Masaya Fukushi

Chromosome Binding Site of Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Is Essential for Persistent Episome Maintenance and Is Functionally Replaced by Histone H1

ABSTRACT Latency-associated nuclear antigen 1 (LANA1) of Kaposis sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) persistently maintains a plasmid containing the KSHV latent origin of replication (oriP) as a closed circular episome in dividing cells. In this study, we investigated the involvement of chromosome binding activity of LANA1 in persistent episome maintenance. Deletion of the N-terminal 22 amino acids of LANA1 (ΔN-LANA) inhibited the interaction with mitotic chromosomes in a human cell line, and the mutant concomitantly lost activity for the long-term episome maintenance of a plasmid containing viral oriP in a human B-cell line. However, a chimera of ΔN-LANA with histone H1, a cellular chromosome component protein, rescued the association with mitotic chromosomes as well as the long-term episome maintenance of the oriP-containing plasmid. Our results suggest that tethering of KSHV episomes to mitotic chromosomes by LANA1 is crucial in mediating the long-term maintenance of viral episomes in dividing cells.

Human T-Cell Leukemia Virus Type 2 (HTLV-2) Tax Protein Transforms a Rat Fibroblast Cell Line but Less Efficiently than HTLV-1 Tax

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are retroviruses with similar biological properties. Whereas HTLV-1 is the causative agent of an aggressive T-cell leukemia, HTLV-2 has been associated with only a few cases of lymphoproliferative disorders. Tax1 and Tax2 are the transcriptional activators of HTLV-1 and HTLV-2, respectively. Here we show that Tax2 transformed a Rat-1 fibroblast cell line to form colonies in soft agar, but the size and number of the colonies were lower than those of Tax1. Use of a chimeric Tax protein showed that the C-terminal amino acids 300 to 353 were responsible for the high transforming activity of Tax1. Activation of cellular genes by Tax1 through transcription factor NF-κB is reportedly essential for the transformation of Rat-1 cells. Tax2 also activated the transcription through NF-κB in Rat-1 cells, and such activity was equivalent to that induced by Tax1. Thus, the high transforming activity of Tax1 is mediated by mechanisms other than NF-κB activation. Our results showed that Tax2 has a lower transforming activity than Tax1 and suggest that the high transforming activity of Tax1 is involved in the leukemogenic property of HTLV-1.

Serial Histopathological Examination of the Lungs of Mice Infected with Influenza A Virus PR8 Strain

Avian influenza H5N1 and pandemic (H1N1) 2009 viruses are known to induce viral pneumonia and subsequent acute respiratory distress syndrome (ARDS) with diffuse alveolar damage (DAD). The mortality rate of ARDS/DAD is extremely high, at approximately 60%, and no effective treatment for ARDS/DAD has been established. We examined serial pathological changes in the lungs of mice infected with influenza virus to determine the progress from viral pneumonia to ARDS/DAD. Mice were intranasally infected with influenza A/Puerto Rico/8/34 (PR8) virus, and their lungs were examined both macro- and micro-pathologically every 2 days. We also evaluated general condition, survival rate, body weight, viral loads in lung, and surfactant proteins in serum. As a result, all infected mice died within 9 days postinfection. At 2 days postinfection, inflammation in alveolar septa, i.e., interstitial pneumonia, was observed around bronchioles. From 4 to 6 days postinfection, interstitial pneumonia with alveolar collapse expanded throughout the lungs. From 6 to 9 days postinfection, DAD with severe alveolar collapse was observed in the lungs of all of dying and dead mice. In contrast, DAD was not observed in the live infected-mice from 2 to 6 days postinfection, despite their poor general condition. In addition, histopathological analysis was performed in mice infected with a dose of PR8 virus which was 50% of the lethal dose for mice in the 20-day observation period. DAD with alveolar collapse was observed in all dead mice. However, in the surviving mice, instead of DAD, glandular metaplasia was broadly observed in their lungs. The present study indicates that DAD with severe alveolar collapse is associated with death in this mouse infection model of influenza virus. Inhibition of the development of DAD with alveolar collapse may decrease the mortality rate in severe viral pneumonia caused by influenza virus infection.

A new ERK2 binding protein, Naf1, attenuates the EGF/ERK2 nuclear signaling.

Extracellular signal regulated kinase1/2 (ERK1/2), an important factor in signal transduction, controls cell growth, differentiation, and death. To elucidate the details of the mechanism of ERK1/2 signaling in human cells, we isolated Nef-associated factor 1 alpha (Naf1 alpha) by a yeast two-hybrid system, which bound to human ERK2. The binding was confirmed by a pull-down assay in vitro and immunoprecipitation in vivo. Upon EGF treatment, Naf1 alpha was phosphorylated by the EGF/MEK/ERK2 signal transduction pathway. To identify the role of Naf1 alpha in the ERK2 signaling, Naf1 alpha-expressing Saos-2 cells were analyzed for ERK2 nuclear translocation and activation of its downstream target. Overexpression of Naf1 alpha suppressed ERK2 entering into the nucleus and inhibited the ERK2-dependent Elk1-driven luciferase transcription, suggesting Naf1 alpha to be an attenuator of activated ERK2 signaling.

Activation of oncogenic transcription factor AP-1 in T cells infected with human T cell leukemia virus type 1.

Human T cell leukemia virus type 1 (HTLV-1) Tax protein transforms primary human T cells in vitro. We previously showed that Tax induces the expression of various family members of the transcription factor AP-1 such as c-Jun, JunD, c-Fos, and Fra-1 at the mRNA level in T cells. In this study, we have examined the ability of Tax to activate transcription through the AP-1-binding site (AP-1 site). A transient transfection study showed that Tax can activate transcription through the AP-1-binding site in a human T cell line, whereas any combination of AP-1 proteins did so much less than Tax, indicating that the activation of the AP-1 site by Tax may require a mechanism other than the induction of AP-1 mRNA. Fresh peripheral blood leukemia cells of all surveyed ATL patients displayed constitutive AP-1 DNA-binding activity, whereas no normal individuals did. However, the HTLV-1 genes, including tax, are not significantly expressed in fresh leukemia cells from ATL patients. Our present results suggest that activation of AP-1 occurs through Tax-dependent and -independent mechanisms in HTLV-1-infected T cells, which may play some roles in dysregulated phenotypes of HTLV-1-infected cells.

Dissection and identification of regions required to form pseudoparticles by the interaction between the nucleocapsid (N) and membrane (M) proteins of SARS coronavirus.

Abstract When expressed in mammalian cells, the nucleocapsid (N) and membrane (M) proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV) are sufficient to form pseudoparticles. To identify region(s) of the N molecule required for pseudoparticle formation, we performed biochemical analysis of the interaction of N mutants and M in HEK293 cells. Using a peptide library derived from N, we found that amino acids 101–115 constituted a novel binding site for M. We examined the ability of N mutants to interact with M and form pseudoparticles, and our observations indicated that M bound to NΔ(101–115), N1–150, N151–300, and N301–422, but not to N1–150Δ(101–115). However, pseudoparticles were formed when NΔ(101–115) or N301–422, but not N1–150 or N151–300, were expressed with M in HEK293 cells. These results indicated that the minimum portion of N required for the interaction with M and pseudoparticle formation consists of amino acids 301–422.

Monitoring of S Protein Maturation in the Endoplasmic Reticulum by Calnexin Is Important for the Infectivity of Severe Acute Respiratory Syndrome Coronavirus

ABSTRACT Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of SARS, a fatal pulmonary disorder with no effective treatment. We found that SARS-CoV spike glycoprotein (S protein), a key molecule for viral entry, binds to calnexin, a molecular chaperone in the endoplasmic reticulum (ER), but not to calreticulin, a homolog of calnexin. Calnexin bound to most truncated mutants of S protein, and S protein bound to all mutants of calnexin. Pseudotyped virus carrying S protein (S-pseudovirus) produced by human cells that were treated with small interfering RNA (siRNA) for calnexin expression (calnexin siRNA-treated cells) showed significantly lower infectivity than S-pseudoviruses produced by untreated and control siRNA-treated cells. S-pseudovirus produced by calnexin siRNA-treated cells contained S protein modified with N-glycan side chains differently from other two S proteins and consisted of two kinds of viral particles: those of normal density with little S protein and those of high density with abundant S protein. Treatment with peptide-N-glycosidase F (PNGase F), which removes all types of N-glycan side chains from glycoproteins, eliminated the infectivity of S-pseudovirus. S-pseudovirus and SARS-CoV produced in the presence of α-glucosidase inhibitors, which disrupt the interaction between calnexin and its substrates, showed significantly lower infectivity than each virus produced in the absence of those compounds. In S-pseudovirus, the incorporation of S protein into viral particles was obviously inhibited. In SARS-CoV, viral production was obviously inhibited. These findings demonstrated that calnexin strictly monitors the maturation of S protein by its direct binding, resulting in conferring infectivity on SARS-CoV.

Fully Human Monoclonal Antibody Directed to Proteolytic Cleavage Site in Severe Acute Respiratory Syndrome (SARS) Coronavirus S Protein Neutralizes the Virus in a Rhesus Macaque SARS Model

Abstract Background. There is still no effective method to prevent or treat severe acute respiratory syndrome (SARS), which is caused by SARS coronavirus (CoV). In the present study, we evaluated the efficacy of a fully human monoclonal antibody capable of neutralizing SARS-CoV in vitro in a Rhesus macaque model of SARS. Methods. The antibody 5H10 was obtained by vaccination of KM mice bearing human immunoglobulin genes with Escherichiacoli–producing recombinant peptide containing the dominant epitope of the viral spike protein found in convalescent serum samples from patients with SARS. Results. 5H10, which recognized the same epitope that is also a cleavage site critical for the entry of SARS-CoV into host cells, inhibited propagation of the virus and pathological changes found in Rhesus macaques infected with the virus through the nasal route. In addition, we analyzed the mode of action of 5H10, and the results suggested that 5H10 inhibited fusion between the virus envelope and host cell membrane. 5H10 has potential for use in prevention and treatment of SARS if it reemerges. Conclusions. This study represents a platform to produce fully human antibodies against emerging infectious diseases in a timely and safe manner.

Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Interacts with Human Myeloid Cell Nuclear Differentiation Antigen Induced by Interferon α

Kaposis sarcoma-associated herpesvirus (KSHV)/human herpes virus type 8 (HHV-8) is tightly linked to the development of Kaposis sarcoma, primary effusion lymphoma (PEL) and some cases of multicentric Castlemans disease. Latency-associated nuclear antigen (LANA) is one of a limited number of KSHV genes consistently expressed in these diseases as well as in KSHV-infected cell lines derived from PEL, and has been shown to play crucial role in persistence of KSHV genomes in the infected cells. In this study, we explored the cellular factors that interact with LANA using yeast two-hybrid screening, and isolated a part of gene encoding human myeloid cell nuclear differentiation antigen (MNDA). MNDA is a hematopoietic interferon-inducible nuclear proteins with a HIN-200 family member with conserved 200-amino acid repeats. Immunoprecipitation assay revealed that LANA interacted with MNDA in a mammalian embryonic kidney cell line. MNDA transcript was undetectable in three PEL cell lines by reverse-transcription polymerase chain reaction, but it was induced by interferon α (IFNα). Moreover, LANA and MNDA were co-localized in the nuclei of MNDA-expressing PEL cells. Our results suggest that LANA interacts with MNDA in KSHV-infected cells exposed to IFNα. Such interaction may modulate IFN-mediated host defense activities.

Visualization of a Functional KSHV Episome-Maintenance Protein LANA in Living Cells

The latency-associated nuclear antigen (LANA) of Kaposis saroma-associated herpesvirus (KSHV) can maintain a plasmid containing the KSHV origin of DNA replication (oriP) as episomes in dividing human cells. Hence, LANA is considered to play crucial roles in persistent KSHV infection in human cells. In this study, we characterized a LANA fusion protein of green fluorescent protein (GFP-LANA). Like the wild-type LANA, GFP-LANA interacted tightly with mitotic chromosomes, and maintained the plasmid selectively with the KSHV oriP for more than three weeks in a human B cell line. Moreover, equivalent amount of GFP-LANA protein was segregated into two daughter cells in living metaphase cells. Our results suggested that the activity of LANA serves the segregation of equivalent amounts of viral genomes tethered with LANA into two daughter progeny cells during cell division. Thus, GFP-LANA is a useful tool for the analyses of the functions and dynamics of LANA in living cells.