Tetsuo Koshizuka

Identification and Characterization of the UL56 Gene Product of Herpes Simplex Virus Type 2

ABSTRACT The UL56 gene product of herpes simplex virus (HSV) has been shown to play an important role in viral pathogenicity. However, the properties and functions of the UL56 protein are little understood. We raised rabbit polyclonal antisera specific for the UL56 protein of HSV type 2 (HSV-2) and examined its expression and properties. The gene product was identified as three polypeptides with apparent molecular masses ranging from 32 to 35 kDa in HSV-2-infected cells, and at least one species was phosphorylated. Studies of their origins showed that the UL56 protein of HSV-2 is also translated from the upstream in-frame methionine codon that is not present in the HSV-1 genome. Synthesis was first detected at 6 h postinfection and was not abolished by the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies revealed that the UL56 protein localized to both the Golgi apparatus and cytoplasmic vesicles in HSV-2-infected and single UL56-expressing cells. Deletion mutant analysis showed that the C-terminal hydrophobic region of the protein was required for association with the cytoplasmic membrane and that the N-terminal proline-rich region was important for its translocation to the Golgi apparatus and cytoplasmic vesicles. Moreover, the results of protease digestion assays and sucrose gradient fractionation strongly suggested that UL56 is a tail-anchored type II membrane protein associated with lipid rafts. We thus hypothesized that the UL56 protein, as a tail-anchored type II membrane protein, may be involved in vesicular trafficking in HSV-2-infected cells.

Herpes simplex virus encodes a virion-associated protein which promotes long cellular processes in over-expressing cells

Background Herpes simplex virus (HSV) possesses a number of accessory genes which are dispensable for replication in cell culture. A previous study showed that the UL21 gene product of HSV type 1 is a virion component that is not necessary for viral replication. The function of the gene product remains unknown.

Subcellular Localization of Herpes Simplex Virus Type 1 UL51 Protein and Role of Palmitoylation in Golgi Apparatus Targeting

ABSTRACT The herpes simplex virus type 1 (HSV-1) UL51 gene products are virion-associated phosphoproteins with apparent molecular masses of 27, 29, and 30 kDa in HSV-1-infected cells. In this study, we have investigated the intracellular localization and distribution of UL51 protein both in infected cells and in transfected cells expressing only UL51. We found that this protein colocalized closely with Golgi marker proteins such as the Golgi-58K protein and GM130 in transfected cells expressing only UL51. However, in infected cells, the UL51 protein localized to the juxtanuclear region but only partially colocalized with the Golgi maker proteins. Mutant protein analysis revealed that the N-terminal 15 amino acid residues of the UL51 protein sufficed for this Golgi localization property. The UL51 protein redistributed on addition of brefeldin A. This was prevented by pretreatment with 2-deoxyglucose and sodium azide, which results in ATP depletion, but not by pretreatment with NaF and AlCl3, which activates heterotrimeric G proteins. Moreover, we found that palmitoylation of the UL51 protein through the N-terminal cysteine at position 9 was necessary for its Golgi localization. Protease digestion analysis suggested that the UL51 protein localized on the cytoplasmic face of the membrane in UL51-transfected cells, while in infected cells it localized mainly to the inside of cytoplasmic vesicles and/or the viral envelope. Transmission immunoelectron microscopy revealed an association of UL51 protein-specific labeling with cytoplasmic virions and also with some membranous structure. We infer from these observations that internalization of UL51 protein into the cytoplasmic vesicle and/or virion may occur in association with viral envelopment in HSV-infected cells.

Herpes simplex virus type 2 US3 blocks apoptosis induced by sorbitol treatment

Previously, we established HEp2 cell lines which express the US3 protein kinase of herpes simplex virus type 2 upon induction with IPTG. Using these cells, we examined whether expression of US3 is sufficient to protect cells from apoptotic cell death induced by sorbitol. Cells expressing US3 showed significantly reduced nuclear fragmentation in the degree that DNA fragmentation and caspase-3 activation were suppressed. It is known that stressors such as osmotic shock and UV irradiation induce the activation of the JNK (c-Jun N-terminal kinase), which can lead to apoptotic cell death. Expression of US3 resulted in the suppression of sorbitol-induced phosphorylation of JNK and MKK4/SEK1, suggesting that the suppression of apoptotic cell death was due to the attenuation of JNK activity.

Herpes Simplex Virus Type 2 UL56 Interacts with the Ubiquitin Ligase Nedd4 and Increases Its Ubiquitination

ABSTRACT The herpes simplex virus UL56 gene is conserved among most members of the Alphaherpesvirinae family and plays a critical role in viral pathogenicity in vivo. The HSV-2 UL56 protein (UL56) is a C-terminally anchored type II membrane protein that is predicted to be inserted into the virion envelope, leaving its N-terminal domain in the tegument. UL56 interacts with KIF1A and UL11. Here we report that UL56 also interacts with the ubiquitin ligase Nedd4 and increases its ubiquitination. Nedd4 was identified as a UL56-interacting protein by a yeast two-hybrid screen. UL56 bound to Nedd4 via its PY motifs. Nedd4 was phosphorylated and degraded in wild-type HSV-2-infected cells but not in cells infected with a UL56-deficient mutant. Ubiquitination assays revealed that UL56 increased ubiquitinated Nedd4, which was actively degraded in infected cells. UL56 also caused a decrease in Nedd4 protein levels and the increased ubiquitination in cotransfected cells. However, UL56 itself was not ubiquitinated, despite its interaction with Nedd4. Based on these findings, we propose that UL56 regulates Nedd4 in HSV-2-infected cells, although deletion of UL56 had no apparent effect on viral growth in vitro.

Identification and characterization of the UL24 gene product of herpes simplex virus type 2.

The UL24 gene of herpes simplex virus type 2 (HSV-2) is predicted to encode a 281 amino acid protein with a molecular mass of 30.5 kDa. In this study, the HSV-2 UL24 gene product has been identified by using a rabbit polyclonal antiserum produced against a recombinant protein containing the full-length UL24 gene product of HSV-2 fused to glutathione-S-transferase. The antiserum reacted specifically with a 32 kDa protein in HSV-2 186-infected Vero cells and with 31 and 32 kDa proteins in UL24-expressing Cos-7 cells. Accumulation of UL24 protein to detectable levels required viral DNA synthesis, indicating that the protein was regulated as a late gene. UL24 protein was found to be associated with purified HSV-2 virions and C capsids. Indirect immunofluorescence analysis demonstrated that the UL24-specific fluorescence was detected in perinuclear regions of the cytoplasm and/or in the nucleus as small discrete granules from 9 h post infection (hpi). Furthermore, the UL24 protein expressed singly was detected predominantly in the nucleus and slightly in the cytoplasm at 24 h after transfection, with branch-like cytoplasmic protruding structures. Strong nucleolus staining was visible in partial cells.

A Single Amino Acid Substitution in the ICP27 Protein of Herpes Simplex Virus Type 1 Is Responsible for Its Resistance to Leptomycin B

ABSTRACT Leptomycin B (LMB) is a specific inhibitor of Crm1-dependent nuclear export of proteins. The replication of herpes simplex virus (HSV) is normally highly sensitive to LMB; a resistant HSV variant, however, was isolated by serial passages of the virus. Analysis of marker transfer and viral DNA sequences revealed that a single amino acid substitution within the ICP27 gene is responsible for conferring this resistance.

The US3 protein kinase of herpes simplex virus attenuates the activation of the c-Jun N-terminal protein kinase signal transduction pathway in infected piriform cortex neurons of C57BL/6 mice

Stereotaxic microinjection of herpes simplex virus (HSV) into the mouse olfactory bulb resulted in infection of neurons of the piriform cortex. Neurons infected with the wildtype HSV showed no evident phosphorylation of c-Jun N-terminal protein kinase (JNK)/c-Jun. In contrast, neurons infected with a US3 gene-disrupted mutant of the L1BR1 virus displayed phosphorylated JNK/c-Jun in a nuclear staining fashion. Induction of neuronal apoptosis by the wildtype HSV was partially suppressed when compared with that of the L1BR1 virus. A US3-rescued isolate of the L1B(-)11 virus behaved as did the wildtype virus. Collectively, the US3 protein kinase of HSV plays a role in attenuating the virus-induced activation of the JNK signal transduction pathway in the central nervous system and may contribute, at least in part, to controlling neuronal apoptosis.

Association of Two Membrane Proteins Encoded by Herpes Simplex Virus Type 2, UL11 and UL56

Herpes simplex virus (HSV) acquires envelope by budding into trans-Golgi network (TGN)-derived vesicles. Previous studies showed that the UL11 gene product enables efficient virion envelopment and export from infected cells and is incorporated into virions as tegument protein. At its N-terminus, UL11 is dually acylated by myristoic and palmitoic acids. Fatty acylation of UL11 provides both membrane binding strength and Golgi-targeting specificity. We show here that UL11 interacts with UL56 protein, a tail-anchored type II membrane protein encoded by HSV, which associated with the Golgi apparatus and cytoplasmic vesicles. We previously showed that UL56 is involved in vesicular transport in infected cells. The UL11–UL56 complex localized to the perinuclear region of the cytoplasm in infected cells. Fatty acylation of UL11 was important for the formation of the UL11–UL56 protein complex. Taken together, our results identify a novel interaction between two HSV proteins facilitated by mutual interactions with Golgi-derived vesicles.

Intercellular trafficking of herpes simplex virus type 2 UL14 deletion mutant proteins

The UL14 gene product of herpes simplex virus is a 32kDa protein expressed late in infection and is a minor component of the virion tegument. We recently showed that the wild-type UL14 protein has heat shock protein (HSP)-like and/or molecular chaperone-like functions. In this study, the intracellular localization of UL14 wild-type and deletion mutant proteins was examined in transfected cells by immunofluorescence. We found that N-terminus deleted but not wild-type/C-terminus deleted mutant proteins showed a significant number of cytoplasmic, multi-cellular stains in transfected Vero cells. The effect was greatly intensified by subjecting cells to heat shock at 43 degrees C, whereas it was obstructed by treatment with the microfilament-disrupting drug cytochalasin D. The staining patterns of UL14 antigen-positive cells after heat shock suggested a cell-to-cell spread of the protein. Although the mechanism is unclear, the phenomenon seems to be an unprecedented type of intercellular trafficking.