Yuhei Maruzuru

Roles of p53 in Herpes Simplex Virus 1 Replication

ABSTRACT p53 is a critical factor in the cellular response to a broad range of stress factors through its ability to regulate various cellular pathways. In this study, tandem affinity purification of transiently expressed herpes simplex virus 1 (HSV-1) regulatory protein ICP22 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that ICP22 interacted with p53 in HSV-1-infected cells. In p53−/− cells, replication of wild-type HSV-1 was reduced compared to that in parental p53+/+ cells, indicating that p53 had a positive effect on HSV-1 replication. In contrast, the levels of viral replication of an ICP22-null mutant virus were similar in both p53−/− and p53+/+ cells. At 2 h postinfection, the level of expression of ICP27, an essential viral regulatory protein, in p53−/− cells infected with wild-type HSV-1 or the ICP22-null mutant virus was lower than in p53+/+ cells. In contrast, at 18 h postinfection, the level of expression of ICP0, a critical viral regulatory protein, in p53−/− cells infected with the ICP22-null mutant virus was higher than in p53+/+ cells, although the levels of ICP0 expression in p53−/− and p53+/+ cells infected with wild-type HSV-1 were almost identical. These results suggested that p53 overall promoted HSV-1 replication and that p53 played both positive and negative roles in HSV-1 replication: upregulating ICP27 expression very early in infection and downregulating ICP0 expression later in infection, which was antagonized by ICP22.

Role of Herpes Simplex Virus 1 Immediate Early Protein ICP22 in Viral Nuclear Egress

ABSTRACT In order to investigate the novel function(s) of the herpes simplex virus 1 (HSV-1) immediate early protein ICP22, we screened for ICP22-binding proteins in HSV-1-infected cells. Our results were as follows. (i) Tandem affinity purification of ICP22 from infected cells, coupled with mass spectrometry-based proteomics and subsequent analyses, demonstrates that ICP22 forms a complex(es) with the HSV-1 proteins UL31, UL34, UL47 (or VP13/14), and/or Us3. All these proteins were previously reported to be important for viral egress through the nuclear membrane. (ii) ICP22 colocalizes with UL31 and UL34 at the nuclear membrane in wild-type HSV-1-infected cells. (iii) The UL31-null mutation prevents the targeting of ICP22 to the nuclear membrane. (iv) The ICP22-null mutation resulted in UL31 and UL34 being mislocalized in the endoplasmic reticulum (in addition to the nuclear membrane) and significantly reduced numbers of primary enveloped virions in the perinuclear space, although capsids accumulated in the nuclei. Collectively, these results suggest that (i) ICP22 interacts with HSV-1 regulators of nuclear egress, including UL31, UL34, UL47, and Us3 in HSV-1-infected cells; (ii) UL31 mediates the recruitment and anchorage of ICP22 at the nuclear membrane; and (iii) ICP22 plays a regulatory role in HSV-1 primary envelopment, probably by interacting with and regulating UL31 and UL34. Here we report a previously unknown function for ICP22 in the regulation of HSV-1 nuclear egress. IMPORTANCE The herpes simplex virus 1 (HSV-1) immediate early protein ICP22 is recognized primarily as a regulator of viral gene expression. In this study, we show that ICP22 interacts with the HSV-1 proteins UL31 and UL34, which play crucial roles at the nuclear membrane in HSV-1 primary envelopment during viral nuclear egress. We also demonstrate that UL31 is required for the targeting of ICP22 to the nuclear membrane and that ICP22 is required for the correct localization of UL31 and/or UL34. Furthermore, we confirm that ICP22 is required for efficient HSV-1 primary envelopment during viral nuclear egress. Thus, we report, for the first time, that ICP22 plays a regulatory role in HSV-1 nuclear egress.

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

ABSTRACT Herpes simplex virus 1 (HSV-1) encodes Us3 protein kinase, which is critical for viral pathogenicity in both mouse peripheral sites (e.g., eyes and vaginas) and in the central nervous systems (CNS) of mice after intracranial and peripheral inoculations, respectively. Whereas some Us3 substrates involved in Us3 pathogenicity in peripheral sites have been reported, those involved in Us3 pathogenicity in the CNS remain to be identified. We recently reported that Us3 phosphorylated HSV-1 dUTPase (vdUTPase) at serine 187 (Ser-187) in infected cells, and this phosphorylation promoted viral replication by regulating optimal enzymatic activity of vdUTPase. In the present study, we show that the replacement of vdUTPase Ser-187 by alanine (S187A) significantly reduced viral replication and virulence in the CNS of mice following intracranial inoculation and that the phosphomimetic substitution at vdUTPase Ser-187 in part restored the wild-type viral replication and virulence. Interestingly, the S187A mutation in vdUTPase had no effect on viral replication and pathogenic effects in the eyes and vaginas of mice after ocular and vaginal inoculation, respectively. Similarly, the enzyme-dead mutation in vdUTPase significantly reduced viral replication and virulence in the CNS of mice after intracranial inoculation, whereas the mutation had no effect on viral replication and pathogenic effects in the eyes and vaginas of mice after ocular and vaginal inoculation, respectively. These observations suggested that vdUTPase was one of the Us3 substrates responsible for Us3 pathogenicity in the CNS and that the CNS-specific virulence of HSV-1 involved strict regulation of vdUTPase activity by Us3 phosphorylation. IMPORTANCE Herpes simplex virus 1 (HSV-1) encodes a viral protein kinase Us3 which is critical for pathogenicity both in peripheral sites and in the central nervous systems (CNS) of mice following peripheral and intracranial inoculations, respectively. Whereas some Us3 substrates involved in Us3 pathogenicity in peripheral sites have been reported, those involved in Us3 pathogenicity in the CNS remain to be identified. Here, we report that Us3 phosphorylation of viral dUTPase (vdUTPase) at serine 187 (Ser-187), which has been shown to promote the vdUTPase activity, appears to be critical for viral virulence in the CNS but not for pathogenic effects in peripheral sites. Since HSV proteins critical for viral virulence in the CNS are, in almost all cases, also involved in viral pathogenicity at peripheral sites, this phosphorylation event is a unique report of a specific mechanism involved in HSV-1 virulence in the CNS.

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

ABSTRACT 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. IMPORTANCE Upon 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.

Herpes Simplex Virus 1 UL34 Protein Regulates the Global Architecture of the Endoplasmic Reticulum in Infected Cells

ABSTRACT Upon herpes simplex virus 1 (HSV-1) infection, the CD98 heavy chain (CD98hc) is redistributed around the nuclear membrane (NM), where it promotes viral de-envelopment during the nuclear egress of nucleocapsids. In this study, we attempted to identify the factor(s) involved in CD98hc accumulation and demonstrated the following: (i) the null mutation of HSV-1 UL34 caused specific dispersion throughout the cytoplasm of CD98hc and the HSV-1 de-envelopment regulators, glycoproteins B and H (gB and gH); (ii) as observed with CD98hc, gB, and gH, wild-type HSV-1 infection caused redistribution of the endoplasmic reticulum (ER) markers calnexin and ERp57 around the NM, whereas the UL34-null mutation caused cytoplasmic dispersion of these markers; (iii) the ER markers colocalized efficiently with CD98hc, gB, and gH in the presence and absence of UL34 in HSV-1-infected cells; (iv) at the ultrastructural level, wild-type HSV-1 infection caused ER compression around the NM, whereas the UL34-null mutation caused cytoplasmic dispersion of the ER; and (v) the UL34-null mutation significantly decreased the colocalization efficiency of lamin protein markers of the NM with CD98hc and gB. Collectively, these results indicate that HSV-1 infection causes redistribution of the ER around the NM, with resulting accumulation of ER-associated CD98hc, gB, and gH around the NM and that UL34 is required for ER redistribution, as well as for efficient recruitment to the NM of the ER-associated de-envelopment factors. Our study suggests that HSV-1 induces remodeling of the global ER architecture for recruitment of regulators mediating viral nuclear egress to the NM. IMPORTANCE The ER is an important cellular organelle that exists as a complex network extending throughout the cytoplasm. Although viruses often remodel the ER to facilitate viral replication, information on the effects of herpesvirus infections on ER morphological integrity is limited. Here, we showed that HSV-1 infection led to compression of the global ER architecture around the NM, resulting in accumulation of ER-associated regulators associated with nuclear egress of HSV-1 nucleocapsids. We also identified HSV-1 UL34 as a viral factor that mediated ER remodeling. Furthermore, we demonstrated that UL34 was required for efficient targeting of these regulators to the NM. To our knowledge, this is the first report showing that a herpesvirus remodels ER global architecture. Our study also provides insight into the mechanism by which the regulators for HSV-1 nuclear egress are recruited to the NM, where this viral event occurs.

Herpes simplex virus-1 evasion of CD8+ T cell accumulation contributes to viral encephalitis

Herpes simplex virus-1 (HSV-1) is the most common cause of sporadic viral encephalitis, which can be lethal or result in severe neurological defects even with antiviral therapy. While HSV-1 causes encephalitis in spite of HSV-1-specific humoral and cellular immunity, the mechanism by which HSV-1 evades the immune system in the central nervous system (CNS) remains unknown. Here we describe a strategy by which HSV-1 avoids immune targeting in the CNS. The HSV-1 UL13 kinase promotes evasion of HSV-1-specific CD8+ T cell accumulation in infection sites by downregulating expression of the CD8+ T cell attractant chemokine CXCL9 in the CNS of infected mice, leading to increased HSV-1 mortality due to encephalitis. Direct injection of CXCL9 into the CNS infection site enhanced HSV-1-specific CD8+ T cell accumulation, leading to marked improvements in the survival of infected mice. This previously uncharacterized strategy for HSV-1 evasion of CD8+ T cell accumulation in the CNS has important implications for understanding the pathogenesis and clinical treatment of HSV-1 encephalitis.

p53 is a Host Cell Regulator during Herpes Simplex Encephalitis

ABSTRACT p53 is a critical host cell factor in the cellular response to a broad range of stress factors. We recently reported that p53 is required for efficient herpes simplex virus 1 (HSV-1) replication in cell culture. However, a defined role for p53 in HSV-1 replication and pathogenesis in vivo remains elusive. In this study, we examined the effects of p53 on HSV-1 infection in vivo using p53-deficient mice. Following intracranial inoculation, p53 knockout reduced viral replication in the brains of mice and led to significantly reduced rates of mortality due to herpes simplex encephalitis. These results suggest that p53 is an important host cell regulator of HSV-1 replication and pathogenesis in the central nervous system (CNS). IMPORTANCE HSV-1 causes sporadic cases of encephalitis, which, even with antiviral therapy, can result in severe neurological defects and even death. Many host cell factors involved in the regulation of CNS HSV-1 infection have been investigated using genetically modified mice. However, most of these factors are immunological regulators and act via immunological pathways in order to restrict CNS HSV-1 infection. They therefore provide limited information on intrinsic host cell regulators that may be involved in the facilitation of CNS HSV-1 infection. Here we demonstrate that a host cell protein, p53, which has generally been considered a host cell restriction factor for various viral infections, is required for efficient HSV-1 replication and pathogenesis in the CNS of mice. This is the first report showing that p53 positively regulates viral replication and pathogenesis in vivo and provides insights into its molecular mechanism, which may suggest novel clinical treatment options for herpes simplex encephalitis.

Herpes Simplex Virus 1 Small Capsomere-Interacting Protein VP26 Regulates Nucleocapsid Maturation

ABSTRACT VP26 is a herpes simplex virus 1 (HSV-1) small capsomere-interacting protein. In this study, we investigated the function of VP26 in HSV-1-infected cells with the following results. (i) The VP26 null mutation significantly impaired incorporation of minor capsid protein UL25 into nucleocapsids (type C capsids) in the nucleus. (ii) The VP26 mutation caused improper localization of UL25 in discrete punctate domains containing multiple capsid proteins (e.g., the VP5 major capsid protein) in the nucleus; these domains corresponded to capsid aggregates. (iii) The VP26 mutation significantly impaired packaging of replicated viral DNA genomes into capsids but had no effect on viral DNA concatemer cleavage. (iv) The VP26 mutation reduced the frequency of type C capsids, which contain viral DNA but not scaffolding proteins, and produced an accumulation of type A capsids, which lack both viral DNA and scaffold proteins, and had no effect on accumulation of type B capsids, which lack viral DNA but retain cleaved scaffold proteins. Collectively, these results indicated that VP26 was required for efficient viral DNA packaging and proper localization of nuclear capsids. The phenotype of the VP26 null mutation was similar to that reported previously of the UL25 null mutation and of UL25 mutations that preclude UL25 binding to capsids. Thus, VP26 appeared to regulate nucleocapsid maturation by promoting incorporation of UL25 into capsids, which is likely to be required for proper capsid nuclear localization. IMPORTANCE HSV-1 VP26 has been reported to be important for viral replication and virulence in cell cultures and/or mouse models. However, little is known about the function of VP26 during HSV-1 replication, in particular, in viral nucleocapsid maturation although HSV-1 nucleocapsids are estimated to contain 900 copies of VP26. In this study, we present data suggesting that VP26 promoted packaging of HSV-1 DNA genomes into capsids by regulating incorporation of capsid protein UL25 into capsids, which was reported to increase stability of the capsid structure. We also showed that VP26 was required for proper localization of capsids in the infected cell nucleus. This is the first report showing that HSV-1 VP26 is a regulator for nucleocapsid maturation.

ESCRT-III mediates budding across the inner nuclear membrane and regulates its integrity

Vesicle-mediated nucleocytoplasmic transport is a nuclear pore-independent mechanism for the nuclear export of macromolecular complexes, but the molecular basis for this transport remains largely unknown. Here we show that endosomal sorting complex required for transport-III (ESCRT-III) is recruited to the inner nuclear membrane (INM) during the nuclear export of herpes simplex virus 1 (HSV-1). Scission during HSV-1 budding through the INM is prevented by depletion of ESCRT-III proteins. Interestingly, in uninfected human cells, the depletion of ESCRT-III proteins induces aberrant INM proliferation. Our results show that HSV-1 expropriates the ESCRT-III machinery in infected cells for scission of the INM to produce vesicles containing progeny virus nucleocapsids. In uninfected cells, ESCRT-III regulates INM integrity by downregulating excess INM.The endosomal sorting complex required for transport-III (ESCRT-III) has been implicated in the packaging of HIV and HSV-1 viruses in the cytoplasm. Here the authors show that ESCRT-III proteins are required for the transport of HSV-1 nucleocapsids from nucleoplasm to cytosol through the nuclear envelope and confirm that the same mechanism is also used for the nucleocytoplasmic transport of RNP in Drosophila cells.