Hayato Yamada

Characterization of the UL16 gene product of herpes simplex virus type 2

SummaryWe have raised rabbit polyclonal antisera against a His-tagged herpes simplex virus type 1 (HSV-1) UL16 fusion protein, one of which very specifically reacted with 40 kDa and 41 kDa proteins in the lysates of HSV-1 and HSV-2-infected cells, respectively. Since its reactivity to the 41 kDa protein was clearly eliminated by pre-adsorption with E. coli lysates expressing the UL16 fusion protein, the antiserum was used to characterize the UL16 products of HSV-2. The HSV-2 UL16 protein was produced at the late phase of infection in a manner highly dependent on viral DNA synthesis and was distributed in both the nuclei and the cytoplasma of infected cells. In immunofluorescence studies, the UL16-specific fluorescence in the nuclei was shown to be detected as small discrete granules. On the other hand, the cytoplasmic fluorescence was diffusely distributed around the nucleus at 8 h postinfection but, at later times of infection, it was mainly detected as a mass at a perinuclear region. The analysis on its association with capsids has revealed that the UL16 protein copurified with C capsids but not B and A capsids, and that the association with C capsids was not tight. Moreover, our experiments have shown that a detectable level of the UL16 protein was not associated with extracellular virions, and that the partially purified UL16 proteins had a DNA-binding activity. These observations are consistent with the hypothesis that the UL16 protein plays a role in capsid maturation including DNA packaging/cleavage. We have also determined the complete nucleotide sequence of the HSV-2 UL16 gene and found that a nonstandard initiation codon may be used for its translation.

Intracellular localization of the UL31 protein of herpes simplex virus type 2

Summary. We studied intracellular localization of the UL31 protein of herpes simplex virus type 2 (HSV-2) in infected and transfected cells. The UL31 protein localized diffusely throughout the nucleus in infected Vero cells and the distribution patterns of the UL31 protein appeared to be different from those of either replication protein ICP8 or capsid protein ICP35. In transfected Vero cells it localized diffusely throughout the nucleus except the nucleolus at early times after transfection. At very low efficiency, it accumulated in the nucleolus. At intermediate times after transfection, the UL31 protein showed punctate staining in the nucleus. These punctate forms fused and became larger. At later times after transfection, granular forms further fused and a nuclear diffuse pattern virtually disappeared. We also constructed five N and C terminal deletion mutants of the UL31 protein for transfection assays and showed that the region containing amino acids 44 to 110 was important for nuclear and nucleolar localization. Moreover, green fluorescent protein (GFP)-targeting experiments showed that the UL31 protein was able to transport nonnuclear GFP to the nucleus and nucleolus as a fusion protein.

Baculovirus genes modulating intracellular innate antiviral immunity of lepidopteran insect cells.

Innate immunity is essential for insects to survive infectious pathogens. In baculovirus-infected lepidopteran cells, apoptosis and global protein synthesis shutdown are major mechanisms of intracellular innate immunity that inhibit viral replication. In contrast, baculoviruses have evolved diverse genes and mechanisms to counter the antiviral immunity activated in infected cells. In this review, we summarize the current knowledge of the cellular antiviral pathways and the baculovirus genes that modulate antiviral immunity. The studies highlighted illustrate a high degree of diversity in both the cellular responses against viral infections and viral responses against intracellular antiviral immunity, providing an important basis of further studies in this field.

Baculovirus IAP1 induces caspase-dependent apoptosis in insect cells

Baculoviruses encode inhibitors of apoptosis (IAPs), which are classified into five groups, IAP1-5, based on their sequence homology. Most of the baculovirus IAPs with anti-apoptotic functions belong to the IAP3 group, with certain exceptions. The functional roles of IAPs from other groups during virus infection have not been well established. We have previously shown that Hyphantria cunea multiple nucleopolyhedrovirus (HycuMNPV) encodes three iap genes, hycu-iap1, hycu-iap2 and hycu-iap3, and that only Hycu-IAP3 has anti-apoptotic activity against actinomycin D-induced apoptosis of Spodoptera frugiperda Sf9 cells. In the present study, we demonstrate that transient expression of Hycu-IAP1 is capable of inducing apoptosis and/or stimulating caspase-3-like protease activity in various lepidopteran and dipteran cell lines. Transient-expression assay analysis also demonstrates that not only Hycu-IAP1 but also IAP1s from Autographa californica MNPV, Bombyx mori NPV and Orgyia pseudotsugata MNPV (OpMNPV) are capable of inducing apoptosis, and that apoptosis induced by Hycu-IAP1 is precluded by the functional anti-apoptotic baculovirus protein Hycu-IAP3. In HycuMNPV-infected Spilosoma imparilis (SpIm) cells and OpMNPV-infected Ld652Y cells, caspase-3-like protease activity is markedly stimulated during the late stages of infection, and the caspase-3-like protease activity stimulated in HycuMNPV-infected SpIm cells is repressed by RNA interference-mediated silencing of hycu-iap1. In addition, initiator caspase Bm-Dronc, the B. mori homologue of Dronc, is cleaved upon transfection of BM-N cells with a plasmid expressing Hycu-IAP1. These results provide the first evidence that baculovirus IAP1s act to induce caspase-dependent apoptosis, possibly by replacing the cellular IAP1 that prevents Dronc activation.

Cloning and characterization of a dronc homologue in the silkworm, Bombyx mori

We cloned and characterized a novel Bombyx mori homologue (bm-dronc) of Drosophila melanogaster dronc (dm-dronc), which could encode a polypeptide of 438 amino acid residues. Bm-Dronc shares relatively low amino acid sequence identities of 25% and 26% with Dm-Dronc and Aedes aegypti Dronc (Aa-Dronc), respectively. Bm-Dronc has the sequence QACRG surrounding the catalytic site (C), which is consistent with the QAC(R/Q/G)(G/E) consensus sequence in most caspases but distinct from the sequences PFCRG and SICRG of Dm-Dronc and Aa-Dronc, respectively. Bm-Dronc possesses a long N-terminal prodomain containing a caspase recruitment domain (CARD), a p20 domain and a p10 domain, exhibiting cleavage activities on synthetic substrates Ac-VDVAD-AMC, Ac-IETD-AMC and Ac-LEHD-AMC, which are preferred by human initiator caspases-2, -8 and -9, respectively. Bm-Dronc transiently expressed in insect cells and Escherichia coli cells underwent spontaneous cleavage and caused apoptosis and stimulation of caspase-3-like protease activity in various lepidopteran cell lines, but not in the dipteran cell line D. melanogaster S2. The apoptosis and the stimulation of caspase-3-like protease activity induced by Bm-Dronc overexpression were abrogated upon transfection with either a double-stranded RNA against bm-dronc or a plasmid expressing functional anti-apoptotic protein Hycu-IAP3 encoded by the baculovirus Hyphantria cunea multiple nucleopolyhedrovirus (MNPV). Apoptosis induction in BM-N cells by infection with a p35-defective Autographa californica MNPV or exposure to actinomycin D and UV promoted the cleavage of Bm-Dronc. These results indicate that Bm-Dronc serves as the initiator caspase responsible for the induction of caspase-dependent apoptosis.

IDENTIFICATION AND CHARACTERIZATION OF THE UL14 GENE PRODUCT OF HERPES SIMPLEX VIRUS TYPE 2

The UL14 gene of herpes simplex virus type 2 (HSV-2) is predicted to encode a 219 amino acid protein with a molecular mass of 23 kDa. In this study, the HSV-2 UL14 gene product has been identified by using a rabbit polyclonal antiserum raised against a recombinant 6 x His-UL14 fusion protein expressed in E. coli. The antiserum reacted specifically with 34, 33 and 28 kDa proteins in HSV-2-infected cell lysates and also with a 34 kDa protein produced by in vitro transcription and translation reactions, suggesting that the 34 kDa protein is the primary translation product of the UL14 gene. The protein was synthesized at late times post-infection (p.i.) and was not detectable in the presence of the viral DNA synthesis inhibitor acycloguanosine. Indirect immunofluorescence studies localized the UL14 protein both to the nucleus and to perinuclear regions of the cytoplasm, and the nuclear UL14 protein was found to co-localize with the scaffolding protein ICP35 at 9 h p.i. However, the protein accumulated in a perinuclear region of the cytoplasm at 12 h p.i., while most of the ICP35 protein localized within assemblons in the nucleus. Although no detectable UL14 protein was associated with intracellular capsids isolated in the presence of 0.5 M NaCl, it was detected in purified virions. Furthermore, the UL14 protein expressed alone was detected both in the nucleus and in the cytoplasm at 24 h after transfection, but was mainly localized to the cytoplasm at later times.

CHARACTERIZATION OF THE UL55 GENE PRODUCT OF HERPES SIMPLEX VIRUS TYPE 2

We have identified the herpes simplex virus type 2 (HSV-2) UL55 gene product using a rabbit polyclonal antiserum raised against a recombinant 6 x His-UL55 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with a 23 kDa protein in HSV-2 186-infected cell lysates. The protein was not detectable in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies localized the UL55 protein within and at the periphery of the nucleus as discrete granules at late times post-infection, and nuclear fractionation studies showed that the protein was associated with the nuclear matrix of infected cells. Moreover, these discrete regions containing the UL55 protein were found to be adjacent to compartments, designated assemblons, containing the capsid protein ICP35. However, the UL55 protein was not detected in purified virions. These results suggest that the UL55 protein of HSV-2 may play an accessory role in virion assembly or maturation.

Overexpression, purification and helix-destabilizing properties of Epstein-Barr virus ssDNA-binding protein.

The Epstein-Barr virus (EBV) ssDNA-binding protein (SSB) encoded by the BALF2 gene is one of the essential replication proteins in the lytic phase of EBV DNA replication. In order to obtain the amount of EBV SSB required for characterization, a recombinant baculovirus containing the complete sequence of the BALF2 open reading frame under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 130 kDa, recognized by anti-BALF2 protein-specific polyclonal antibody. The overexpressed EBV SSB was purified homogeneously from the cytosolic fraction of the recombinant virus-infected cells. The purified protein displaced short DNA strands from their complementary sequences in the single-stranded form of M13. The helix-destabilizing activity was neutralized by the anti-BALF2 protein-specific antibody. Maximum unwinding occurred at EBV SSB concentrations exceeding saturation level of the DNA substrate. The DNA unwinding reaction mediated by the EBV SSB was highly cooperative and extremely rapid. The reaction displayed no directionality and required neither ATP nor MgCl2, two essential cofactors for DNA helicase activity. The helix-destabilizing property of the EBV SSB may function to melt out secondary structures on the ssDNA template, thereby facilitating the movement of the EBV DNA polymerase.

Identification of a Novel Apoptosis Suppressor Gene from the Baculovirus Lymantria dispar Multicapsid Nucleopolyhedrovirus

ABSTRACT Ld652Y cells from Lymantria dispar readily undergo apoptosis upon infection with a variety of nucleopolyhedroviruses (NPVs), while L. dispar multicapsid NPV (LdMNPV) infection of Ld652Y cells results in the production of a high titer of progeny viruses. Here, we identify a novel LdMNPV apoptosis suppressor gene, apsup, which functions to suppress apoptosis induced in Ld652Y cells by infection with vAcΔp35, a p35-defective recombinant Autographa californica MNPV. apsup also suppresses apoptosis of Ld652Y cells induced by actinomycin D and UV exposure. Apsup is expressed in LdMNPV-infected Ld652Y cells late in infection, and RNA interference-mediated apsup ablation induces apoptosis of LdMNPV-infected Ld652Y cells.

Subcellular localization of the US3 protein kinase of herpes simplex virus type 2

SummaryOne supposes that herpes simplex virus US3 gene product possessing serine/threonine protein kinase activity is a cytoplasmic enzyme. To determine its subcellular localization during viral replication we prepared an antiserum to a synthetic oligopeptide corresponding to the N-terminal region of the US3 protein of HSV type 2 strain 186. The US3 protein first appeared in the cytoplasm of infected cell at 4 h postinfection but strong fluorescence was detected in the nuclei at 8 h postinfection. At 12 h postinfection fluorescence was mainly detected in the cytoplasm, again. Further, the US3 protein expressed alone was widely distributed throughout the cell, indicating that the US3 protein by itself can be localized in the nuclei even in the absence of any other viral proteins. These observations suggest that the HSV-2 US3 protein kinase may function not only in the cytoplasm but also in the nuclei.