Norio Yamamoto

Overexpressed NF-κB inducing kinase contributes to the tumorigenesis of adult T-cell leukemia and Hodgkin Reed-Sternberg cells

The nuclear factor-kappaB (NF-kappaB) transcription factors play important roles in cancer development by preventing apoptosis and facilitating the tumor cell growth. However, the precise mechanisms by which NF-kappaB is constitutively activated in specific cancer cells remain largely unknown. In our current study, we now report that NF-kappaB-inducing kinase (NIK) is overexpressed at the pretranslational level in adult T-cell leukemia (ATL) and Hodgkin Reed-Sternberg cells (H-RS) that do not express viral regulatory proteins. The overexpression of NIK causes cell transformation in rat fibroblasts, which is abolished by a super-repressor form of IkappaBalpha. Notably, depletion of NIK in ATL cells by RNA interference reduces the DNA-binding activity of NF-kappaB and NF-kappaB-dependent transcriptional activity, and efficiently suppresses tumor growth in NOD/SCID/gammac(null) mice. These results indicate that the deregulated expression of NIK plays a critical role in constitutive NF-kappaB activation in ATL and H-RS cells, and suggest also that NIK is an attractive molecular target for cancer therapy.

HIV protease inhibitor nelfinavir inhibits replication of SARS-associated coronavirus

Abstract A novel coronavirus has been identified as an etiological agent of severe acute respiratory syndrome (SARS). To rapidly identify anti-SARS drugs available for clinical use, we screened a set of compounds that included antiviral drugs already in wide use. Here we report that the HIV-1 protease inhibitor, nelfinavir, strongly inhibited replication of the SARS coronavirus (SARS-CoV). Nelfinavir inhibited the cytopathic effect induced by SARS-CoV infection. Expression of viral antigens was much lower in infected cells treated with nelfinavir than in untreated infected cells. Quantitative RT-PCR analysis showed that nelfinavir could decrease the production of virions from Vero cells. Experiments with various timings of drug addition revealed that nelfinavir exerted its effect not at the entry step, but at the post-entry step of SARS-CoV infection. Our results suggest that nelfinavir should be examined clinically for the treatment of SARS and has potential as a good lead compound for designing anti-SARS drugs.

Malignant transformation of B lymphoma cell line BJAB by Epstein–Barr virus-encoded small RNAs

EBV‐encoded small RNAs (EBERs) are non‐polyadenylated and abundantly transcribed RNAs, whose functions have not yet been fully elucidated. Here, we report that the EBV‐negative B lymphoma cell line, BJAB, was rendered more malignant and resistant to apoptosis by EBERs. EBER‐transfected cells exhibited enhanced growth potential in SCID mice as well as in soft agar, and showed resistance to apoptotic stimuli in comparison with the vector control. EBERs inhibited the activity of the double‐stranded RNA‐dependent protein kinase, PKR, which is reputed to act as a tumor‐suppressor. These results suggest that EBERs play an important role in the pathogenesis of EBV‐associated malignancies through the inhibition of PKR.

Modulation of TNF-α-converting enzyme by the spike protein of SARS-CoV and ACE2 induces TNF-α production and facilitates viral entry

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a high-risk infectious pathogen. In the proposed model of respiratory failure, SARS-CoV down-regulates its receptor, angiotensin-converting enzyme 2 (ACE2), but the mechanism involved is unknown. We found that the spike protein of SARS-CoV (SARS-S) induced TNF-α-converting enzyme (TACE)-dependent shedding of the ACE2 ectodomain. The modulation of TACE activity by SARS-S depended on the cytoplasmic domain of ACE2, because deletion mutants of ACE2 lacking the carboxyl-terminal region did not induce ACE2 shedding or TNF-α production. In contrast, the spike protein of HNL63-CoV (NL63-S), a CoV that uses ACE2 as a receptor and mainly induces the common cold, caused neither of these cellular responses. Intriguingly, viral infection, judged by real-time RT-PCR analysis of SARS-CoV mRNA expression, was significantly attenuated by deletion of the cytoplasmic tail of ACE2 or knock-down of TACE expression by siRNA. These data suggest that cellular signals triggered by the interaction of SARS-CoV with ACE2 are positively involved in viral entry but lead to tissue damage. These findings may lead to the development of anti-SARS-CoV agents.

Analysis of human immunodeficiency virus type 1 integration by using a specific, sensitive and quantitative assay based on real-time polymerase chain reaction.

A novel real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type-1 (HIV-1) DNA with high specificity and sensitivity. This assay reproducibly allowed the detection of three copies of integrated HIV DNA in a background of 100,000 cell equivalents of human chromosomal DNA. The non-specific amplification of unintegrated HIV-1 DNA was significantly inhibited in this assay and the specificity of this assay was much higher than the previously reported method. This assay showed that kinetics in viral DNA sysnthesis was cell-type dependent and that the kinetics of HIV-1 DNA integration was very rapid in Jurkat T cell line. This method may provide new insights into the integration processes and be useful in evaluating future integrase inhibitors.

Anti-RNA virus activity of polyoxometalates.

Abstract The anti-RNA virus activity of polyoxometalates (POM) is reviewed, with a special emphasis on the anti-respiratory virus activities. There are many causative agents of acute viral respiratory infections; and it is rather difficult to identify the relevant agent in a given case by rapid clinical means. During acute progress of infection before the definitive diagnosis is obtained physicians need to prescribe certain broad spectrum anti-viral drugs. A titanium containing polyoxotungstate, PM-523 exhibited potent anti-influenza virus (FluV) A and anti-respiratory syncytial virus (RSV) activities in vitro. Therapeutic effect of FluV A infected mice with aerosol inhalation of PM-523 was proven. A vanadium substituted polyoxotungstate, PM-1001 has antiviral activity against FluV A, RSV, parainfluenza virus (PfluV) type 2, Dengue fiver virus, HIV-1 and SARS coronavirus in vitro. Thus, POMs have been proven to be broad spectrum and non-toxic anti-RNA virus agents in both in vitro and in vivo experiments and are promising candidates for first-line therapeutics in acute respiratory diseases.

Genetic analysis of attachment glycoprotein (G) gene in new genotype ON1 of human respiratory syncytial virus detected in Japan

We studied the evolution of the G gene in the new genotype ON1 of RSV detected from patients with acute respiratory infection in Japan. Phylogenetic analyses and the evolutionary timescale were obtained by the Bayesian MCMC method. We also analyzed p‐distance and positive selection sites. A new genotype ON1 emerged around 2001. The evolution rate was rapid (3.57 × 10−3 substitutions/site per year). The p‐distance was short and no positive selection site was found in the present strains. These results suggested that a new genotype ON1 of RSV‐A emerged approximately10 years ago and spread to some countries with a high evolution rate.

Inhibition of human immunodeficiency virus type 1 (HIV-1) nuclear import via Vpr–Importin α interactions as a novel HIV-1 therapy

The development of multidrug-resistant viruses compromises the efficacy of anti-human immunodeficiency virus (HIV) therapy and limits treatment options. Therefore, new targets that can be used to develop novel antiviral agents need to be identified. One such target is the interaction between Vpr, one of the accessory gene products of HIV-1 and Importin alpha, which is crucial, not only for the nuclear import of Vpr, but also for HIV-1 replication in macrophages. We have identified a potential parent compound, hematoxylin, which suppresses Vpr-Importin alpha interaction, thereby inhibiting HIV-1 replication in a Vpr-dependent manner. Analysis by real-time PCR demonstrated that hematoxylin specifically inhibited nuclear import step of pre-integration complex. Thus, hematoxylin is a new anti-HIV-1 inhibitor that targets the nuclear import of HIV-1 via the Vpr-Importin alpha interaction, suggesting that a specific inhibitor of the interaction between viral protein and the cellular factor may provide a new strategy for HIV-1 therapy.

An HIV protease inhibitor, ritonavir targets the nuclear factor-kappaB and inhibits the tumor growth and infiltration of EBV-positive lymphoblastoid B cells

Epstein‐Barr Virus (EBV)‐associated immunoblastic lymphoma occurs in immunocompromised patients such as those with AIDS or transplant recipients after primary EBV infection or reactivation of a preexisting latent EBV infection. In the present study, we evaluated the effect of ritonavir, an HIV protease inhibitor, on EBV‐positive lymphoblastoid B cells in vitro and in mice model. We found that it induced cell‐cycle arrest at G1‐phase and apoptosis through down‐regulation of cell‐cycle gene cyclin D2 and antiapoptotic gene survivin. Furthermore, ritonavir suppressed transcriptional activation of NF‐κB in these cells. Ritonavir efficiently prevented growth and infiltration of lymphoma cells in various organs of NOD/SCID/γcnull mice at the same dose used for treatment of patients with AIDS. Our results indicate that ritonavir targets NF‐κB activated in tumor cells and shows anti‐tumor effects. These data also suggest that this compound may have promise for treatment or prevention of EBV‐associated lymphoproliferative diseases that occur in immunocompromised patients.

Molecular evolution of attachment glycoprotein (G) gene in human respiratory syncytial virus detected in Japan 2008–2011

We investigated the evolution of the C-terminal 3rd hypervariable region of G gene in the prevalent human respiratory syncytial virus (RSV) subgroups A (RSV-A) and B (RSV-B) in Japan in 2008-2011. Phylogenetic analysis and the evolutionary timescale was obtained by the Bayesian Markov Chain Monte Carlo method. All 38 RSV-A strains detected were classified into genotype NA1 and the 17 RSV-B strains detected belonged to genotypes BA and GB2. NA1 subdivided around 1998 in the present phylogenetic tree. Genotype BA subdivided around 1994. The evolutionary rates for RSV-A and RSV-B were estimated at 3.63×10⁻³ and 4.56×10⁻³ substitutions/site/year, respectively. The mean evolutionary rate of RSV-B was significantly faster than that of RSV-A during all seasons. The pairwise distance was relatively short (less than 0.06). In addition, some unique sites under positive selection were found. The results suggested that this region of the RSV strains rapidly evolved with some unique amino acid substitutions due to positive pressure.