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.

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.

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

While human T-cell leukemia virus type 1 (HTLV-1) is associated with the development of adult T-cell leukemia (ATL), HTLV-2 has not been reported to be associated with such malignant leukemias. HTLV-1 Tax1 oncoprotein transforms a rat fibroblast cell line (Rat-1) to form multiple large colonies in soft agar, and this activity is much greater than that of HTLV-2 Tax2. We have demonstrated here that the increased number of transformed colonies induced by Tax1 relative to Tax2 was mediated by a PDZ domain-binding motif (PBM) in Tax1, which is absent in Tax2. Tax1 PBM mediated the interaction of Tax1 with the discs large (Dlg) tumor suppressor containing PDZ domains, and the interaction correlated well with the transforming activities of Tax1 and the mutants. Through this interaction, Tax1 altered the subcellular localization of Dlg from the detergent-soluble to the detergent-insoluble fraction in a fibroblast cell line as well as in HTLV-1-infected T-cell lines. These results suggest that the interaction of Tax1 with PDZ domain protein(s) is critically involved in the transforming activity of Tax1, the activity of which may be a crucial factor in malignant transformation of HTLV-1-infected cells in vivo.

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-B2/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-B2/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-B2/p100 plays a crucial role in the Tax1-mediated transformation of T cells and that NF-B2/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.

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's 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 (⌬N-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 ⌬N-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.Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is a gamma 2 herpesvirus and is associated with the development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease (5,6,10,17,21,24). KSHV can establish long-term persistent infections in tumor cells and lymphoma-derived cell lines. In such cells, the double-stranded KSHV DNA genome can persist as multiple copies of closed circular episomes (5, 9), like the genome of its close relative, Epstein-Barr virus (EBV).Two viral components can mediate the long-term episome maintenance of KSHV in infected cells. One is latency-associated nuclear antigen 1 (LANA1), encoded by open reading frame 73, and the other is a cis-acting DNA sequence (latent origin of replication [oriP]) in the 5Ј end of the KSHV genome (terminal repeat [TR] sequence) (1, 2, 7). LANA1 persistently maintains a plasmid containing multiple TRs as an episome in a human B-cell line (1, 2).Previous studies showed that LANA1 was directly bound to the KSHV TR sequence (2,8,11). Furthermore, an imperfect 20-bp palindrome in the TR sequence exhibited binding activity for LANA1, and a plasmid containing three TRs was persistently maintained as an episome in a human cell line (2, 11). Domain map analysis revealed that the C-terminal region of LANA1 bound to this 20-bp palindrome in the TR sequence (11). Since this region contains the LANA1 dimerization domain (22), it seems likely that LANA1 binds to the TR sequence as a dimer, like EBNA1, the episome maintenance protein of EBV.About 40 to 80 copies of episomes are stably maintained in KSHV-infected lymphoma cells in vivo (1, 4). The constant KSHV copy number in dividing cells indicates that KSHV has an efficient system for segregating viral episomes into two daughter cells in every cell division. In the present study, we examined the involvement of the chromosome interaction activity of LANA1 in long-term episome maintenance. A 22-amino-acid deletion from the N terminus of LANA1 (⌬N-LANA) inhibit...

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-B is reportedly essential for the transformation of Rat-1 cells. Tax2 also activated the transcription through NF-B 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-B 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.

The monoclonal antibody 2D5 neutralized vaccinia virus by preventing penetration of the virus and reacting with VP23-29K. The conformation of the VP23-29K was maintained by a disulfide bond(s), and the 2D5mAb reacted stronger with the nonreduced 23-kDa form than with the reduced 29-kDa form. We selected several escape mutants. Sequences of the A17L genes, which were thought to encode the VP23-29K, did not show cognate mutation. Genomic DNA of a 2D5mAb-resistant mutant (M4) was cleaved with HindIII, and all the fragments were introduced into parental IHD-J strain vaccinia virus by transfection. Only the L fragment produced a 2D5mAb-resistant virus. Dissection of the L fragment and subsequent transfection revealed that the L1R gene induced the 2D5mAb-resistant virus. The 2D5mAb-resistant mutants showed a consensus G to A conversion at nucleotide 101 of their LIRs which would replace asparatic acid 35 with asparagine. Ishibashi-111 strain mousepox virus spontaneously resistant to 2D5mAb also had the same sequence at this region. Moreover, the VP23-29K was myristoylated as predicted by the L1R gene. The coding gene of the VP23-29K was L1R.