Herpes Simplex Virus 1 VP22 Regulates Translocation of Multiple Viral and Cellular Proteins and Promotes Neurovirulence

Numerous studies have focused on the regulatory functions of ICP27, an immediate-early (IE) protein of herpes simplex virus 1 (HSV-1). However, its homolog in HSV-2, termed ICP27t2, has been little studied. Here, we used two different approaches to functionally compare ICP27t2 and ICP27. In transfection-based assays, ICP27t2 closely resembled ICP27 in its capacity to enhance HSV-1 late gene expression, suppress the splicing of a viral intron, and complement the growth of an HSV-1 ICP27 null mutant. To study ICP27t2 in the context of viral infection, we engineered K2F1, an HSV-1 mutant that encodes ICP27t2 in place of ICP27. In Vero cells, K2F1 replicated with wild-type (WT) kinetics and yields, expressed delayed-early and late proteins normally, and was fully capable of activating several cellular signal transduction pathways that are ICP27 dependent. Thus, we conclude that ICP27t2 and ICP27 are functionally very similar and that ICP27t2 can mediate all ICP27 activities that are required for HSV-1 replication in cell culture. Surprisingly, however, we found that K2F1 forms plaques that are morphologically different from those of WT HSV-1. Investigation of this trait demonstrated that it results from the decreased release of progeny virions into the culture medium. This appears to be due to a reduction in the detachment of K2F1 progeny from the extracellular surface of the infected cell. We identified two HSV-1 ICP27 amino-terminal deletion mutants with a similar release defect. Together, these results demonstrate that ICP27 plays a heretofore-unappreciated role in modulating the efficiency of progeny virion release. IMPORTANCEICP27 is an essential, multifunctional regulatory protein that has a number of critical roles in the HSV-1 life cycle. Although ICP27 homologs are encoded by all known members of the Herpesviridae, previous work with several of these homologs has shown that they cannot substitute for ICP27 in the context of HSV-1-infected cells. Here, we identify ICP27t2 as the first homolog that can efficiently replace ICP27 in HSV-1 infection. Unexpectedly, our results also reveal that the sequence of the ICP27 gene can affect the release of HSV-1 progeny virions from the infected cell. Thus, our comparative study has revealed a novel function for ICP27 in the regulation of virus release. Herpes simplex virus 1 (HSV-1), a human alphaherpesvirus, is widespread in the human population, with greater than 57% of U.S. adults seropositive (1). The interaction of HSV-1 with the human host has been well studied (reviewed in reference 2). The virus is transmitted to new hosts via body secretions such as saliva, with the primary site of infection often being the oral mucosa. Following replication in epithelial cells, HSV-1 infects sensory neurons, where it establishes a lifelong latent infection in which the viral genome persists in the trigeminal ganglion as a semiquiescent episome. Periodically, the virus reactivates and returns to the oral cavity, where it can cause lesions (cold sores) and be transmitte...

Section: Discussionsupporting

confidence: 69%

Functional Comparison of Herpes Simplex Virus 1 (HSV-1) and HSV-2 ICP27 Homologs Reveals a Role for ICP27 in Virion Release

Park

Lalli

Sedlackova-Slavikova

et al. 2015

“…The more striking feature in the present study was that the phosphorylation, as well as the enzymatic activity, of vdUTPase played no role in viral replication and pathogenicity in the eyes and vaginas of mice following ocular and vaginal inoculation, respectively. To our knowledge, HSV proteins or their domains critical for viral replication and virulence in the CNS of mice following intracranial inoculation are, in almost all cases, also involved in pathogenicity in peripheral sites, such as eyes and vaginas, following peripheral (ocular and vaginal) inoculation (7,10,11,15,27,41,(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59). Therefore, this phosphorylation reaction, which promoted the vdUTPase activity, is a unique indication of a specific mechanism involved in HSV-1 replication and virulence in and replication in mice.…”

Section: Fig 5 Effect Of Mutations In Vdutpase or The Kinase-dead Mutmentioning

confidence: 99%

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

Shindo

Maruzuru

Kawaguchi

2014

Self Cite

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. IMPORTANCEHerpes 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.

“…Commercial mouse monoclonal antibodies to Myc (PL14; MBL), ␣-tubulin (DM1A; Sigma), ICP0 (1112; Goodwin Institute), ICP4 (58S; ATCC), VP5 (3B6; Virusys), gB (H1817; Virusys), gD (DL6; Danta Cruz Biotechnology), gH (52-S; ATCC), gC (H1A022; Virusys), gG (7F5; Virusys), and gE (9H3; Abcam) and commercial rabbit polyclonal antibodies to VP23 (CAC-CT-HSV-UL18; CosmoBio) and UL11 (CAC-CT-HSV-UL11; CosmoBio) were used. Rabbit polyclonal antibodies to UL51, UL14, UL7, UL31, UL34, VP26, UL41, UL46, VP13/ 14, VP16, VP22, UL50, Us3, and Us9, a mouse monoclonal antibody to UL13, and a mouse polyclonal antibody to gN were described previously (7,26,35,(38)(39)(40)(41)(42). Rabbit polyclonal antibody to gM (43) was kindly provided by J. Baines.…”

Section: Cells and Virusesmentioning

confidence: 99%

The Interaction between Herpes Simplex Virus 1 Tegument Proteins UL51 and UL14 and Its Role in Virion Morphogenesis

Oda

Arii

Koyanagi

et al. 2016

Self Cite

To investigate the molecular mechanism(s) by which herpes simplex virus 1 (HSV-1) tegument protein UL51 promotes viral replication, we screened for viral proteins that interact with UL51 in infected cells. Affinity purification of tagged UL51 in HSV-1-infected Vero cells was coupled with immunoblotting of the purified UL51 complexes with various antibodies to HSV-1 virion proteins. Subsequent analyses revealed that UL51 interacted with another tegument protein, UL14, in infected cells. Mutational analyses of UL51 showed that UL51 amino acid residues Leu-111, Ile-119, and Tyr-123 were required for interaction with UL14 in HSV-1-infected cells. Alanine substitutions of these UL51 amino acid residues reduced viral replication and produced an accumulation of unenveloped and partially enveloped nucleocapsids in the cytoplasm at levels comparable to those of UL51-null, UL14-null, and UL51/UL14 double-null mutations. In addition, although UL51 and UL14 colocalized at juxtanuclear domains in HSV-1-infected cells, the amino acid substitutions in UL51 produced aberrant localization of UL51 and UL14. The effects of these substitutions on localization of UL51 and UL14 were similar to those of the UL51-null and UL14-null mutations on localization of UL14 and UL51, respectively. These results suggested that the interaction between UL51 and UL14 was required for proper localization of these viral proteins in infected cells and that the UL51-UL14 complex regulated final viral envelopment for efficient viral replication. IMPORTANCEHerpesviruses contain a unique virion structure designated the tegument, which is a protein layer between the nucleocapsid and the envelope. HSV-1 has dozens of viral proteins in the tegument, which are thought to facilitate viral envelopment by interacting with other virion components. However, although numerous interactions among virion proteins have been reported, data on how these interactions facilitate viral envelopment is limited. In this study, we have presented data showing that the interaction of HSV-1 tegument proteins UL51 and UL14 promoted viral final envelopment for efficient viral replication. In particular, prevention of this interaction induced aberrant accumulation of partially enveloped capsids in the cytoplasm, suggesting that the UL51-UL14 complex acted in the envelopment process but not in an upstream event, such as transport of capsids to the site for envelopment. This is the first report showing that an interaction between HSV-1 tegument proteins directly regulated final virion envelopment. Herpesviruses have a unique virion structure designated the tegument, which is a proteinaceous layer consisting of a number of different viral proteins and is located between the nucleocapsid and the envelope (1). Herpes simplex virus 1 (HSV-1), classified in the Alphaherpesvirinae subfamily of the Herpesviridae family, is one of the best-studied members in the family and has at least 23 different viral proteins in the tegument (2).In HSV-1-infected cells, packaging of nascent progeny...