3.Proapoptotic and pronecrosis effect of different truncated hepatitis C virus core proteins.
Xue-bing YAN ; Zhi CHEN ; Dong-hui LUO ; Xiao-yan XU ; Wei WU ; Lin-fu ZHOU
Journal of Zhejiang University. Science. B 2005;6(4):295-300
OBJECTIVETo study the roles of different truncated hepatitis C virus (HCV) core proteins (CORE) in the pathogenesis of HCV persistent infection and hepatocellular carcinoma (HCC) and to assess intracellular localization in transiently transfected cells.
METHODSSeven truncated GFP (green fluorescent protein)-CORE fusion protein expression plasmids were constructed, which contained HCV CORE sequences derived from tumor tissues (BT) and non-tumor tissues (BNT) from one patient infected with HCV. Amino acid (aa) lengths were BT: 1-172 aa, 1-126 aa, 1-58 aa, 59-126 aa, 127-172 aa; BNT: 1-172 aa and C191: 1-172 aa respectively. Subcellular localization of CORE-GFP was analyzed by con-focal laser scanning microscope. Apoptosis and necrosis were quantified by flow cytometry.
RESULTSDifferent truncated CORE-GFP localized mainly in the cytoplasm, but nuclear staining was also observed. HCV CORE could induce apoptosis and necrosis, and different truncated COREs could induce cell apoptosis and necrosis at different levels. Among the same length 1-172 aa of BT, BNT and C191, the cell apoptosis and necrosis percentage of BT is highest, and C191 is the lowest (BT>BNT>C191). To the different fragment COREs of BT, N-terminal of CORE induced apoptosis and necrosis higher, compared with that of C-terminal (1-172 aa>1-126 aa>1-58 aa>127-172 aa>59-126 aa).
CONCLUSIONThese results suggest HCV CORE could induce apoptosis and necrosis of cells, which might play an important role in the pathogenesis of HCV persistent infection and HCC and the different CORE domains of different HCV quasi-species might have some difference in their pathogenesis.
Apoptosis ; Cell Line, Tumor ; Hepacivirus ; genetics ; pathogenicity ; physiology ; Humans ; Necrosis ; virology ; Sequence Deletion ; genetics ; Viral Core Proteins ; chemistry ; genetics ; metabolism
4.A study on endocytosis of hepatitis C virus mediated by CD81.
Zhan-sheng JIA ; De-wei DU ; Qiu-ping LIU ; Chun-yu WANG ; Hong-yan QIN ; Xin WEI ; Fu-tao ZHAO ; Guang-yu LI ; Hua HAN
Chinese Journal of Hepatology 2006;14(7):545-547
Animals
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Antigens, CD
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metabolism
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CHO Cells
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Cricetinae
;
Cricetulus
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Endocytosis
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Hepacivirus
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genetics
;
pathogenicity
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physiology
;
Tetraspanin 28
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Viral Envelope Proteins
;
genetics
5.Construction of an engineered M1GS-HCV/C141 ribozyme and determination of its antiviral activity in vitro.
Xifang LI ; Wenjun ZHANG ; Zhiwen HUANG ; Chengcheng ZHANG ; Guifei LUO
Chinese Journal of Biotechnology 2013;29(12):1786-1795
Hepatitis C virus (HCV), one of the major pathogens of viral hepatitis, causes significant hazards in humans. Interferon treatment in combination with ribavirin is used as the first line clinical treatment for HCV infection. However, good response to this treatment has only been observed in few patients and repeated recurrence has also been reported frequently. Therefore, new antiviral agents and therapies are in urgent demand. Here, we report a newly constructed Escherichia coli RNase P based M1GS ribozyme that can specifically and efficiently target the core gene of HCV. The guide sequence (GS) of this M1IGS was designed according to the sequence of the core coding region of HCV genome. The GS was then covalently linked to the 3' terminus of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. The specification of this sequence-specific ribozyme, M1GS, was then examined using an in vitro cleavage assay. The cytotoxicity and its activity in inhibition of HCV gene expression and viral proliferation were further studied in vivo. Our results show that the reconstructed M1GS ribozyme displayed obvious catalytic activity in cleaving target mRNAs fragment in vitro. Notable reduction in the expression of HCV core protein and a 1 000-fold reduction in viral growth were also observed in cultured HCV infected Huh7.5.1 cells expressing the functional M1GS ribozyme. This study demonstrated a direct evidence for the antiviral activity of the customized M1GS-HCV/C141 ribozyme, and thus provided a promising new strategy for clinical treatment of HCV infection.
Antiviral Agents
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pharmacology
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Escherichia coli
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genetics
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Genetic Engineering
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Hepacivirus
;
genetics
;
physiology
;
RNA, Catalytic
;
genetics
;
pharmacology
;
RNA, Guide
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genetics
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Ribonuclease P
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genetics
;
Viral Core Proteins
;
genetics
6.Recent advances in the study of mechanism of APOBEC3G against virus.
Yan-Ping ZHU ; Jian-Dong JIANG ; Zong-Gen PENG
Acta Pharmaceutica Sinica 2014;49(1):30-36
APOBEC3 is a class of cytidine deaminase, which is considered as a new member of the innate immune system, and APOBEC3G belongs to this family. The research about APOBEC3G is a new direction of innate immune defense mechanism against virus. APOBEC3G has the restrictive activity on many viral replications, which deaminates dC to dU in the viral genome and then induces extensive hypermutation. APOBEC3G can also interrupt viral replication at several phases such as reverse transcription, replication, nucleocapsid and so on by non-deamination mechanisms. However, virus can encode viral proteins to counteract the restriction activity of APOBEC3G. Elucidation of the antagonistic interaction between APOBEC3G and the virus will be contributed to development of new antiviral drugs in the future.
APOBEC-3G Deaminase
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Animals
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Cytidine Deaminase
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genetics
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metabolism
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DNA Replication
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Deamination
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HIV-1
;
physiology
;
Hepacivirus
;
genetics
;
physiology
;
Hepatitis B virus
;
genetics
;
physiology
;
Humans
;
Paramyxoviridae
;
genetics
;
physiology
;
Retroviridae
;
physiology
;
Virus Replication
;
vif Gene Products, Human Immunodeficiency Virus
;
metabolism
7.siRNAs targeting La, hVAP-33, eIF2Bgamma, and HCV IRES inhibit the replication and expression of HCV in Huh7 cells.
Mei-xia WANG ; Bin XU ; Jin DUAN ; Xiao-qing FU ; Ming JIN
Chinese Journal of Hepatology 2012;20(10):769-773
OBJECTIVETo investigate the in vivo functional roles of the La autoantigen (La), the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein (hVAP-33), and the subunit gamma of the human eukaryotic initiation factors 2B (eIF2Bgamma) as co-infection factors supporting chronic infection with hepatitis C virus (HCV).
METHODSSmall interfering (si)RNAs were designed against the HCV internal ribosome entry site (IRES) and transfected into Huh7 cells chronically infected with the HCV pseudovirus (designated as Huh7-HCV cells). The IRES siRNA producing the most effective silencing was selected for further analysis by fluorescence quantitative polymerase chain reaction (qPCR). siRNAs designed against La, hVAP-33, and eIF2Bgamma and the IRES-specific siRNA were then transfected, respectively or in various combinations, into the Huh7-HCV cell line for 48 h. The delta CT values were calculated and used to compare the HCV inhibitive efficacies of the siRNAs in isolation or in combination. Western blotting analysis was used to compare the quantity of core protein expression in each group.
RESULTSThe four gene-specific siRNAs, in isolation or in combination, caused inhibition of HCV replication and gene and protein expressions to varying degrees. The combination of La + IRES siRNAs produced the strongest inhibition of HCV core antigen expression. The combinations of hVAP-33 + IRES siRNAs and eIF2Bgamma + IRES siRNAs produced stronger inhibitions of HCV replication and gene and protein expressions than either hVAP-33 siRNA or eIF2Bgamma siRNA alone.
CONCLUSIONLa, hVAP-33, and eIF2Bgamma act as co-infection factors of HCV chronic infection in vivo. HCV replication and gene and protein expression can be inhibited significantly by RNA interference of these co-infection factors and/or HCV IRES.
Autoantigens ; genetics ; Cell Line ; Eukaryotic Initiation Factor-2B ; genetics ; Hepacivirus ; immunology ; physiology ; Humans ; RNA, Small Interfering ; genetics ; Ribonucleoproteins ; genetics ; Vesicular Transport Proteins ; genetics ; Virus Replication
8.Distribution of HCV genotypes and its clinical features in patients coinfected with HCV and HBV.
Jian JIAO ; Jiang-bin WANG ; Ping ZHAO
Chinese Journal of Hepatology 2003;11(11):688-688
Genotype
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Hepacivirus
;
genetics
;
physiology
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Hepatitis B
;
complications
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Hepatitis B virus
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Hepatitis C
;
complications
;
Humans
;
RNA, Viral
;
analysis
9.Regulation mechanism of HCV NS5A on p53 protein transactivity.
Guo-zhong GONG ; Yong-fang JIANG ; Ying-hua ZHU ; Xian-shi SU
Chinese Journal of Hepatology 2003;11(3):162-165
OBJECTIVETo study the inhibition effect of HCV NS5A on p53 protein transactivity and its possible mechanism.
METHODSLuciferase reporter gene system was used for the study of p53 transactivity on p21 promoter and electrophorectic mobility-shift assay (EMSA) was applied to observe whether HCV NS5A could suppress the binding ability of p53 protein to its specific DNA sequence.
RESULTSEndogenous p53 protein could stimulate p21 promoter activity, and the relative luciferase activity increased significantly (3.49 x 10(5) vs 0.60 x 10(5), t = 5.92, P<0.01). Exogenous p53 protein also up-regulated p21 promoter driving luciferase expression, comparing to the control group (0.47 x 10(5)), the relative luciferase activity increased (5.63 x 10(5)) obviously (t = 10.12, P<0.01). HCV NS5A protein inhibited both endogenous and exogenous p53 transactivity on p21 promoter in a dose-dependent manner (F > or = 20.71, P<0.01). In the experiment of EMSA, p53 could bind to its specific DNA sequence, but when co-transfected with HCV NS5A expressing vector, the p53 binding affinity to its DNA decreased.
CONCLUSIONHCV NS5A can inhibit p53 protein transactivity on p21 promoter through its inhibiting of p53 binding ability to the specific DNA sequence.
Hepacivirus ; genetics ; Humans ; Promoter Regions, Genetic ; Transcriptional Activation ; drug effects ; Tumor Suppressor Protein p53 ; drug effects ; genetics ; metabolism ; physiology ; Viral Core Proteins ; genetics ; Viral Nonstructural Proteins ; genetics ; pharmacology