Toshiomi Okuno

IDENTIFICATION OF HUMAN HERPESVIRUS-6 AS A CAUSAL AGENT FOR EXANTHEM SUBITUM

A virus was isolated from the peripheral blood lymphocytes of patients with exanthem subitum, cultured successfully in cord blood lymphocytes, and shown to be antigenically related to human herpesvirus-6 (HHV-6). Morphological features, as studied by thin-section electronmicroscopy, resembled those of herpes group viruses. Convalescent-phase serum samples, tested against the new viral antigen and HHV-6 antigen, showed seroconversion. The results strongly suggest that the newly isolated virus is identical or closely related to HHV-6 and the causal agent for exanthem subitum.

Latent human herpesvirus 6 infection of human monocytes/macrophages

Human herpesvirus 6 (HHV-6) DNA was detected in peripheral blood from exanthem subitum patients during the acute and convalescent phases of infection using the polymerase chain reaction. Although DNA could be detected in non-adherent and adherent mononuclear cells during the acute phase, it was detected predominantly in adherent cells during the convalescent phase; furthermore, viral DNA was found in adherent cells of healthy adults. When adherent mononuclear cells were cultured in vitro, virus was found to replicate well in differentiated cells cultured for 7 days in vitro before infection. When cells were cultured for more than 1 month, no detectable antigen and no evidence of virus growth was observed, but viral DNA could be detected. These apparently latently infected monocytes were treated with phorbol ester, after which virus could be recovered from the cultures. Therefore, we have developed an in vitro latency system for HHV-6; our results suggest that HHV-6 may latently infect monocytes in vivo and in vitro and that it may be reactivated in cells by some factors.

Human Herpesvirus 6 Infection In Renal Transplantation

The relationship between renal transplantation and human herpesvirus 6 (HHV-6) infection was studied. All 21 kidney donors examined had antibody to HHV-6 at the time of transplantation. The 21 kidney recipients also had detectable antibody to HHV-6 before transplantation--and, of these, 8 patients showed a significant increase of serum antibody titer against HHV-6 after transplantation. All these 8 recipients suffered severe kidney rejection. Furthermore, virus isolation from peripheral blood lymphocytes of 2 recipients who suffered rejection was attempted, and in both cases HHV-6 was isolated. Biopsy specimens of rejected kidneys of 9 other patients were examined for the presence of HHV-6 antigens, and in 5 of these specimens antigens were detected in the tubular epithelium, as well as in infiltrating histiocytes and lymphocytes. These results suggest that HHV-6 can infect renal tissues and that the infection may be correlated with rejection or with immunosuppressive therapy.

Octamer-Binding Sequence Is a Key Element for the Autoregulation of Kaposi's Sarcoma-Associated Herpesvirus ORF50/Lyta Gene Expression

ABSTRACT The expression of the Kaposis sarcoma-associated herpesvirus (KSHV) open reading frame 50 (ORF50) protein, Lyta (lytic transactivator), marks the switch from latent KSHV infection to the lytic phase. ORF50/Lyta upregulates several target KSHV genes, such as K8 (K-bZip), K9 (vIRF1), and ORF57, finally leading to the production of mature viruses. The auto-upregulation of ORF50/Lyta is thought to be an important mechanism for efficient lytic viral replication. In this study, we focused on this autoregulation and identified the promoter element required for it. An electrophoretic mobility shift assay indicated that the octamer-binding protein 1 (Oct-1) bound to this element. Mutations in the octamer-binding motif resulted in refractoriness of the ORF50/Lyta promoter to transactivation by ORF50/Lyta, and Oct-1 expression enhanced this transactivation. These results suggest that the autoregulation of ORF50/Lyta is mediated by Oct-1.

Activation of latent Kaposi's sarcoma-associated herpesvirus by demethylation of the promoter of the lytic transactivator

Kaposis sarcoma-associated herpesvirus (KSHV) is strongly linked to Kaposis sarcoma, primary effusion lymphomas, and a subset of multicentric Castlemans disease. The mechanism by which this virus establishes latency and reactivation is unknown. KSHV Lyta (lytic transactivator, also named KSHV/Rta), mainly encoded by the ORF 50 gene, is a lytic switch gene for viral reactivation from latency, inasmuch as it is both essential and sufficient to drive the entire viral lytic cycle. Here we show that the Lyta promoter region was heavily methylated in latently infected cells. Treatment of primary effusion lymphoma-delivered cell lines with tetradecanoylphorbol acetate caused demethylation of the Lyta promoter and induced KSHV lytic phase in vitro. Methylation cassette assay shows demethylation of the Lyta promoter region was essential for the expression of Lyta. In vivo, biopsy samples obtained from patients with KSHV-related diseases show the most demethylation in the Lyta promoter region, whereas samples from a latently infected KSHV carrier remained in a methylated status. These results suggest a relationship among a demethylation status in the Lyta promoter, the reactivation of KSHV, and the development of KSHV-associated diseases.

Transcriptional Regulation of the Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor Gene

ABSTRACT The Kaposis sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, open reading frame (ORF) K9 encodes a viral interferon regulatory factor (vIRF) that functions as a repressor for interferon-mediated signal transduction. Consequently, this gene is thought to play an important role in the tumorigenicity of KSHV. To understand the molecular mechanisms underlying vIRF expression, we studied the transcriptional regulation of this gene. Experiments using 5′ rapid amplification of cDNA ends and primer extension revealed that vIRF had different transcriptional patterns during the latent and lytic phases. The promoter region of the minor transcript, which was mainly expressed in uninduced BCBL-1 cells, did not contain a canonical TATA box, but a cap-like element and an initiator element flanked the transcription start site. The promoter of the major transcript, which was mainly expressed in tetradecanoyl phorbol acetate-induced BCBL-1 cells, contained a canonical TATA box. A luciferase reporter assay using a deletion mutant of the vIRF promoter and a mutation in the TATA box showed that the TATA box was critical for the lytic activity of vIRF. The promoter activity in the latent phase was eight times stronger than that of the empty vector but was less than 10% of the activity in the lytic phase. Therefore, KSHV may use different functional promoter elements to regulate the expression of vIRF and to antagonize the cells interferon-mediated antiviral activity. We have also identified a functional domain in the ORF 50 protein, an immediate-early gene product that is mainly encoded by ORF 50. The ORF 50 protein transactivated the vIRF and DNA polymerase promoters in BCBL-1, 293T, and CV-1 cells. Deleting one of its two putative nuclear localization signals (NLSs) resulted in failure of the ORF 50 protein to localize to the nucleus and consequently abrogated its transactivating activity. We further confirmed that the N-terminal region of the ORF 50 protein included an NLS domain. We found that this domain was sufficient to translocate β-galactosidase to the nucleus. Analysis of deletions within the vIRF promoter suggested that two sequence domains were important for its transactivation by the ORF 50 protein, both of which included putative SP-1 and AP-1 binding sites. Competition gel shift assays demonstrated that SP-1 bound to these two domains, suggesting that the SP-1 binding sites in the vIRF promoter are involved in its transactivation by ORF 50.

Human Herpesvirus 6 Variant A Glycoprotein H-Glycoprotein L-Glycoprotein Q Complex Associates with Human CD46

ABSTRACT Human CD46 is a cellular receptor for human herpesvirus 6 (HHV-6). Virus entry into host cells requires a glycoprotein H (gH)-glycoprotein L (gL) complex. We show that the CD46 ectodomain blocked HHV-6 infection and bound a complex of gH-gL and the 80-kDa U100 gene product, designated glycoprotein Q, indicating that the complex is a viral ligand for CD46.

Accumulation of Heterochromatin Components on the Terminal Repeat Sequence of Kaposi's Sarcoma-Associated Herpesvirus Mediated by the Latency-Associated Nuclear Antigen

ABSTRACT In the latent infection of Kaposis sarcoma-associated herpesvirus (KSHV), its 160-kb circularized episomal DNA is replicated and maintained in the host nucleus. KSHV latency-associated nuclear antigen (LANA) is a key factor for maintaining viral latency. LANA binds to the terminal repeat (TR) DNA of the viral genome, leading to its localization to specific dot structures in the nucleus. In such an infected cell, the expression of the viral genes is restricted by a mechanism that is still unclear. Here, we found that LANA interacts with SUV39H1 histone methyltransferase, a key component of heterochromatin formation, as determined by use of a DNA pull-down assay with a biotinylated DNA fragment that contained a LANA-specific binding sequence and a maltose-binding protein pull-down assay. The diffuse localization of LANA on the chromosomes of uninfected cells changed to a punctate one with the introduction of a bacterial artificial chromosome containing most of the TR region, and SUV39H1 clearly colocalized with the LANA-associated dots. Thus, the LANA foci in KSHV-infected cells seemed to include SUV39H1 as well as heterochromatin protein 1. Furthermore, a chromatin immunoprecipitation assay revealed that the TR and the open reading frame (ORF) K1 and ORF50/RTA genes, but not the ORF73/LANA gene, lay within the heterochromatin during KSHV latency. Taken together, these observations indicate that LANA recruits heterochromatin components to the viral genome, which may lead to the establishment of viral latency and govern the transcription program.

Identification and Analysis of the K5 Gene of Kaposi's Sarcoma-Associated Herpesvirus

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8), belongs to the gammaherpesvirus subfamily and encodes ∼80 open reading frames (ORFs). Among them are a few candidates for immediate-early genes (e.g., K5). We developed a monoclonal antibody (MAb), 328C7, against the K5 antigen. This MAb reacted with the K5 gene product by immunoscreening of a cDNA library from BCBL-1 cells, and this result was confirmed by transfection of the K5 ORF into Cos-7 cells. After induction of lytic infection by treatment with 12-O-tetradecanoylphorbol-13-acetate, MAb 328C7 reacted with an antigen in the cytoplasm of BCBL-1 and BC-3 cells as early as after 4 h of induction. Immunoelectron microscopy showed that the K5 antigen was situated mainly in the endoplasmic reticulum but was not present on the virion or in the nucleus. Northern blotting with a K5-specific probe revealed a single transcript of 1.2 kb, while Western blotting showed the antigen to be a 36-kDa polypeptide. The 5′ and 3′ ends were then determined by rapid amplification of cDNA, followed by sequencing of RACE products, and a splice was revealed upstream of the K5 ORF. K5 expression was unaffected by the respective DNA and protein synthesis inhibitors phosphonoformic acid and cycloheximide plus actinomycin D, confirming its immediate-early nature. Transient-transfection assays showed that the K5 promoter was transactivated by ORF 50 (KSHV Rta), a homolog of Epstein-Barr virus Rta, but the K5 gene product exhibited no transregulation of its own promoter or those of DNA polymerase and the human immunodeficiency virus type 1 long terminal repeat. This is the first such analysis of an immediate-early gene product; determination of its specific biological function requires further investigation.

Synthesis and processing of glycoproteins of Varicella-Zoster virus (VZV) as studied with monoclonal antibodies to VZV antigens

Varicella-Zoster virus (VZV) contains four major glycoproteins designated as gp 1(115K), gp 2(100-80K), gp 3(64K), and gp 5(55K) (K. Shiraki, T. Okuno, K. Yamanishi, and M. Takahashi, J. Gen. Virol. 61, 255-269, 1982). The present studies focused on the synthesis and processing of these glycoproteins using monoclonal antibodies. Twenty-seven hybridomas secreting monoclonal antibodies against VZV proteins have been established. The antibodies were characterized further by radioimmunoprecipitation with [35S]-methionine, [3H]glucosamine, [125I]labeled viral antigens followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Twelve clones of these hybridomas produced antibodies to glycosylated polypeptides of VZV, which could be classified into three groups on the basis of the electrophoretic patterns. The first group reacted specifically with polypeptides with apparent molecular weights of 94K and 83K (both presumed to correspond to gp 2) in infected cells, on the surface membrane of infected cells, and on the virions. The second group precipiated polypeptides with molecular weights of 94K, 83K, 55K (corresponding to gp 5) in infected cells, and 94K, 83K, 55K, 45K on the surface membrane of infected cells, and 94K, 83K, and 55K on the virions. Finally, the third group reacted with polypeptides with molecular weights of 116K, 106K, and 64K corresponding to gp 3) in infected cells, 64K on the surface membrane of infected cells, and on the virions. By pulse-chase experiments, antibodies from the former two groups precipitated new polypeptides with molecular weights of 75K and 49K, respectively, which were presumed to be precursor proteins. These data suggest that the precursor proteins of gp 2, gp 3, and gp 5 are synthesized in infected cells, glycosylated, and the product proteins are expressed on the surface membrane of infected cells as well as on the virions. It was also found that a glycoprotein of 45K detected in culture fluid of infected cells (K. Shiraki and M. Takahashi, J. Gen. Virol. 61, 271-275, 1982) was derived from the polypeptide with molecular weight of 55K (gp 5).