Yuji Isegawa

Human Herpesvirus 7 Open Reading Frame U12 Encodes a Functional β-Chemokine Receptor

ABSTRACT Human herpesvirus 7 (HHV-7), which belongs to the betaherpesvirus subfamily, infects mainly CD4+ T cells in vitro and infects children during infancy. After the primary infection, HHV-7 becomes latent. HHV-7 contains two genes (U12 and U51) that encode putative homologs of cellular G-protein-coupled receptors. To analyze the biological function of the U12 gene, we cloned the gene and expressed the U12 protein in cells. The U12 gene encoded a calcium-mobilizing receptor for the EBI1 ligand chemokine-macrophage inflammatory protein 3β (ELC/MIP-3β) but not for other chemokines, suggesting that the chemokine selectivity of the U12 gene product is distinct from that of the known mammalian chemokine receptors. These studies revealed that U12 activates distinct transmembrane signaling pathways that may mediate biological functions by binding with a β-chemokine, ELC/MIP-3β.

Selective amplification of cDNA sequence from total RNA by cassette-ligation mediated polymerase chain reaction (PCR): Application to sequencing 6·5 kb genome segment of hantavirus strain B-1

A method, referred to as cassette-ligation mediated polymerase chain reaction (PCR), has been developed to permit selective and specific amplification of cDNA sequence from total cellular RNA. This technique comprises (i) digestion of cDNA with multiple restriction enzymes, (ii) ligation of cleavage products to double-stranded DNA cassettes possessing a corresponding restriction site and (iii) amplification of cassette-ligated restriction fragments containing a short, known sequence (but not all the other ligation products) by PCR using the specific and cassette primers; the specific primer is designed to prime synthesis from the known sequence of the cDNA whereas the cassette primer anneals to one strand of the cassette. Sequencing from the cassette primer provides information to design a new primer for the next walking step. The amplified cDNA fragments are often larger than the maximum DNA fragments (500-600 bp) that can be sequenced without the need of synthesizing internal sequencing primer. Each of such large cDNA fragments is dissected into smaller DNA fragments by repeating cassette-ligation mediated PCR exploiting different restriction sites and different sets of cassette primers. This dissection process reduces the number of specific primers to a minimum, thereby increasing the speed of sequencing and minimizing the overall cost. We have successfully applied this cDNA walking and sequencing by the cassette-ligation mediated PCR to the sequencing of an entire 6.5 kb genome segment of hantavirus strain B-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of an avirulent mutant of Sendai virus with two amino acid mutations from a highly virulent field strain through adaptation to LLC-MK2 cells.

A field strain of Sendai virus (SeV) Ohita-M1 (M1) was isolated from an epidemic in an animal laboratory by passaging in mice. A mutant strain, Ohita-MVC11 (MVC11), was then obtained by passaging M1 in rhesus monkey (LLC-MK2) cells. MVC11 was adapted to LLC-MK2 cells and produced 20 times higher levels of infectious virus than M1. This increased production of infectious virus in LLC-MK2 cells was associated with enhanced viral gene expression. However, MVC11 could not replicate efficiently in mouse lung and was not lethal to mice even when inoculated at a titre of 8 x 10(5) cell-infecting units (CIU) per mouse. On the other hand, with an inoculum of only 4 x 10(1) CIU per mouse, corresponding to 1 LD50, M1 replicated well in mouse lung and was highly virulent to mice. Nucleotide and deduced amino acid sequence analyses of the entire genomes of M1 and MVC11 revealed that adaptation to LLC-MK2 cells and the attenuation of mouse pathogenicity of MVC11 were associated with only two amino acid substitutions; one on the C protein (Phe substituted by Ser at position 170) and the other on the RNA polymerase, the L protein (Glu substituted by Ala at position 2050).

Human herpesvirus 6 and human herpesvirus 7 infections in renal transplant recipients and healthy adults in Turkey

SummaryWe explored the prevalence of human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) infections in 16 renal transplant recipients and 16 healthy controls by virus isolation, serology, polymerase chain reaction (PCR) followed by dot blot hybridization. HHV-6 variant A was isolated from one renal transplant recipient. Seven patients (44%) and six controls (38%) had HHV-6 variant B DNA in their peripheral blood mononuclear cells. The prevalence of HHV-7 DNA was found to be the same in patients and controls (19%).

The immunological activity of a deletion mutant of influenza virus haemagglutinin lacking the globular region

A deletion mutant of influenza virus haemagglutinin (HA; headless HA) lacking the globular region was expressed in CV-1 cells and detected with a monoclonal antibody, C179, which recognizes a conformational epitope in the middle of the stem region of HA and neutralizes all H1 and H2 subtypes. The cDNA coding for the headless HA was constructed from influenza virus A/Okuda/57 (H2N2), which was also used to select C179. The conformational epitope recognized by C179 was highly stable even after removal of the globular region. The survival rate of mice immunized with the headless HA and challenged with lethal influenza virus A/FM/l/47 (H1N1) was significantly higher than that of the control mice. The headless HA has the potential to induce cross-protection against influenza virus infection.

A VP4 sequence highly conserved in human rotavirus strain AU-1 and feline rotavirus strain FRV-1

The primary amino acid sequence of the VP4 proteins of human rotavirus strain AU-1 and feline rotavirus strain FRV-1 was deduced from nucleotide sequence analysis of full-length genome segment 4 cDNAs produced by a combined reverse transcription-polymerase chain reaction. The VP4 genes were 2359 nucleotides in length and contained one long open reading frame capable of encoding a protein of 775 amino acids. Strain AU-1 and FRV-1 VP4s were 98.8% similar at both the nucleotide sequence and amino acid level. Given that most of the genome segments of strains AU-1 and FRV-1 formed hybrids under stringent hybridization conditions, the relationship between their VP4 gene sequences is best explained by feline rotavirus being transmitted to human hosts as whole virions relatively recently. Of added interest is that AU-1 and FRV-1 VP4 both exhibit high degrees of similarity (96.0% nucleotide identity and 97.2 to 97.5% amino acid identity) with serotype G1 human rotavirus strain K8 VP4, which is distinct from any other sequenced VP4 allele. This suggests that strain K8 VP4 was derived by natural gene reassortment from a feline rotavirus or a strain AU-1-like human rotavirus.

Human herpesvirus 6 induces IL-8 gene expression in human hepatoma cell line, Hep G2

The infectivity of human herpesvirus 6 (HHV‐6) in a human hepatoma cell line, Hep G2 cells, and the effect of HHV‐6 on production of inflammatory cytokines in these cells were examined to analyze pathogenesis of HHV‐6 in the liver. We demonstrated that Hep G2 cells were susceptible to infection with HHV‐6, and produced infectious virus. Moreover, infection of Hep G2 cells by HHV‐6 induced the expression of IL‐8 mRNA, but not IL‐1β. The effect on induction of IL‐8 gene expression was observed only in Hep G2 cells infected with infectious virus, whereas both heat‐inactivated HHV‐6 and UV‐irradiated HHV‐6 did not change the IL‐8 mRNA level in these cells. These data suggest that HHV‐6 may induce the cytokine‐mediated inflammatory response by infecting liver cells, which could result in liver dysfunction in vivo.

The R3 Region, One of Three Major Repetitive Regions of Human Herpesvirus 6, Is a Strong Enhancer of Immediate-Early Gene U95

ABSTRACT An immediate-early (IE) gene of human herpesvirus 6 (HHV-6), U95, has similarity at the amino acid level to the murine cytomegalovirus (MCMV) IE2 gene and is related to the human cytomegalovirus (HCMV) US22 gene family. Sequence analyses of U95 cDNA clones revealed that the transcription start site was located about 1.6 kbp upstream of the putative initiating ATG and that the transcript consisted of two exons. A single intron extended from nucleotides 142589 to 144229, which contained ORF U94. A protein with a molecular mass of about 120 kDa was translated from this cDNA clone in an in vitro transcription-translation assay. The transcription start site was found to be 220 bp downstream of the R3 region by primer extension analysis. HHV-6 has three repetitive elements, R1, R2, and R3, in or near the IE-A locus. R3 is composed of 24 copies of a 104- to 107-bp sequence element, which contains multiple putative binding sites for cellular transcription factors such as AP2 and NF-κB, and its biological significance has yet to be elucidated. The region between −710 and +46 relative to the transcription start site of U95 was analyzed in this study. Deletion from −710 to −396, corresponding to three copies of an R3 unit, decreased the promoter activity by 15-fold, and coexpression of IκBα(S32A/S36A) repressed it to almost the same level. Electrophoretic mobility shift assays showed that NF-κB family members p50 and c-Rel bound to NF-κB sites derived from the R3 region. These results demonstrate that R3 strongly enhances the U95 promoter activity and that NF-κB and binding sites for NF-κB in the R3 region play an important role in its activation. Because U95 promoter activity correlated with the number of R3 units, which each contained an NF-κB site, the repetitive organization of R3 is important for regulating U95 transcription.

Kaposi’s sarcoma-associated herpesvirus (KSHV)-encoded vMIP-I and vMIP-II induce signal transduction and chemotaxis in monocytic cells

Summary. Kaposi’s sarcoma-associated herpesvirus (KSHV)/Human herpesvirus 8 encodes three chemokines, which are called viral macrophage inflammatory protein (vMIP)-I, -II, and -III. Here, we expressed the KSHV vMIP-I and vMIP-II proteins and analyzed their biological functions. Both vMIP-I and vMIP-II had an apparent molecular mass of 7.8 kDa and were localized to the cytoplasm in a body cavity-based lymphoma cell line BC-3, stimulated with phorbol ester. We next treated a human monocytic leukemia cell line, THP-1, with purified recombinant vMIP-I and vMIP-II, or vMIP-I and vMIP-II fused with alkaline phosphatase to study Ca2+ signalling and in vitro chemotaxis in response to these proteins. Calcium mobilization was induced by both vMIP-I and vMIP-II. Furthermore, vMIP-I and vMIP-II induced Ca2+ mobilization in K562 cells expressing the CC chemokine receptor 5 (CCR5), suggesting that both may be agonistic for CCR5. Additionally, vMIP-I induced Ca2+ mobilization through the intermediary of CCR8. These viral MIPs were also capable of chemotactically activating the THP-1 cells. These results imply that vMIP-I and vMIP-II may play important roles in the propagation of KS and primary effusion lymphoma by inducing the chemotaxis of CCR5-expressing monocytes.

Human herpesvirus 6 ganciclovir-resistant strain with amino acid substitutions associated with the death of an allogeneic stem cell transplant recipient

BACKGROUND Viral resistance to antiviral drugs can cause serious complications in immunosuppressed patients. We isolated from an allogeneic stem cell transplant (SCT) recipient an antiviral-resistant human herpesvirus 6 (HHV-6) strain with mutations that caused amino acid substitutions. OBJECTIVE To study the impact of mutations in the U38 and U69 genes of the ganciclovir (GCV)-resistant HHV-6 strain associated with the death of the SCT recipient. STUDY DESIGN Viruses were obtained from blood taken during symptomatic disease. Mutations in the genes for U69 protein kinase and U38 DNA polymerase were analyzed and the effects of the U69 mutations on GCV resistance were assayed using a recombinant baculovirus system. RESULTS Increasing HHV-6 antigenemia occurred after 2-3 months of preemptive GCV therapy, followed by symptomatic HHV-6 disease that ended in fatal fungus-related septic shock. The HHV-6 strain isolated from the patient was 100-fold more resistant to GCV than was a wild-type strain. New mutations were found in HHV-6 genes U38 (P462S and A565V) and U69 (L202I and L213I). The mutation of U38 P462S corresponds to a mutation in the UL54 gene (P522S) of a GCV-resistant HCMV. The U69 mutations did not alter GCV sensitivity in baculovirus GCV-resistant assay system. CONCLUSIONS Drug-resistant mutations arising during preemptive therapy may complicate post-transplant HHV-6 disease in SCT recipients. The increased copy number during GCV treatment of this new GCV-resistant HHV-6 strain correlated with mutations in the U69 and U38 genes. Since the kinase mutation did not alter sensitivity to GCV when tested in the in vitro system, it is likely that the substitutions in the polymerase related to GCV resistance.