PubMed · 7874788
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K R Huber, M Knapp, K Bauer. 1995. Subtyping hepatitis C virus.. https://pubmed.ncbi.nlm.nih.gov/7874788/
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Hepatitis C virus (HCV) establishes persistent infection by rewiring host stress-response pathways. Chaperone-mediated autophagy (CMA) contributes to HCV replication, but it remains unclear whether HCV regulates lysosome-associated membrane protein 2 A (LAMP-2A), the rate-limiting receptor for CMA. Here, we examined LAMP-2A regulation in HCV-infected Huh-7.5 cells. HCV infection increased LAMP-2A promoter activity, mRNA, and protein abundance, indicating transcriptional upregulation. Among candidate stress-responsive transcription factors, nuclear factor erythroid 2-related factor 2 (NRF2), hypoxia-inducible factor 1α (HIF-1α), and nuclear factor of activated T cells 1 (NFAT1) were elevated in infected cells. However, promoter mutagenesis identified NRF2 as the principal direct regulator. Mutation of the NRF2-responsive antioxidant response element markedly reduced basal and HCV-induced LAMP-2A promoter activity. Chromatin immunoprecipitation assays revealed NRF2 association with the LAMP-2A promoter, and HCV infection increased nuclear accumulation and Ser40 phosphorylation of NRF2. Functionally, shRNA-mediated knockdown of LAMP-2A reduced intracellular HCV RNA and protein levels. These findings identify an NRF2-LAMP-2A regulatory axis engaged during HCV infection and support a model in which HCV upregulates LAMP-2A to establish a cellular environment favorable for viral replication.
The biochemical properties of the RNA-dependent RNA polymerase (RdRp) of the hepatitis C virus were analyzed. A hexahistidine affinity-tagged NS5B fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. Enzymatic activity of the purified protein was inhibited by KCl or high concentrations of NaCl and was absolutely dependent on Mg2+, which could be replaced by Mn2+. NS5B was found to be processive and able to copy long heteropolymeric templates with an elongation rate of 150-200 nucleotides/min at 22 degreesC. Kinetic constants were determined for all four nucleoside triphosphates and different templates. In case of a heteropolymeric RNA template corresponding to the last 319 nucleotides of the hepatitis C virus genome, Km values for UTP, GTP, ATP, and CTP were approximately 1.0, approximately 0.5, approximately 10, and approximately 0.3 microM, respectively. The profile of several inhibitors of RdRp activity and substrate analogs indicated that the enzyme has a strong preference for ribonucleoside 5'-triphosphates and that it closely resembles 3Dpol of picornaviruses.
BACKGROUND: The presence of hepatitis C virus (HCV) replication in peripheral blood mononuclear cells (PBMCs) remains controversial. We determined the presence of the negative HCV RNA strand in PBMCs from a group of HCV-positive patients before and after liver transplantation. METHODS: Nine patients receiving orthotopic liver transplantation for end-stage HCV-related liver disease were studied. PBMCs were collected on the day of transplantation and 1 month later. The negative HCV RNA strand was detected by highly strand-specific Tth-based reverse transcriptase polymerase chain reaction. RESULTS: All nine patients were positive for the presence of the HCV RNA-positive strand in serum and PBMCs both before and after transplantation. The presence of the negative HCV RNA strand was documented in three PBMC samples after transplantation but in none of the samples collected before transplantation. CONCLUSION: Our results suggest that under circumstances of impaired immunity associated with pharmacological immunosuppression, HCV may be lymphotropic in vivo.