Fig 1.
Structure of telomerase reverse transcriptase (TERT).
TERT contains a long NTE1 linked with a central catalytic RT domain and a short carboxy CTE, (TEN, yellow; TRBD, orange; fingers, gray; RT, blue; CTE, magenta) with conserved motifs in TRBD (T, green; CP, red) or RT motifs (IFD, cyan) or black. The specific T and CP motifs in the TRBD recognize essential components of telomerase RNA. The specific motifs 3 and IFD are conserved areas located on surface of RT that likely interact with the active site through helical arrangements. 1Abbreviations: NTE = N-terminal extension; TEN = Telomerase essential N-terminal domain; TRBD = Telomerase RNA binding domain; RT = reverse transcriptase; CTE = C-terminal extension; IFD = insertion in fingers domain.
Fig 2.
TERT activation and expression after HCVcc infection.
(A) Primary human hepatocytes (PHH) infected with J6JFH (HCVcc) Right panel: HCV RNA and TERT mRNA were assayed in RNA isolates using quantitative RT-PCR. Telomerase activity was assayed in cellular lysates by Real-time Telomeric Repeat Amplification Protocol-Reverse Transcriptase PCR (TRAP-RT PCR) from cells collected at the indicated days post infection. HCV RNA p<0.05; day 2 vs 4 and p<0.01 day 7 vs 4. Real-time Telomerase enzyme activity p<0.01 all points as compared to day 2. TERT mRNA p<0.01 all points as compared to day 1. Left panel: WB analyses of cellular lysates for TERT protein and HCV NS3 taken on the indicated days post infection. (B) Huh-7.5 cells infected with HCVcc. Right panel: HCV RNA and TERT mRNA were assayed in RNA isolates using quantitative RT-PCR. Telomerase activity was assayed in cellular lysates by Real-time TRAP-RT PCR from cells collected at the indicated days post infection. HCV RNA p<0.01 all points as compared to day 2. TRAP activity p<0.01 all points as compared to day 0. TERT mRNA p<0.01 all points as compared to day 1. Left panel: WB analyses of cellular lysates for TERT protein and HCV NS3 taken on the indicated days post infection. (C) WB and TRAP analysis in Non-structural (NS) or Full length (FL) HCV replicon lines. Left panel: Log phase cultures were processed for WB analysis. Protein-blotted membranes were probed with anti-TERT (C-terminal specific) or anti-NS3 antibodies. Passage of replicons without G-418 selection medium for > 5 days (right two lanes) led to loss of HCV infection (absence of NS3-4A) and expression of 45 kD TERT fragment. Right panel: TRAP activity was assayed in replicon or control (Huh-7 or Huh-7.5) cell lysates by Real-time RT-PCR in 2–3 day log phase cultures. (TRAP activity of control cells < replicons p<0.01). (D) HCVcc infection enhances TERT promoter function. pBT255-luc (100ng/ml) and pRL-CMV (Renilla) (2ng/ml) plasmid were co-transfected into permissive Huh-7.5 cells. After 24 hrs 1.0 Multiplicity of Infection (MOI) of HCVcc was added to the cultures. The luciferase activity in cell lysates was determined at various times thereafter with dual luciferase reporter assay. p<0.01 all bars, as compared with day 0.
Fig 3.
Immunoprecipitation: NS3-4A binds TERT.
(A) N-FLAG-labelled TERT plasmid was transfected into HEK-293 cells together with vector only or vectors containing NS3-4A, N-FLAG NS5A, or core. FLAG-NS5A served as internal FLAG control to ensure TERT did not bind FLAG. Cellular lysates were then immunoprecipitated *(IP) with the indicated antibodies and the IPs evaluated on WB visualized with anti-FLAG antibody. Right panel shows sizing and verification of input protein on WB stained with indicated Immunoblot antibody (IB). (B) Log-phase replicons (Huh 5-15NS) or (C) vector transfected HEK-293 cells were harvested, lysed in cell lysis buffer, and then incubated with the indicated antibodies for immunoprecipitation. The IPs were subjected to gel SDS electrophoresis and the products assessed on WB using specific antibodies. (D) Cell lysates from Huh 5.15NS replicons were treated with RNAase A, DNAase I, or ethidium bromide prior to immunoprecipitation. The IPs were evaluated on WB stained with the indicated IB antibody. RNAase H (not shown) also had no effect. *Abbreviations: (IP) = immunoprecipitation antibody. (IB) = Immunoblot antibody. Anti-TERT antibody = rabbit monoclonal specific to C-terminal end of TERT. Anti-NS3 antibody = rabbit polyclonal antibody. H = heavy Immunoglobulin chain, L = Light immunoglobulin chain. Immunoglobulin bands were expected in some cases from reactivity of second antibody for immunoglobulin in the IP.
Fig 4.
(A) Binding of NS3-4A fragments to endogenous TERT. (i). Fragments of NS3-4A were constructed in 3xFLAG-CMV vectors with an N-terminal FLAG tag. ii). Following transfection of FLAG-NS3-4A fragments into HEK-293 cells, immunoprecipitation (IP) for endogenous TERT was performed using anti-TERT antibody and the complexes evaluated on WB using anti-FLAG antibodies (upper blot). Sizing of N-FLAG NS3-4A fragments after transfection on immunoblots stained with anti-FLAG antibody (lower blot). Abbreviations are as listed in Fig 3. (B) Binding of TERT fragments to NS3-4A. (i) Fragments of TERT were constructed in p3XFLAG-CMV-10 vectors with an N-terminal FLAG tag. (ii) The vectors were transfected into HEK-293 cells together with pcDNA3.1 NS3-4A and immunoprecipitation (IP) was performed using anti-NS3-4A antibody. IPs were evaluated on immunoblots using anti-FLAG antibodies (upper panel). Appropriate sizing of N-FLAG TERT fragments was on WB stained with anti-FLAG antibody (lower panel). Abbreviations are as listed in Fig 3.
Fig 5.
Binding of NS3-4A to TERT holoenzyme complexes.
(A) NS3-4A association with TERT holoenzyme complexes. NP-40 buffer extracts from Huh-5.15NS replicons were subjected to 10–30% glycerol gradient centrifugation as described in Methods. Gradients were then fractionated into 400ul fractions and 10ul aliquots of each were analyzed on WB using specific antibodies to the indicated proteins. (B) Telomerase activity in holoenzyme gradient fractions. 10 μl aliquots of each gradient fraction (A) were subjected to TRAP-RT-PCR assay in triplicate and quantified relative to input signal telomerase activity. Each bar represents the mean +/- SEM. Relative telomerase activity of fractions > 11 vs fractions < 11, p < 0.001.
Fig 6.
HCV infection triggers Caspase degradation of TERT.
(A) Left panel, log phase Huh 5.15NS replicons were incubated with the pancaspase inhibitor (z-vad-fmk) (200uM) and at various times cellular lysates were assayed on WB for TERT or NS3-4A using C-terminal specific antibody. In right panel, log phase Huh 5.15NS replicons were incubated with indicated amounts of the specific Caspase 7 inhibitor (Millipore 28832) for 48 hr. Cellular lysates were then assayed on WB for TERT using C-terminal specific antibody. In the lower panel, log phase Huh 5.15NS replicons were incubated with pancaspase inhibitor or vehicle control for the indicated times and relative telomerase activity was determined in the cellular lysates as compared to day 0 using Real-time TRAP-RT PCR. *[Pancaspase inhibitor > control days 2, 3, and 4, p< 0.01] (B) Huh-7.5 cells were either infected with HCVcc (right panel) or mock control (left panel) and on various days WB were performed to detect TERT, NS3, and un-activated full length caspases 6 and 7 as well as cleaved fragments as indicated. #Antibodies recognizing both full length and upper cleaved caspase fragments were from Cell Signaling (Caspase 7 #9492 and Caspase 6 #9762). (C) Huh-7.5 or Huh-7 cells were infected with HCVcc and on various days assayed for TERT as well as lower kD cleaved fragments of caspase 6 and 7 by WB. Cell Signaling *ASP 162 antibody (#9761) was used to detect cleaved caspase 6 and **ASP 198 antibody (#8438) was used to detect cleaved caspase 7 with products appearing at 18 kD in both cases.
Fig 7.
Immunocytochemical localization of TERT and NS3.
(A) Uninfected or HCV infected log-phase Huh-7.5 cells were reacted with anti-TERT or anti-NS3 antibodies, then with Alexa Fluor 488 (green) or Alexa Fluor 568 (red) labelled second antibodies respectively. TO-PRO was used to visualize nuclei (lower panel). (B) Uninfected log phase Huh 7 cells (upper panel), were labelled with anti-TERT then co-labelled with MitoTracker (red). Confocal microscopy to merge images was performed on a Zeiss LSM710 confocal fluorescence microscope. Merged fluorescence is yellow. Log-phase Huh-5.15 replicons were labelled with anti-TERT (middle panel) or anti-NS3 (lower panel) antibodies, (green), then co-labelled with MitoTracker. Confocal images were generated as above.
Fig 8.
NS3-4A stimulates telomerase activity.
(A) NS3-4A stimulates activity of cell free mixtures of TERT and TERC. NS3-4A, TERT and TERC were prepared from pcDNA 3.1 vectors using RRL cell free incubations in independent tubes. For TERC, T7 runoff transcripts were prepared from TERC DNA vector and RNA was gel purified from RRL incubations. Equivalent amounts of TERT and TERC were incubated with increasing amounts of NS3-4A or empty vector and subjected to real-time TRAP assay (Left panel) or TRAP products visualized on non-denaturing 12% PAGE using TRAPeze (Right Panel). WB, lower left shows TRAP loading controls. Abbreviations: bp = base pairs, IC = internal control = 36 bp. Initial product on gel is 54 bp. Note label of TRAP products. (B) NS3-4A stimulates telomerase activity of TERT holoenzyme complexes. Lysates from log-phase uninfected Huh-7 cells were separated on 10–30% glycerol gradients. 10 ul aliquots of gradient fractions 17–19 were mixed with various amounts of NS3-4A which were generated from cell-free RRL incubations of NS3-4A pcDNA3.1 vector. The mixtures were either assayed by real-time TRAP assay (Left panel) or TRAP products visualized with TRAPeze (Right panel) as described for Fig 8A. WB, lower left shows TRAP loading controls. Abbreviations: as in Fig 8A. (C) NS3-4A transfection. HEK-293 cells were transfected with 1ug (as DNA) of vector containing NS3-4A, empty vector, or mock control. 48 hr later, cells were lysed and telomerase activity determined with real time TRAP assay (Left panel) or TRAP products visualized with TRAPeze (Right panel). WB, middle panel shows TRAP loading controls. Abbreviations: as in Fig 8A. Gel lanes shown for Fig 8C were from the same gel as Fig 8B, consequently, the 10 bp ladder is identical. (D) HCV infection. Huh-7.5 cells were infected with HCVcc and 2 days later telomerase activity was determined with real time TRAP assay (Left panel) or TRAP products visualized with TRAPeze (Right panel). (E) Specificity of NS3-4A stimulation of telomerase. All probes were constructed as we reported previously [24]. Translational products were prepared from pcDNA 3.1 vectors using RRL cell free system in independent tubes. TERC was prepared as described in 8C, then equal amounts were added to a constant amount of TERT and various amounts of NS3-4A, core, NS5A, or NS3-4A with primuline (5 uM) (NS3 helicase inhibitor) or danoprevir (1nM) (NS3 protease inhibitor). Telomerase activity was quantified by realtime TRAP- RT PCR assay (upper panel). Input RRL protein products were verified by immunoblots (lower panel). [NS3-4A and TERT aliquots were from same RRL incubation as used for Fig 8A]. [NS3-4A mutant = catalytically silent protease from serine-alanine mutation (HCV H strain serine 139 to alanine)]. (F) Inhibition of NS3-4A-holoenzyme complexes. Holoenzyme complexes were prepared from 10–30% glycerol gradient fractions of cellular extracts obtained from HCV replicons (Huh-5-15NS Upper panel) or control HEK-293 cells. Telomerase activity was assayed in each fraction using realtime TRAP- RT PCR with or without 1 nm danoprevir or 5 uM primuline. [*p< 0.05 **p < 0.01 from uninhibited control reaction.]