HCV Induces Telomerase Reverse Transcriptase, Increases Its Catalytic Activity, and Promotes Caspase Degradation in Infected Human Hepatocytes

Abstract

IntroductionTelomerase repairs the telomeric ends of chromosomes and is active in nearly all malignant cells. Hepatitis C virus (HCV) is known to be oncogenic and potential interactions with the telomerase system require further study. We determined the effects of HCV infection on human telomerase reverse transcriptase (TERT) expression and enzyme activity in primary human hepatocytes and continuous cell lines.ResultsPrimary human hepatocytes and Huh-7.5 hepatoma cells showed early de novo TERT protein expression 2–4 days after infection and these events coincided with increased TERT promoter activation, TERT mRNA, and telomerase activity. Immunoprecipitation studies demonstrated that NS3-4A protease-helicase, in contrast to core or NS5A, specifically bound to the C-terminal region of TERT through interactions between helicase domain 2 and protease sequences. Increased telomerase activity was noted when NS3-4A was transfected into cells, when added to reconstituted mixtures of TERT and telomerase RNA, and when incubated with high molecular weight telomerase ‘holoenzyme’ complexes. The NS3-4A catalytic effect on telomerase was inhibited with primuline or danoprevir, agents that are known to inhibit NS3 helicase and protease activities respectively. In HCV infected cells, NS3-4A could be specifically recovered with telomerase holoenzyme complexes in contrast to NS5A or core protein. HCV infection also activated the effector caspase 7 which is known to target TERT. Activation coincided with the appearance of lower molecular weight carboxy-terminal fragment(s) of TERT, chiefly sized at 45 kD, which could be inhibited with pancaspase or caspase 7 inhibitors.ConclusionsHCV infection induces TERT expression and stimulates telomerase activity in addition to triggering Caspase activity that leads to increased TERT degradation. These activities suggest multiple points whereby the virus can influence neoplasia. The NS3-4A protease-helicase can directly bind to TERT, increase telomerase activity, and thus potentially influence telomere repair and host cell neoplastic behavior.

Zinc Protoporphyrin Binding to Telomerase Complexes and Inhibition of Telomerase Activity

“…6B left panels). As we reported previously, TERT is sparsely present in the nucleus of unsynchronized, log phase Huh7 cells, but is chie y found at perinuclear sites which co-localize with mitochondria (21). Interestingly, even perinuclear TERT, likely lacking telomeric DNA, showed avid TERT-ZnPP co-localization (Fig.…”

“…The effects of ZnPP on TERT expression were apparent in different Huh 7 constructs and, interestingly, in a NS 5.15 HCV replicon, ZnPP promoted disappearance of both 120 KD telomerase monomer as well as the 45 KD C-terminal TERT fragment that we previously reported to be speci c for HCV infected cells (Fig. 2B), (21).…”

“…Plasmid pCl neo-hEST2 together with TERC (pBS U3-hTR-500) were transfected into log phase HEK293 cells and non-denaturing cellular lysates were prepared 48 hr later in lysis buffer (Cell Signaling Technology, Beverly, MA). Immunoprecipitation was performed as described previously (21). Brie y, transfected cells were harvested, washed in PBS, lysed in cell lysis buffer, and clari ed by cold centrifugation (14,000xg for 10 min).…”

“…Telomerase RNA component (TERC) plasmid (pBS U3-hTR-500) was also obtained from Addgene (plasmid # 28170), a gift from Dr. Kathleen Collins (20). Construction and characterization of Flag-labelled human TERT and wild type human TERT plasmids were as described (21).…”

<p>Hepatocellular Carcinoma Mechanisms Associated with Chronic HCV Infection and the Impact of Direct-Acting Antiviral Treatment</p>

“…Recent publications by Zhu et al suggest that HCV core and NS3 proteins can activate TERT expression and reverse transcriptase activity. 130,131 The p53-Rb Pathway…”