Masayasu Oie

Isolation and Characterization of Novel Human Parechovirus from Clinical Samples

Identification of HPeV-6 will advance HPeV diagnosis and epidemiology.

Cooperation of NF-κB2/p100 Activation and the PDZ Domain Binding Motif Signal in Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax1 but Not HTLV-2 Tax2 Is Crucial for Interleukin-2-Independent Growth Transformation of a T-Cell Line

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia, and the distinct pathogenicity of these two closely related viruses is thought to stem from the distinct biological functions of the respective transforming proteins, HTLV-1 Tax1 and HTLV-2 Tax2. In this study, we demonstrate that Tax1 but not Tax2 interacts with NF-κB2/p100 and activates it by inducing the cleavage of p100 into the active transcription factor p52. Using RNA interference methods, we further show that NF-κB2/p100 is required for the transformation induced by Tax1, as determined by the ability to convert a T-cell line (CTLL-2) from interleukin-2 (IL-2)-dependent to -independent growth. While Tax2 shows a reduced transforming activity relative to Tax1, Tax2 fused with a PDZ domain binding motif (PBM) present only in Tax1 shows transforming activity equivalent to that of Tax1 in CTLL-2 cells expressing an inducer of p52 processing. These results reveal that the activation of NF-κB2/p100 plays a crucial role in the Tax1-mediated transformation of T cells and that NF-κB2/p100 activation and PBM function are both responsible for the augmented transforming activity of Tax1 relative to Tax2, thus suggesting that these Tax1-specific functions play crucial roles in HTLV-1 leukemogenesis.

Both G3BP1 and G3BP2 contribute to stress granule formation

Upon exposure to various environmental stresses such as arsenite, hypoxia, and heat shock, cells inhibit their translation and apoptosis and then repair stress‐induced alterations, such as DNA damage and the accumulation of misfolded proteins. These types of stresses induce the formation of cytoplasmic RNA granules called stress granules (SGs). SGs are storage sites for the many mRNAs released from disassembled polysomes under these stress conditions and are essential for the selective translation of stress‐inducible genes. Ras‐GTPase‐activating protein SH3 domain‐binding protein 1 (G3BP1) is a component of SGs that initiates the assembly of SGs by forming a multimer. In this study, we examined the role of G3BP2, a close relative of G3BP1, in SG formation. Although single knockdown of either G3BP1 or G3BP2 in 293T cells partially reduced the number of SG‐positive cells induced by arsenite, the knockdowns of both genes significantly reduced the number. G3BP2 formed a homo‐multimer and a hetero‐multimer with G3BP1. Moreover, like G3BP1, the overexpression of G3BP2 induced SGs even without stress stimuli. Collectively, these results suggest that both G3BP1 and G3BP2 play a role in the formation of SGs in various human cells and thereby recovery from these cellular stresses.

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.

Stress Granules Inhibit Apoptosis by Reducing Reactive Oxygen Species Production

ABSTRACT Cells can undergo two alternative fates following exposure to environmental stress: they either induce apoptosis or inhibit apoptosis and then repair the stress-induced alterations. These processes minimize cell loss and prevent the survival of cells with aberrant DNA and protein alterations. These two alternative fates are partly controlled by stress granules (SGs). While arsenite, hypoxia, and heat shock induce the formation of SGs that inhibit apoptosis, X-ray irradiation and genotoxic drugs do not induce SGs, and they are more prone to trigger apoptosis. However, it is unclear precisely how SGs control apoptosis. This study found that SGs suppress the elevation of reactive oxygen species (ROS), and this suppression is essential for inhibiting ROS-dependent apoptosis. This antioxidant activity of SGs is controlled by two SG components, GTPase-activating protein SH3 domain binding protein 1 (G3BP1) and ubiquitin-specific protease 10 (USP10). G3BP1 elevates the steady-state ROS level by inhibiting the antioxidant activity of USP10. However, following exposure to arsenite, G3BP1 and USP10 induce the formation of SGs, which uncovers the antioxidant activity of USP10. We also found that the antioxidant activity of USP10 requires the protein kinase activity of ataxia telangiectasia mutated (ATM). This work reveals that SGs are critical redox regulators that control cell fate under stress conditions.

Hemadsorption and fusion inhibition activities of hemagglutinin analyzed by vaccinia virus mutants

Vaccinia virus IHD-J strain induces hemagglutinin (HA) on the surface membrane of infected cells and does not elicit cell-cell fusion (F-). We isolated 21 independent hemadsorption-negative (HAD-) mutant viruses from IHD-J and five HAD+ revertants from one of these mutants. Of the 21 mutants, 19 that synthesized either no or little HA at the cell surface caused cell-cell fusion (F+), whereas none of the five revertants that synthesized HA at the cell surface induced cell-cell fusion. Furthermore, anti-HA monoclonal antibody B2D10 induced extensive polykaryocytosis of IHD-J-infected cells and suppressed the ability of the IHD-J-infected cell extract to inhibit the polykaryocytosis induced by IHD-W. The other 2 of the 21 HAD- mutants, B1 and A2, which induced HAs at the cell surface, showed F- and F+ phenotype, respectively. The HA molecule of mutant B1 had a single amino acid substitution of Lys for Glu-121 in its extracellular domain, whereas that of mutant A2 had a single substitution mutation of Tyr for Cys-103. We conclude that the vaccinia HA is a fusion inhibition protein, that the active sites for the two activities reside separately in its extracellular domain, and that cysteine-103 is important in forming the proper tertiary structure of the protein to exert both activities.

PDZ domain-binding motif of human T-cell leukemia virus type 1 Tax oncoprotein is essential for the interleukin 2 independent growth induction of a T-cell line

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL), whereas HTLV type 2 (HTLV-2), is not associated with ATL or any other leukemia. HTLV-1 encodes the transforming gene tax1, whose expression in an interleukin (IL)-2-dependent T-cell line (CTLL-2) induces IL-2-independent growth.ResultsIn this study, we demonstrated that IL-2-independent growth induction by Tax1 was abrogated by mutations of the PDZ domain-binding motif (PBM) at the Tax1 C-terminus. HTLV-2 Tax2, which shares 75% amino acid identity with Tax1 but does not have a PBM, was not able to induce IL-2-independent growth of CTLL-2.ConclusionOur results suggest that Tax1, through interaction with PDZ domain protein(s) induces IL-2-independent growth, which may be a factor in multi-step leukemogenesis caused by HTLV-1.

Identification of a novel motif responsible for the distinctive transforming activity of human T-cell leukemia virus (HTLV) type 1 Tax1 protein from HTLV-2 Tax2

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia (ATL), whereas its relative HTLV-2 is not associated with any malignancies including ATL. HTLV-1 Tax1 transformed a T-cell line from interleukin (IL)-2-dependent growth to IL-2-independent growth, with an activity that was much more potent in comparison to HTLV-2 Tax2. This distinction was mediated by at least two Tax1 specific functions, an interaction with host cellular factors through the PDZ domain binding motif (PBM) and the activation of NF-kappaB2 (NF-κB2)/p100.ResultsUsing a series of Tax1 chimeric proteins with Tax2, we found that amino acids 225-232 of Tax1, the Tax1(225-232) region, was essential for the activation of NF-κB2 as well as for the high transforming activity. The strict amino acid conservation of Tax1(225-232) among HTLV-1 and simian T-cell leukemia virus type 1 (STLV-1), but not HTLV-2 and STLV-2, indicates that function(s) through the Tax1(225-232) region are biologically significant. Interestingly, another HTLV-1 relative, HTLV-3, has a PBM, but does not conserve the Tax1(225-232) motif in Tax3, thus indicating that these two motifs classify the three HTLVs into the separate groups.ConclusionThese results suggest that the combinatory functions through Tax1(225-232) and PBM play crucial roles in the distinct biological properties of the three HTLVs, perhaps also including their pathogenesis.

Trends in transmitted drug-resistant HIV-1 and demographic characteristics of newly diagnosed patients: Nationwide surveillance from 2003 to 2008 in Japan

The emergence and transmission of drug-resistant human immunodeficiency virus-1 (HIV-1) compromises antiretroviral treatment for HIV-1. Thus, testing for drug resistance is recommended at diagnosis and before initiating highly active antiretroviral treatment. We conducted an epidemiological study enrolling newly diagnosed patients between 2003 and 2008 in our nationwide surveillance network. In the 6-year study period, the prevalence of drug-resistant HIV-1 among 2573 patients, consisting mainly of Japanese men in their late-30s and infected through male-to-male sexual contacts, followed an increasing trend from 5.9% (16/273) in 2003 to 8.3% (50/605) in 2008. Nucleoside reverse transcriptase inhibitor-associated mutations predominated in each year, with T215 revertants being the most abundant. The predictive factor for drug-resistant HIV-1 transmission was subtype B (OR=2.36; p=0.004), and those for recent HIV-1 infection were male gender (OR=3.79; p=0.009), MSM behavior (OR=1.67; p=0.01), Japanese nationality (OR=2.31; p=0.008), and subtype B (OR=5.64; p<0.05). Continued activities are needed to raise awareness of the risks of HIV-1 infection and complications of drug-resistant strains. Continued surveillance is also needed to understand trends in the HIV-1 epidemic.