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

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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...

“…We recently reported data supporting this hypothesis more directly (22). First, YK751 (vdUTPaseS187A), an HSV-1 mutant with an alanine substitution for vdUTPase Ser-187 (S187A) (Fig.…”

mentioning

confidence: 79%

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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“…Plasmid pSSCH-Luc encoding shRNA against firefly luciferase (Luc) mRNA was described previously (51). Plasmids were constructed for this study as follows.…”

Section: Methodsmentioning

confidence: 99%

Role of Host Cell p32 in Herpes Simplex Virus 1 De-Envelopment during Viral Nuclear Egress

Liu

Kato

Oyama

et al. 2015

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To clarify the function(s) of the herpes simplex virus 1 (HSV-1) major virion structural protein UL47 (also designated VP13/14), we screened cells overexpressing UL47 for UL47-binding cellular proteins. Tandem affinity purification of transiently expressed UL47 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that UL47 interacted with cell protein p32 in HSV-1-infected cells. Unlike in mock-infected cells, p32 accumulated at the nuclear rim in HSV-1-infected cells, and this p32 recruitment to the nuclear rim required UL47. p32 formed a complex(es) with HSV-1 proteins UL31, UL34, Us3, UL47, and/or ICP22 in HSV-1-infected cells. All these HSV-1 proteins were previously reported to be important for HSV-1 nuclear egress, in which nucleocapsids bud through the inner nuclear membrane (primary envelopment) and the enveloped nucleocapsids then fuse with the outer nuclear membrane (de-envelopment). Like viral proteins UL31, UL34, Us3, and UL47, p32 was detected in primary enveloped virions. p32 knockdown reduced viral replication and induced membranous invaginations adjacent to the nuclear rim containing primary enveloped virions and aberrant localization of UL31 and UL34 in punctate structures at the nuclear rim. These effects of p32 knockdown were reduced in the absence of UL47. Therefore, the effects of p32 knockdown in HSV-1 nuclear egress were similar to those of the previously reported mutation(s) in HSV-1 regulatory proteins for HSV-1 de-envelopment during viral nuclear egress. Collectively, these results suggested that p32 regulated HSV-1 de-envelopment and replication in a UL47-dependent manner. IMPORTANCEIn this study, we have obtained data suggesting that (i) the HSV-1 major virion structural protein UL47 interacted with host cell protein p32 and mediated the recruitment of p32 to the nuclear rim in HSV-1-infected cells; (ii) p32 was a component of the HSV-1 nuclear egress complex (NEC), whose core components were UL31 and UL34; and (iii) p32 regulated HSV-1 de-envelopment during viral nuclear egress. It has been reported that p32 was a component of human cytomegalovirus NEC and was required for efficient disintegration of nuclear lamina, which has been thought to facilitate HSV-1 primary envelopment during viral nuclear egress. Thus, p32 appeared to be a core component of herpesvirus NECs, like UL31 and UL34 homologs in other herpesviruses, and to play multiple roles in herpesvirus nuclear egress. H erpesvirus nucleocapsids are too large to traverse the nuclear lamina or cross the inner (INM) and outer (ONM) nuclear membranes through nuclear pores. Therefore, herpesviruses appear to have evolved a unique nuclear egress mechanism in which progeny nucleocapsids assembled in the nucleus acquire primary envelopes by budding through the INM into the perinuclear space (primary envelopment), the space between the INM and ONM, and enveloped nucleocapsids then fuse with the ONM to release de-enveloped nucleocapsids into the cytoplasm (de-envelopment) (1, 2). A heterodim...

“…sh-Luc-HEp-2 cells expressing short hairpin RNA (shRNA) against firefly luciferase were described previously (36). U2OS cells (ATCC HTB-96) were purchased from the ATCC and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…pcDNA-HA-RanBP10, an expression plasmid for RanBP10 fused to a hemagglutinin (HA) tag with an influenza virus hemagglutinin epitope, was constructed by amplifying the RanBP10 ORF by PCR from cDNA synthesized from the total RNA of HEp-2 cells using primers 5=-CGGAATTCACCATGTACCCATACGATGT T C C G G A T T A C G C T G G A T C C A C C A T G G C G G C A G C G A C G GCAGAC-3= and 5=-CGGATATCCTAGTGCAAGTAGTCATC AAC-3= and inserting the amplicon into the EcoRI and EcoRV sites of pcDNA3.1 (Invitrogen). Total RNA was isolated, and cDNAs were synthesized from the isolated RNA as described previously (36). To construct pcDNA-RanBP10, in which the HA tag in pcDNA-HARanBP10 was excised, pcDNA-HA-RanBP10 was digested with BamHI, treated with T4 DNA polymerase, and religated.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…Oligonucleotides 5=-TTTGTTACATTGGTTTATAGCATCG CTTCCTGTCACGATGCTATAAACCAATGTAACTTTTTTG-3= and 5=-AATTCAAAAAAGTTACATTGGTTTATAGCATCGTGACAG GAAGCGATGCTATAAACCAATGTAA-3= were annealed and cloned into BbsI and EcoRI sites of pmU6 (37). The BamHI-SalI fragment of the resulting plasmid, containing the U6 promoter and the sequence encoding shRNA against the 3=-UTR of RanBP10 mRNA, was cloned into the BamHI and SalI sites of pSSCH, which is a derivative of the retrovirus vector pMX containing a hygromycin B resistance gene (36), to produce pSSCH-RanBP10. Retrovirus vectors pMXs-HARanBP10 and pMXs-RanBP10, expressing HA-tagged RanBP10 and RanBP10, respectively, were constructed by cloning the EcoRI-NotI fragment of pcDNA-HA-RanBP10 and the BamHI-NotI fragment of pcDNA-RanBP10 into the EcoRI-NotI and BamHI-NotI sites of pMxs-puro (37), respectively.…”

Section: Cells and Virusesmentioning

confidence: 99%

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Cellular Transcriptional Coactivator RanBP10 and Herpes Simplex Virus 1 ICP0 Interact and Synergistically Promote Viral Gene Expression and Replication

Sato

Maruzuru

Oyama

et al. 2016

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To investigate the molecular mechanism(s) by which herpes simplex virus 1 (HSV-1) regulatory protein ICP0 promotes viral gene expression and replication, we screened cells overexpressing ICP0 for ICP0-binding host cell proteins. Tandem affinity purification of transiently expressed ICP0 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that ICP0 interacted with cell protein RanBP10, a known transcriptional coactivator, in HSV-1-infected cells. Knockdown of RanBP10 in infected HEp-2 cells resulted in a phenotype similar to that observed with the ICP0-null mutation, including reduction in viral replication and in the accumulation of viral immediate early (ICP27), early (ICP8), and late (VP16) mRNAs and proteins. In addition, RanBP10 knockdown or the ICP0-null mutation increased the level of histone H3 association with the promoters of these viral genes, which is known to repress transcription. These effects observed in wild-type HSV-1-infected HEp-2 RanBP10 knockdown cells or those observed in ICP0-null mutant virus-infected control HEp-2 cells were remarkably increased in ICP0-null mutant virus-infected HEp-2 RanBP10 knockdown cells. Our results suggested that ICP0 and RanBP10 redundantly and synergistically promoted viral gene expression by regulating chromatin remodeling of the HSV-1 genome for efficient viral replication. IMPORTANCEUpon entry of herpesviruses into a cell, viral gene expression is restricted by heterochromatinization of the viral genome. Therefore, HSV-1 has evolved multiple mechanisms to counteract this epigenetic silencing for efficient viral gene expression and replication. HSV-1 ICP0 is one of the viral proteins involved in counteracting epigenetic silencing. Here, we identified RanBP10 as a novel cellular ICP0-binding protein and showed that RanBP10 and ICP0 appeared to act synergistically to promote viral gene expression and replication by modulating viral chromatin remodeling. Our results provide insight into the mechanisms by which HSV-1 regulates viral chromatin remodeling for efficient viral gene expression and replication. H erpes simplex virus 1 (HSV-1) has more than 80 different genes that fall into three major classes, designated immediate early (IE) or ␣, early (E) or ␤, and late (L) or ␥, which are expressed in a regulated cascade during the HSV-1 lytic infection cycle (1). ICP0, the subject of this study, is an IE protein with a RING finger domain that confers E3 ubiquitin ligase activity, thereby mediating the ubiquitination and proteasome-dependent degradation of target proteins in HSV-1-infected cells (1-4). Based on studies using ICP0-null mutant viruses, ICP0 has been shown to be required for efficient HSV-1 gene expression and replication in cell cultures (3-7). Numerous studies of ICP0 have gradually identified the mechanisms by which ICP0 acts in HSV-1-infected cells as follows. (i) ICP0 induces the disruption of nuclear structures, designated ND10, by degrading promyelocytic leukemia protein (PML) and Sp100, major cellula...