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

Herpesviruses are nuclear-replicating viruses that have successfully evolved to evade the immune system of humans, establishing lifelong infections. ICP27 from herpes simplex virus is a multifunctional regulatory protein that is functionally conserved in all known human herpesviruses. It has the potential to interact with an array of cellular proteins, as well as intronless viral RNAs. ICP27 plays an essential role in viral transcription, nuclear export of intronless RNAs, translation of viral transcripts, and virion host shutoff function. It has also been implicated in several signaling pathways and the prevention of apoptosis. Although much is known about its central role in viral replication and infection, very little is known about the structure and mechanistic properties of ICP27 and its homologs. We present the first crystal structure of ICP27 C-terminal domain at a resolution of 2.0 Å. The structure reveals the C-terminal half of ICP27 to have a novel fold consisting of ␣-helices and long loops, along with a unique CHCC-type of zinc-binding motif. The two termini of this domain extend from the central core and hint to possibilities of making interactions. ICP27 essential domain is capable of forming self-dimers as seen in the structure, which is confirmed by analytical ultracentrifugation study. Preliminary in vitro phosphorylation assays reveal that this domain may be regulated by cellular kinases. IMPORTANCE ICP27 is a key regulatory protein of the herpes simplex virus and has functional homologs in all known human herpesviruses.Understanding the structure of this protein is a step ahead in deciphering the mechanism by which the virus thrives. In this study, we present the first structure of the C-terminal domain of ICP27 and describe its novel features. We critically analyze the structure and compare our results to the information available form earlier studies. This structure can act as a guide in future experimental designs and can add to a better understanding of mechanism of ICP27, as well as that of its homologs. Herpes simplex virus (HSV) and other human herpesviruses (HHVs; e.g., Epstein-Barr virus and varicella-zoster virus) have evolved a unique strategy of virion host shutoff, in which they efficiently suppress the host protein synthesis. At the onset of the lytic phase, viral immediate-early (IE) genes are expressed first. These IE proteins are responsible for inducing the expression of viral early and late genes. The key IE protein of HSV, infectious cell protein 27 (ICP27), helps in the host shutoff function (1, 2). This 63-kDa protein consists of 512 amino acids and is involved in multiple functions, such as stalling host pre-RNA processing, exporting intronless viral mRNAs out of the nucleus, shuttling between host nucleus and cytoplasm facilitated by its nuclear export signal (NES) and nuclear localization signal (NLS), and interacting with RNA via its RGG box (3, 4). Most of these functions are conserved across the Herpesviridae family, and ICP27 homologs are present in all kno...

Section: Resultsmentioning

confidence: 99%

Structure of the C-Terminal Domain of the Multifunctional ICP27 Protein from Herpes Simplex Virus 1

Patel

Dahlroth

Rajakannan

et al. 2015

“…We also analyzed plaque sizes in SK-N-SH cell monolayers infected with YK333(EGFP), YK491(⌬VP26/EGFP), YK492 (VP26T111A/EGFP),YK493(VP26⌬/TA-Repair/EGFP),orYK494 (VP26T111D/EGFP) under plaque assay conditions described previously (41). In agreement with the growth properties of these viruses demonstrated in this and previous studies (42), YK491(⌬VP26/EGFP) and YK492(VP26T111A/EGFP) produced plaques similar in size but that were smaller than those observed with YK333(EGFP) and YK493(VP26⌬/TA-Repair/EGFP) (Fig.…”

mentioning

confidence: 99%

Function of the Herpes Simplex Virus 1 Small Capsid Protein VP26 Is Regulated by Phosphorylation at a Specific Site

Kobayashi

Oda

Koyanagi

et al. 2015

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c Replacement of the herpes simplex virus 1 small capsid protein VP26 phosphorylation site Thr-111 with alanine reduced viral replication and neurovirulence to levels observed with the VP26 null mutation. This mutation reduced VP26 expression and mislocalized VP26 and its binding partner, the major capsid protein VP5, in the nucleus. VP5 mislocalization was also observed with the VP26 null mutation. Thus, we postulate that phosphorylation of VP26 at Thr-111 regulates VP26 function in vitro and in vivo. V iral capsids encase and protect viral genomes, and they enable release of viral genomes into newly infected cells (1). In infected cells, viral capsid proteins also perform important additional roles in regulating viral replicative processes, such as viral gene expression, genome replication, trafficking and maturation of virions, and inhibiting host defenses (i.e., immune response and apoptosis), and in modifying host cellular machinery to establish an environment for effective viral replication, i.e., host cellular signaling and cell cycle control (2-6). The fact that the capsid proteins of various viruses are phosphorylated in infected cells is well established (7-13). It has been reported that phosphorylation of viral capsid proteins regulates both subcellular localization and functions including transcriptional regulation, viral uncoating, and nucleic acid chaperone activity (7-13). These observations hold true in the life cycle of various viruses, including human immunodeficiency virus, hepatitis B virus, hepatitis C virus, and severe acute respiratory syndrome virus (7-13).Herpes simplex virus 1 (HSV-1), the subject of this study, is one of the best-studied members of the Herpesviridae family (herpesviruses) (14). All herpesvirus capsids, which contain genomic DNA and are able to mature into infectious virus (C capsids), share common structural features: an icosahedral shape with a diameter of 125 nm and consist of 161 capsomers (150 hexons and 11 pentons), a portal complex which has an axial channel through which viral genome DNA enters and exits the capsid, 320 triplexes which connect each of the capsomers and the portal complex, small capsomere-interacting proteins (SCP), and capsid vertexspecific components (CVSC) (15, 16). In HSV-1, the hexons and pentons consist of 6 and 5 molecules of VP5, respectively; the triplexes consist of one VP19C and two VP23 molecules, the portal complex consists of 12 molecules of UL6, the SCP is VP26 (which interacts with each of the VP5 molecules in the hexon), and the CVSC consists of one molecule of UL17 and one of UL25 (15-21). All of these HSV-1 capsid proteins are conserved in the Herpesviridae family, and all have been reported to be phosphorylated in HSV-1-infected cells (22)(23)(24)(25). However, there is a lack of information on the significance of phosphorylation in HSV-1 capsid proteins, and those of other herpesviruses, in viral replication and pathogenesis.Recently, Bell et al. reported that a large-scale, phosphoproteomic analysis of HSV-1-infected murine ...

“…Immunoblotting, immunofluorescence, and determination of plaque size were performed as described previously (62)(63)(64).…”

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confidence: 99%

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

Shindo

Koyanagi

Sagara

et al. 2016

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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.