Mugitani

Kashima

et al. 2014

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The herpes simplex virus 1 (HSV-1) UL12 protein (pUL12) is a nuclease that is critical for viral replication in vitro and neurovirulence in vivo. In this study, mass spectrometric analysis of pUL12 and phosphate-affinity SDS-polyacrylamide gel electrophoresis analysis identified tyrosine at pUL12 residue 371 (Tyr-371) as a pUL12 phosphorylation site: Tyr-371 is conserved in pUL12 homologs in herpesviruses in all Herpesviridae subfamilies. Replacement of Tyr-371 with phenylalanine (Y371F) in pUL12 (i) abolished its exonuclease activity in HSV-1-infected Vero, HEL, and A549 cells, (ii) reduced viral replication, cell-cell spread, and pUL12 expression in infected cells in a cell type-dependent manner, (iii) led to aberrant subcellular localization of pUL12 in infected cells in a cell type-dependent manner, and (iv) reduced HSV-1 neurovirulence in mice. The effects of the pUL12 Y371F mutation in cell cultures and mice were similar to those of a nuclease-dead double mutation in pUL12, although the Y371F mutation reduced viral replication severalfold more than the nuclease-dead double mutation in a cell type-and multiplicity-of-infection-dependent manner. Replacement of Tyr-371 with glutamic acid, which mimics constitutive phosphorylation, restored the wild-type phenotype in cell cultures and mice. These results suggested that phosphorylation of pUL12 Tyr-371 was essential for pUL12 to express its nuclease activity in HSV-1-infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice mainly by upregulating pUL12 nuclease activity and, in part, by regulating the subcellular localization and expression of pUL12 in HSV-1-infected cells. IMPORTANCEHerpesviruses encode a considerable number of enzymes for their replication. Like cellular enzymes, the viral enzymes need to be properly regulated in infected cells. Although the functional aspects of herpesvirus enzymes have gradually been clarified, information on how most of these enzymes are regulated in infected cells is lacking. In the present study, we report that the enzymatic activity of the herpes simplex virus 1 alkaline nuclease pUL12 was regulated by phosphorylation of pUL12 Tyr-371 in infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice, mainly by upregulating pUL12 nuclease activity. Interestingly, pUL12 and tyrosine at pUL12 residue 371 appeared to be conserved in all herpesviruses in the family Herpesviridae, raising the possibility that the herpesvirus pUL12 homologs may also be regulated by phosphorylation of the conserved tyrosine residue.