In normal human cells, telomeres shorten with successive rounds of cell division, and immortalization correlates with stabilization of telomere length. These observations suggest that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression. Here, we demonstrate that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling primary presenescent human fibroblasts, previously believed to lack hTERT expression and telomerase activity. Disruption of telomerase activity in normal human cells slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3' single-stranded telomeric overhang without changing the rate of overall telomere shortening. Together, these observations support the view that telomerase and telomere structure are dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence.

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The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRP), which is the central catalytic enzyme of HCV replicase. We established a new method to purify soluble HCV NS5B in the glutathione S-transferase-fused form NS5Bt from Escherichia coli which lacks the C-terminal 21 amino acid residues encompassing a putative anchoring domain (anino acids 2990 -3010). The recombinant soluble protein exhibited RdRP activity in vitro which was dependent upon the template and primer, but it did not exhibit the terminal transferase activity that has been reported to be associated with the recombinant NS5B protein from insect cells. The RdRP activity of purified glutathione S-transferaseNS5Bt and thrombin-cleavaged non-fused NS5Bt shares most of the properties. Substitution mutations of NS5Bt at the GDD motif, which is highly conserved among viral RdRPs, and at the clustered basic residues (amino acids 2919 -2924 and 2693-2699) abolished the RdRP activity. The C-terminal region of NS5B, which is dispensable for the RdRP activity, dramatically affected the subcellular localization of NS5B retaining it in perinuclear sites in transiently overexpressed mammalian cells. These results may provide some clues to dissecting the molecular mechanism of the HCV replication and also act as a basis for developing new anti-viral drugs.

Chronic infection of hepatitis B virus (HBV) isH epatitis B virus (HBV) infection is a worldwide health problem and is one of the major causes of hepatocellular carcinoma (HCC) in the world. However, new incidences of HBV infection have been dramatically reduced by several prevention programs. The crucial role of HBV in hepatocarcinogenesis is beyond doubt, however, the mechanism by which HBV causes transformation of hepatocytes remains uncertain.(1-3) Hepatitis B virus X protein (HBx) has long been suspected to play a positive role in hepatocarcinogenesis because HCC incidence has been reported in animals infected with mammalian hepadnaviruses. Among these hepadnaviruses, open reading frame-X (X-ORF) is conserved in the genomes. However, hepatocarcinogenesis has not been found in avian infected with avian hepadnaviruses where X-ORF is absent. In addition to the putative contribution of HBx, other oncogenic mechanisms of the HBV-related hepatocarcinogenesis have also been proposed, such as the integration-dependent scenario of HBV DNA in host genomes, (4) and the inflammation-dependent scenario.(5) Since no higher incidence of HCC has been reported in HBV-expressing transgenic mice, (6) host immunological responses to HBV infection (but not HBV proliferation by itself ) might be primarily contributing to the HBV-related hepatocarcinogenesis. HBx, a small protein of 154 amino acids, is a multifunctional regulator that modulates a variety of host processes through directly or indirectly interacting with virus and host factors. (2,3) In this short review, we briefly summarize the life cycle of HBV, expression of HBx, and the molecular functions of HBx. We then focus on the recent progress on the biological roles of HBx in HBV replication and cellular transformation. Expression and functions of HBx. HBV is a prototype of Hepadnaviridae, hepatotropic small DNA viruses, of which hosts are among limited species of water birds, squirrels and primates. The HBV genome is a 3.2-kb circular, partially double-stranded DNA molecule with four overlapping ORFs, PC-C, PS-S, P, and X-ORFs (Fig. 1). (7) Once HBV infection of hepatocytes occurs, its genome is converted to covalently closed circular (CCC) DNA, which serves as the template for transcription by the host RNA polymerase II, generating the 3.5-, 2.4-, 2.1-, and 0.7-kb transcripts under control of four HBV promoters (Cp, PS1p, Sp, and Xp), respectively (Fig. 1). Two HBV enhancers (Enh I and Enh II) positively regulate transcription of the HBV promoters in combination with the transcription factors that bind these promoters.(7-9) HBV replicates by reverse transcription of the viral pregenomic 3.5-kb RNA (pgRNA) using the HBV polymerase that catalyzes RNA-dependent DNA synthesis and DNA-dependent DNA synthesis. (7,10) The reverse transcription of hepadnaviruses is unique among DNA viruses, and the primer of the reverse transcription is a tyrosine residue of an N-terminal domain of Pol that is distinct from reverse transcription of retroviruses. (10)

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