Yasuko Mori

Human Herpesvirus 6 Variant A but Not Variant B Induces Fusion from Without in a Variety of Human Cells through a Human Herpesvirus 6 Entry Receptor, CD46

ABSTRACT Human herpesvirus 6 (HHV-6) is a lymphotropic betaherpesvirus that productively infects T cells and monocytes. HHV-6 isolates can be differentiated into two groups, variants A and B (HHV-6A and HHV-6B). Here, we show a functional difference between HHV-6A and -6B in that HHV-6A induced syncytium formation of diverse human cells but HHV-6B did not. The syncytium formation induced by HHV-6A was observed 2 h after infection; moreover, it was found in the presence of cycloheximide, indicating that HHV-6A induced fusion from without (FFWO) in the target cells. Furthermore, the fusion event was dependent on the expression of the HHV-6 entry receptor, CD46, on the target cell membrane. In addition, we determined that short consensus repeat 2 (SCR2), -3, and -4 of the CD46 ectodomain were essential for the formation of the virus-induced syncytia. Monoclonal antibodies against glycoproteins B and H of HHV-6A inhibited the fusion event, indicating that the syncytium formation induced by HHV-6A required glycoproteins H and B. These findings suggest that FFWO, which HHV-6A induced in a variety of cell lines, may play an important role in the pathogenesis of HHV-6A, not only in lymphocytes but also in various tissues, because CD46 is expressed ubiquitously in human tissues.

Human herpesvirus 8-encoded interleukin-6 homologue (viral IL-6) induces endogenous human IL-6 secretion.

We found that human herpesvirus 8‐encoded IL‐6 (vIL‐6) induced endogenous human IL‐6 (huIL‐6) secretion from various cell lines (MT‐4, THP‐1, U937, Raji, and CESS) including patients with multicentric Castlemans disease. Especially, in MT‐4 cells, huIL‐6 was enhanced with vIL‐6 by 30‐fold compared with that of control. In addition, reverse transcriptase‐polymerase chain reaction confirmed that vIL‐6 induced huIL‐6 expression in MT‐4 cells. Our novel finding of the huIL‐6 induction by vIL‐6 indicates that vIL‐6 may play a significant role in the pathogenesis of HHV‐8 associated diseases. J. Med. Virol. 61:332–335, 2000.

The Human Herpesvirus 6 U100 Gene Product Is the Third Component of the gH-gL Glycoprotein Complex on the Viral Envelope

ABSTRACT The human herpesvirus 6 (HHV-6) variant A U100 gene encodes the third component of the glycoprotein H (gH)-glycoprotein L (gL)-containing complex. Glycosidase digestion analysis showed that the U100 gene products are glycoproteins consisting of an 80-kDa protein with complex N-linked oligosaccharides and a 74-kDa protein with immature, high-mannose N-linked oligosaccharides. Based on these characteristics, we designated the U100 gene products glycoprotein Q (gQ). Only the 80-kDa form of gQ was coimmunoprecipitated with an anti-gH antibody, suggesting that the 80-kDa protein associates with the gH-gL complex in HHV-6-infected cells. Furthermore, the complex was detected in purified virions, suggesting that it may play an important role in viral entry.

Human herpesvirus-6 rep/U94 gene product has single-stranded DNA-binding activity.

The characterization is reported of the human herpesvirus-6B (HHV-6B) rep/U94 gene, which is a homologue of the adeno-associated virus type 2 rep. In this study, a monoclonal antibody was produced against HHV-6B REP (anti-REP mAb). Immunofluorescence staining using the anti-REP mAb showed that REP was localized to the nucleus in HHV-6-infected MT4 cells. It was first detected at 24 h post-infection (p.i.) and accumulated to higher levels by 72 h p.i. REP may be expressed only at very low levels in HHV-6-infected cells: even when the late protein glycoprotein H was detected in nearly 90% of HHV-6-infected cells, REP was detected in only a small percentage of them. Western blot analysis showed that the anti-REP mAb recognized a 56-kDa polypeptide in HHV-6B-infected MT4 cells. Furthermore, the REP protein was shown to bind single-stranded DNA.

Clinical characteristics of acyclovir-resistant herpetic keratitis and experimental studies of isolates

Abstract• Background: We treated two patients with dendritic keratitis that did not respond to acyclovir (ACV) ointment therapy. Their systemic immune status was normal; however, one patient had a long history of atopic disease and the other had previously undergone topical corticosteroid treatment. HSV-1 was isolated from the patients and inoculated into animals to investigate its viral pathogenicity and latent infection. • Methods: HSV-1 isolates from the patients were tested for drug sensitivity to acyclovir, ganciclovir, idoxuridine, trifluridine, foscarnet and interferon-β in vitro. In in vivo studies, bilateral corneas of two New Zealand white rabbits and 10 BALB/c mice in each of four groups were infected by the respective viral isolates. The extent of corneal epithelial and/or stromal lesions produced by the viruses was evaluated. The trigeminal ganglial tissues of the mice were examined for viral latent infection by co-culture with Vero cells. • Results: Herpetic keratitis in both patients was characterized by prolonged clinical course, succeeded by various types of corneal lesions and ocular complications. In in vitro studies, the two HSV-1 isolates demonstrated cross-resistance to ACV, ganciclovir and/or idoxuridine. Both strains demonstrated weakly virulent corneal epithelial and/or stromal lesions in rabbits and mice. One isolate displayed delayed advent but prolonged course of epithelial lesions in rabbits. The latent infection incidences of the isolates in mice trigeminal ganglia were 6.25% (1/16) and 0% (0/18) respectively. • Conclusion: Topical immune depression may induce ACV-resistant HSV-1 infection in the cornea, with a prolonged course in association with ocular complications. The prolonged infectious course of the viral isolates in the animal study partially supported the clinical demonstrations in the patient. The existence of latent infection by one ACV-resistant HSV-1 in its animals may indicate the possibility of its recurrence. Trifluridine may be an alternative choice for treating corneal epithelial lesions caused by ACV-resistant HSV-1.

Branching Morphogenesis of Mouse Embryonic Submandibular Epithelia Cultured under Three Different Conditions

To investigate how the mesenchyme interacts with the epithelium, we employed three different culture systems: System A, in which intact submandibular gland rudiments at the mid 13‐day stage were cultured on Millipore filters; System B, in which the 13‐day epithelium and mesenchyme were separated once with dispase, recombined again, and cultured on the filter; System C, in which the separated 13‐day epithelium was clotted with Matrigel and cultured with the mesenchyme across the filter or in the presence of EGF instead of the mesenchyme. In Systems A and B, 13‐day epithelia expanded and produced similar lobules with narrow clefts and stalk. When the 13‐day epithelium was cultured in System C under the influence of the mesenchyme, it formed rather oval lobules with stalk that were superficially similar to those in System A, but narrow clefts, as seen in the intact early 13‐day gland, were rarely found in System C. Furthermore, no long stalk formation was observed when EGF was introduced in place of the mesenchyme. A bacterial collagenase from Clostridium histolyticum gave a considerable inhibition of branching of the 13‐day epithelium in Systems A and B, but no significant inhibition was observed in System C when the mesenchyme or EGF was employed as the source of diffusible factor(s). In contrast, although the 13‐day epithelium was significantly resistant to the action of heparitinase I from Flavobacterium heparinum in Systems A and B, the enzyme almost completely inhibited the expansion and branching of the epithelium in System C. Judging from these observations, we conclude that the mechanisms of lobular formation in Systems A and B are not the same as those in System C, where the epithelium is clotted with basement membrane matrix components during tissue culture.

Detection of HSV mRNA using reverse transcription-polymerase chain reaction for diagnosis in murine herpetic keratitis model

Reverse transcription polymerase chain reaction (RT-PCR) was applied in the detection of herpes simplex virus-1 (HSV-1) mRNA from tear film and corneal epithelium in a murine herpetic keratitis model. The diagnostic value of this new technique for acute herpetic keratitis was evaluated in comparison with direct PCR for genomic DNA and viral culture. On day 2 postinfection (PI) of HSV, all mice showed dendritic keratitis, and PCR, RT-PCR, and viral culture were positive in all samples. On day 8 PI, no dendritic keratitis was observed in any mouse, PCR was positive in all samples, while RT-PCR was positive in only 5 of 12 samples and viral culture in only 2 of 12. The sensitivity of RT-PCR was lower than that of PCR, and approximately the same as viral culture; however, the findings of RT-PCR more closely concurred with clinical observations than the findings of PCR. These results show the potential of RT-PCR for rapid, specific diagnosis of acute herpetic keratitis.