Genetic variability and diversity of intracellular genome-length hepatitis C virus RNA in long-term cell culture.

Hepatitis C virus (HCV) is known to circulate persistently in vivo as a complex population of different but closely related viral variants. To understand the quasispecies nature of HCV, we performed genetic analysis of intracellular HCV RNAs obtained in long-term cell culture of genome-length HCV-RNA-replicating cells. The results revealed that genetic mutations in HCV RNAs accumulated in a time-dependent manner, and that the mutation rates of HCV RNAs were 3.5-4.8 x 10(-3) base substitutions/site/year. The mutation rates of nonstructural regions that are essential for RNA replication were lower than those of structural regions. The genetic diversity of HCVs was also enlarged in a time-dependent manner. Furthermore, we found that the GC content of HCV RNA was increased in a time-dependent manner. These results suggest that an HCV-RNA-replicating cell culture system would be useful for analysis of the evolutionary dynamics and variations of HCV.

Functional cross-talk of HIV-1 Tat with p53 through its C-terminal domain.

Human immunodeficiency virus type 1 (HIV-1) Tat repressed the p53-dependent gene expression through its C-terminal domain of Tat (amino acid residues 73-86) independent of the involvement of NF-kappaB and coactivator CBP/p300. Although Tat did not directly bind to p53, this repression required the N-terminal domain of p53. In contrast, Tat and p53 cooperated in the activation of HIV-1 gene expression. Thus, the cross-talk between Tat and p53 may be linked with cellular transformation by HIV-1 infection or activation of HIV-1 replication.

HTLV-1 tax oncoprotein represses the p53-mediated trans-activation function through coactivator CBP sequestration.

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein repressed the transcriptional activity of wild-type p53 through its N-terminal trans-activation domain. Although Tax did not directly bind to p53, this repression required the activation of CREB pathway by Tax. In contrast to a recent report by Pise-Masison et al. (1998a, b) we found that the phosphorylation of p53 on Ser 15 is not a major cause of the Tax-mediated inactivation of p53. However, Tax with a mutation in the coactivator CBP-binding site (K88A), which activates NF-kappaB but not the CREB pathway, could not repress the p53 trans-activation function. Moreover, Tax inhibited p53 binding to CBP in vitro and inhibited synergistic activation of transcription by CBP and p53. Thus, Tax is likely to compete with p53 in binding with CBP, thereby repressing its trans-activation function.

Functional impairment of p73 and p51, the p53-related proteins, by the human T-cell leukemia virus type 1 Tax oncoprotein.

We have previously demonstrated that the human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein represses the trans-activation function of p53 tumor suppressor protein. Recently, several proteins with sequence homology to p53 have been identified. In this study, we demonstrated that Tax represses the trans-activation functions of p73alpha, p73beta, and p51A, the p53-related proteins, as well as p53. Moreover, a mutant Tax of coactivator CBP-binding site (K88A), which activated NF-kappaB but not CREB pathway, could not repress the p73 nor p51 trans-activation functions, indicating that CBP-binding domain of Tax is essential for the suppression of their functions. Using proteins of Gal4-fused N-terminal region of p73 and p51, we showed that Tax-mediated inactivation of p73 or p51 requires for their N-terminal trans-activation domains. Furthermore, only the putative N-terminal trans-activation domains of them did not have enough transcriptional activities and their adjacent regions are essential for their full trans-activation, suggesting the existence of their second trans-activation subdomains. Thus, HTLV-1 Tax inactivated the p53-related proteins through their N-terminal trans-activation domains.

Suppression of the poly(ADP-ribose) polymerase activity by DNA-dependent protein kinase in vitro.

It has been suggested that DNA-dependent protein kinase (DNA-PK) is a central component of DNA double-strand-break repair. The mechanism of DNA-PK action, however, has not been fully understood. Poly(ADP-ribose) polymerase (PARP) is another nuclear enzyme which has high affinity to DNA ends. In this study, we analysed the interaction between these two enzymes. First, DNA-PK was found to suppress the PARP activity and alters the pattern of poly(ADP-ribosyl)ation. Although DNA-PK phosphorylates PARP in a DNA-dependent manner, this modification is unlikely to be responsible for the suppression of PARP activity, since this suppression occurs even in the absence of ATP. Conversely, PARP was found to ADP-ribosylate DNA-PK in vitro. However, the auto-phosphorylation activity of DNA-PK was not influenced by this modification. In a competitive electrophoretic mobility shift assay, Ku 70/80 complex, the DNA binding component of DNA-PK, was found to have higher affinity to a short fragment of DNA than does PARP. Furthermore, co-immunoprecipitation analysis suggested direct or close association between Ku and PARP. Thus, DNA-PK suppresses PARP activity, probably through direct binding and/or sequestration of DNA-ends which serve as an important stimulator for both enzymes.

In vivo phosphorylation of poly(ADP-ribose) polymerase is independent of its activation.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme, which is activated by DNA strand breaks. Although PARP is known to be cleaved by the cysteine protease, caspase-3/CPP32, during apoptosis, signal cascade which regulates the PARP activity has not been fully understood. In this study, we investigated post-translational modification of PARP. We found that PARP was phosphorylated by a serine kinase in vivo. PARP was activated temporarily and extensive auto-modification occurred on PARP, possibly by the fragmented DNA during apoptosis induced by etoposide in Jurkat cells. However, the phosphorylation level was not changed for up to 6 h, after PARP cleavage began in apoptosis by the treatment with etoposide. Furthermore, we showed the presence of a PARP-associated kinase in nuclear extracts of the HTLV-I infected T-cell lines but not in uninfected T-cell lines, whereas this kinase did not inhibit the PARP activity even in the presence of ATP. Taken together, in vivo phosphorylation of PARP might be independent of the activation or cleavage of PARP.

Characterization of the internal promoter of human T-cell leukemia virus type I.

The HTLV-I provirus contains two different promoters: the classical retroviral promoter in the 5' long terminal repeat (LTR) and our previously identified second promoter in the pol gene just upstream of the ATG codon of the tax gene. Here, we demonstrated that the internal promoter expresses the gene for Tax but not Rex. As the deletion of upstream of the transcriptional initiation site (nt 5130) caused down-regulation of the promoter activity, we termed the region HTLV-I internal regulatory element (HIRE). We found a cellular sequence-specific DNA binding protein which binds to HIRE. Furthermore, we demonstrated that the 3' LTR regulates Tax expression from the internal promoter. These findings may shed light on a novel mechanism for gene expression in complex retroviruses of the HTLV family.

HIRF: a novel nuclear factor that binds to the human T-cell leukemia virus type I internal regulatory element (HIRE).

The transcription of human T-cell leukemia virus type I (HTLV-I) provirus starts from a promoter located in the 5' long terminal repeat (LTR). We have identified a second promoter at the 3' end of the pol gene. This internal promoter expresses the Tax transactivator protein, but does not require Tax for its activity. Furthermore, we have found the novel enhancer motif AGTTCTGCCC, which are located near the initiation site. We have named the sequence HIRE (HTLV-I internal regulatory element). The HIRE binding protein is a ubiquitous protein. We purified this protein from the HTLV-I producing cell line MT-2 cells by DNA affinity chromatography. SDS-PAGE analysis revealed four major bands (70, 85, 115 and more than 200 kDa) and some minor bands on the gel. We renatured each major protein and showed the 70 and 115 kDa proteins bind to DNA, although the 115 kDa protein seemed to bind nonspecifically. We have designated these components as HIRF (HTLV-I internal regulatory factor).

Novel internal promoter/enhancer of HTLV-I for Tax expression.

The transcription initiation signals in retroviruses lie within the long terminal repeat (LTR) sequences. We have found a new transcriptional promoter in a central portion of the genome of human T-cell leukaemia virus type I (HTLV-I). The transcription start site is located just upstream to the ATG codon of the transcriptional trans-activator molecule, tax protein (Tax). The internal promoter may provide a new insight into gene expression of HTLV-I. The mechanism of leukaemogenesis by the defective HTLV-I is also discussed. Furthermore, we have identified two repeats of a novel enhancer sequence AGTTCT, which are located around the initiation site. We call the sequence HIRE (HTLV-I Internal Regulatory Element).

Cytotoxic activity of rev protein of human immunodeficiency virus type 1 by nucleolar dysfunction.

The rev protein (Rev) of the human immunodeficiency virus type 1 (HIV-1) is known as a post-transcriptional regulator of viral gene expression. It is located in the cell nucleolus. Transiently expressed Rev caused nucleolar ballooning and deformity with aberrant accumulation of rRNAs, and de novo synthesis of rRNAs decreased dramatically in these cells. However, similarly expressed rex protein (Rex) of the human T-cell leukemia virus type I, which is a functional homologue to Rev, did not affect nucleolar structure and function. Rev expression resulted in cell death with nucleolar destruction in an inducible cell line. Analysis of Rev mutants revealed that both the nucleolar targeting signal of Rev and the multimerization domain are prerequisites to the nucleolar disintegration by Rev. Human T-cells acutely infected with HIV-1 contained nucleoli which were deformed and filled with Rev, but chronically infected cells had intact nucleoli. Involvement of Rev in cytopathic effects in HIV-1 infection is discussed.