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Arnab Basu,
Bing Li,
Debra M Mills,
Rekha G Panchal,
Steven C Cardinale,
Michelle M Butler,
Norton P Peet,
Helena Majgier-Baranowska,
John D Williams,
Ishan Patel,
Donald T Moir,
Sina Bavari,
Ranjit Ray,
Michael R Farzan,
Lijun Rong,
Terry L Bowlin
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ABSTRACT: Ebola virus (EBOV) causes severe hemorrhagic fever, for which therapeutic options are not available. Preventing the entry of EBOV into host cells is an attractive antiviral strategy, which has been validated for HIV by the FDA approval of the anti-HIV drug enfuvirtide. To identify inhibitors of EBOV entry, the EBOV envelope glycoprotein (EBOV-GP) gene was used to generate pseudotype viruses for screening of chemical libraries. A benzodiazepine derivative (compound 7) was identified from a high-throughput screen (HTS) of small-molecule compound libraries utilizing the pseudotype virus. Compound 7 was validated as an inhibitor of infectious EBOV and Marburg virus (MARV) in cell-based assays, with 50% inhibitory concentrations (IC(50)s) of 10 μM and 12 μM, respectively. Time-of-addition and binding studies suggested that compound 7 binds to EBOV-GP at an early stage during EBOV infection. Preliminary Schrödinger SiteMap calculations, using a published EBOV-GP crystal structure in its prefusion conformation, suggested a hydrophobic pocket at or near the GP1 and GP2 interface as a suitable site for compound 7 binding. This prediction was supported by mutational analysis implying that residues Asn69, Leu70, Leu184, Ile185, Leu186, Lys190, and Lys191 are critical for the binding of compound 7 and its analogs with EBOV-GP. We hypothesize that compound 7 binds to this hydrophobic pocket and as a consequence inhibits EBOV infection of cells, but the details of the mechanism remain to be determined. In summary, we have identified a novel series of benzodiazepine compounds that are suitable for optimization as potential inhibitors of filoviral infection.
Journal of Virology 01/2011; 85(7):3106-19. · 5.40 Impact Factor
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ABSTRACT: Virus entry into a host cell is an attractive target for therapy because propagation of virus can be blocked at an early stage, minimizing chances for the virus to acquire drug resistance. Anti-infective drug discovery for BSL-4 viruses like Ebola or Lassa hemorrhagic fever virus presents challenges due to the requirement for a BSL-4 laboratory containment facility. Pseudotyped viruses provide a surrogate model in which the native envelope glycoprotein of a BSL-2 level virus (e.g., vesicular stomatitis virus) is replaced with envelope glycoprotein of a foreign BSL-4 virus (e.g., Ebola virus). Because the envelope glycoprotein determines interaction of virus with its cellular receptors, pseudotyped viruses can mimic the viral entry process of the original virus. Moreover, they are competent for only a single cycle of infection, and therefore can be used in BSL-2 facilities. Pseudotyped viruses have been used in high-throughput screening of entry inhibitors for a number of BSL-4 level viruses. This unit includes protocols for preparing pseudotyped viruses using lentiviral vectors and use of pseudotyped viruses for high-throughput screening of viral entry inhibitors.
Current protocols in pharmacology 12/2010; Chapter 13:Unit 13B.3.
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ABSTRACT: Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 were used with MF59 adjuvant as a candidate vaccine for a phase 1 safety and immunogenicity trial. Ten of 41 vaccinee serum samples displayed a neutralization titer of > or =1:20 against vesicular stomatitis virus (VSV)-HCV pseudotype, 15 of 36 serum samples tested had a neutralization titer of > or =1:400 against human immunodeficiency virus (HIV)-HCV pseudotype, and 10 of 36 serum samples tested had a neutralization titer of > or =1:20 against cell culture-grown HCV genotype 1a. Neutralizing serum samples had increased affinity levels and displayed >2-fold higher specific activity levels to well-characterized epitopes on E1/E2, especially to the hypervariable region 1 of E2.
The Journal of Infectious Diseases 09/2010; 202(6):862-6. · 6.41 Impact Factor
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ABSTRACT: The mechanism of entry of hepatitis C virus (HCV) through interactions between the envelope glycoproteins and specific cell surface receptors remains unclear at this time. We have previously shown with the vesicular stomatitis virus (VSV)/HCV pseudotype model that the hypervariable region 1 of the HCV E2 envelope glycoprotein helps in binding with glycosaminoglycans present on the cell surface. In this study, we have examined the binding of HCV envelope glycoproteins with chemically modified derivatives of heparin. Furthermore, we have determined the functional relevance of the interaction of heparin derivatives with HCV envelope glycoproteins for infectivity by using a human immunodeficiency virus (HIV)/HCV pseudotype, a VSV/HCV pseudotype, and cell culture-grown HCV genotype 1a. Taken together, our results suggest that the HCV envelope glycoproteins rely upon O-sulfated esters of a heparin homologue to facilitate entry into mammalian cells.
Journal of Virology 05/2007; 81(8):3933-41. · 5.40 Impact Factor
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ABSTRACT: Activated hepatic stellate cells (HSCs) are the major source of extracellular matrix in fibrosis and cirrhosis. In this study, we have investigated the role of hepatitis C virus (HCV) core protein induced immortalized human hepatocytes (IHH) on HSC growth. Preferential growth of IHH and apoptosis of activated human hepatic stellate cells (LX2) were observed upon coculture of these two cell types in a dual chamber or in the presence of conditioned medium (CM) from IHH. CM did not display a growth inhibitory role on other hepatic (Huh-7, HepG2, Hep3B and THLE) and non-hepatic (HeLa, MCF-7, and BHK) epithelial cells, indicating that the soluble mediator from IHH does not have a generalized effect on cell lines examined in our study. Further studies suggested that CM from IHH increased the expression of TRAIL receptors on LX2 cell surface, and induced apoptosis by a caspase dependent mechanism. Peptide mass fingerprinting of the purified soluble mediator from CM suggested that gelsolin fragments may play a role in apoptosis of LX2 cells. Taken together, our results suggested that a soluble mediator secreted from immortalized human hepatocytes plays an important role in hepatic stellate cell growth regulation.
APOPTOSIS 09/2006; 11(8):1391-400. · 4.79 Impact Factor
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ABSTRACT: Hepatitis C virus (HCV) infection is a major contributor to the development of end-stage liver disease, including cirrhosis and hepatocellular carcinoma (HCC). We have previously shown that HCV core protein promotes immortalization of primary human hepatocytes. To identify molecular changes involved in core protein-mediated immortalization, we have investigated differential gene expression by microarray analyses in primary human hepatocytes and HCV core gene introduced hepatocytes after senescence (early passage), immortalization (middle passage), and anchor-independent growth (late passage). Out of 33,000 human genes screened, 1918 transcripts were differentially expressed (>2-fold) in immortalized human hepatocytes (IHH) as compared to negative controls. Our analyses provided a molecular portrait of changes in gene expression associated with three distinct stages of hepatocytes after introduction of HCV core gene. Many of the overall changes were involved with important cellular pathways, including cell growth regulation, immune regulation, oxidative stress, and apoptosis. We focused on the Stat3 signaling pathway by further verifying selected genes at the protein level relevant to hepatocyte growth regulation. Our data suggested that the introduction of HCV core protein results in an increase in expression of IL-6, gp130, leptin receptor, and Stat3. Upregulation of these genes in turn may regulate c-myc and cyclin D1, downstream of the Stat3 signaling pathway. Identification of these modulated genes with potential roles may help in the selection of targets for therapies against HCV-mediated liver disease progression.
Virology 06/2006; 349(2):347-58. · 3.35 Impact Factor
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ABSTRACT: Activated hepatic stellate cells (HSCs) are the major source of extracellular matrix in fibrosis and cirrhosis. In this study, we have investigated the role of hepatitis C virus (HCV) core protein induced immortalized human hepatocytes (IHH) on HSC growth. Preferential growth of IHH and apoptosis of activated human hepatic stellate cells (LX2) were observed upon coculture of these two cell types in a dual chamber or in the presence of conditioned medium (CM) from IHH. CM did not display a growth inhibitory role on other hepatic (Huh-7, HepG2, Hep3B and THLE) and non-hepatic (HeLa, MCF-7, and BHK) epithelial cells, indicating that the soluble mediator from IHH does not have a generalized effect on cell lines examined in our study. Further studies suggested that CM from IHH increased the expression of TRAIL receptors on LX2 cell surface, and induced apoptosis by a caspase dependent mechanism. Peptide mass fingerprinting of the purified soluble mediator from CM suggested that gelsolin fragments may play a role in apoptosis of LX2 cells. Taken together, our results suggested that a soluble mediator secreted from immortalized human hepatocytes plays an important role in hepatic stellate cell growth regulation.
APOPTOSIS 06/2006; · 4.79 Impact Factor
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ABSTRACT: We have previously shown that hepatitis C virus (HCV) core protein modulates multiple cellular processes, including those that inhibit tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis. In this study, we have investigated the signaling mechanism for inhibition of TNF-alpha-mediated apoptosis in human hepatoma (HepG2) cells expressing core protein alone or in context with other HCV proteins. Activation of caspase-3 and the cleavage of DNA repair enzyme poly(ADP-ribose) polymerase were inhibited upon TNF-alpha exposure in HCV core protein-expressing HepG2 cells. In vivo protein-protein interaction studies displayed an association between TNF receptor 1 (TNFR1) and TNFR1-associated death domain protein (TRADD), suggesting that the core protein does not perturb this interaction. A coimmunoprecipitation assay also suggested that HCV core protein does not interfere with the TRADD-Fas-associated death domain protein (FADD)-procaspase-8 interaction. Further studies indicated that HCV core protein expression inhibits caspase-8 activation by sustaining the expression of cellular FLICE (FADD-like interleukin-1beta-converting enzyme)-like inhibitory protein (c-FLIP). Similar observations were also noted upon expression of core protein in context to other HCV proteins expressed from HCV full-length plasmid DNA or a replicon. A decrease in endogenous c-FLIP by specific small interfering RNA induced TNF-alpha-mediated apoptotic cell death and caspase-8 activation. Taken together, our results suggested that the TNF-alpha-induced apoptotic pathway is inhibited by a sustained c-FLIP expression associated with the expression of HCV core protein, which may play a role in HCV-mediated pathogenesis.
Journal of Virology 06/2006; 80(9):4372-9. · 5.40 Impact Factor
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ABSTRACT: Progress in understanding hepatitis C virus (HCV) biology has remained a challenge due to the lack of an efficient cell culture system for virus growth. In this study, we examined HCV core protein-mediated immortalized human hepatocytes (IHH) for growth of HCV. In vitro-transcribed full-length RNA from HCV genotype 1a (clone H77) was introduced into IHH by electroporation. Reverse transcription-PCR of cellular RNA isolated from HCV genome-transfected IHH suggested that viral RNA replication occurred. IHH transfected with the full-length HCV genome also displayed viral protein expression by indirect immunofluorescence. In contrast, cells transfected with polymerase-defective HCV (H77/GND) RNA as a negative control did not exhibit expression of the viral genome. Immunogold labeling demonstrated localization of E1 protein in the rough endoplasmic reticulum of RNA-transfected IHH. Virus-like particles of approximately 50 nm were observed in the cytoplasm. After being inoculated with culture media of cells transfected with the full-length HCV genome, naïve IHH displayed NS5a protein expression in a dilution-dependent manner, but expression of NS5a was inhibited by prior incubation of culture medium with HCV-infected patient sera. NS5a-positive immunofluorescence of cell culture media of IHH transfected with full-length H77 RNA yielded approximately 4.5 x 10(4) to 1 x 10(5) focus-forming units/ml. A similar level of virus growth was observed upon transfection of RNA from HCV genotype 2a (JFH1) into IHH. Taken together, our results suggest that IHH support HCV genome replication and virus assembly.
Journal of Virology 06/2006; 80(9):4633-9. · 5.40 Impact Factor
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ABSTRACT: Hepatitis C virus (HCV) core protein has multifunctional activities. We have previously reported that the core protein of HCV immortalizes primary human hepatocytes, which may relate to multistage hepatocarcinogenic events. These immortalized human hepatocytes (IHH) served as a model to study the mechanism of HCV core protein-mediated cell growth regulation. Inhibition of core protein expression in earlier stages after hepatocyte immortalization leads to the induction of apoptosis. Here, we have observed that introduction of antisense core (AS-Core) sequences for inhibition of core protein expression enhanced the expression of E2F1 and p53 levels in early passage IHH. Inhibition of core protein expression also altered the expression level of Bcl-2 family proteins, displaying an increase of the proapoptotic Bax and a decrease in the level of the anti-apoptotic Bcl-xL proteins. These alterations, however, did not result in the release of cytochrome c from the mitochondria. Apaf-1 is frequently deregulated under various pathologic conditions, and examination of AS-Core-expressing apoptotic cells indicated a significant increase in the level of Apaf-1, which coincided with caspase-9 activation. Knockdown of Apaf-1 or the transcriptional regulatory proteins, E2F1 or p53, by small interfering RNA (siRNA) duplexes inhibited the activation of caspase-9 and enhanced cell viability in AS-Core-expressing cells. These findings may contribute to the understanding of the pathophysiology of HCV core protein-mediated hepatocyte growth regulation and disease progression.
Virology 07/2005; 336(2):198-207. · 3.35 Impact Factor
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ABSTRACT: We have previously reported that a pseudotype virus generated by reconstitution of hepatitis C virus (HCV) chimeric envelope glycoprotein E1-G or E2-G on the surface of a temperature-sensitive mutant of vesicular stomatitis virus (VSVts045) interacts independently with mammalian cells to initiate infection. Here, we examined whether coexpression of both of the envelope glycoproteins on pseudotype particles would augment virus infectivity and/or alter the functional properties of the individual subunits. Stable transfectants of baby hamster kidney (BHK) epithelial cells expressing either one or both of the chimeric envelope glycoproteins of HCV on the cell surface were generated. The infectious titer of the VSV pseudotype, derived from a stable cell line incorporating both of the chimeric glycoproteins of HCV, was approximately 4- to 5-fold higher than that of a pseudotype bearing E1-G alone or approximately 25- to 30-fold higher than that of E2-G alone when assayed with a number of mammalian cell lines. Further studies suggested that that the E1-G/E2-G or E2-G pseudotype was more sensitive to the inhibitory effect of heparin than the E1-G pseudotype. Treatment of the E1-G/E2-G pseudotype with a negatively charged sulfated sialyl lipid (NMSO3) displayed a approximately 4-fold-higher sensitivity to neutralization than pseudotypes with either of the two individual glycoproteins. In contrast, VSVts045, used as a backbone for the generation of pseudotypes, displayed at least 20-fold-higher sensitivity to NMSO3-mediated inhibition of virus plaque formation. The effect of low-density lipoprotein on the E1-G pseudotype was greater than that apparent for the E1-G/E2-G pseudotype. The treatment of cells with monoclonal antibodies to CD81 displayed an inhibitory effect upon the pseudotype with E1-G/E2-G or with E2-G alone. Taken together, our results indicate that the HCV E1 and E2 glycoproteins have separable functional properties and that the presence of these two envelope glycoproteins on VSV/HCV pseudotype particles increases infectious titer.
Journal of Virology 01/2005; 78(23):12838-47. · 5.40 Impact Factor
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ABSTRACT: Hepatitis C virus (HCV) often causes persistent infection in humans. This could be due in part to the effect of viral proteins on cellular gene expression. Earlier observations suggest that the HCV core protein expressed from genotype 1a modulates important cellular genes at the transcriptional level, affects programmed cell death (apoptosis) and promotes cell growth. Recently, different groups of investigators have reported the translation of an approximately 16 kDa protein (named F/ARFP/core+1 ORF) from an alternate open reading frame of the HCV core-encoding genomic region. The functional significance of this F protein is presently unknown. Thus, whether the F and core proteins have both shared and distinct functions was investigated here. The experimental observations suggested that the F protein does not significantly modulate c-myc, hTERT and p53 promoter activities, unlike the HCV core protein. Interestingly, the F protein repressed p21 expression. Further studies indicated that the F protein does not inhibit tumour necrosis factor alpha-mediated apoptosis of HepG2 cells or promote rat embryo fibroblast growth. Taken together, these results suggest that the F protein does not share major properties identified previously for the HCV core protein, other than regulating p21 expression.
Journal of General Virology 09/2004; 85(Pt 8):2299-306. · 3.36 Impact Factor
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ABSTRACT: We have previously reported a functional role associated with hepatitis C virus (HCV) E1 glycoprotein using vesicular stomatitis virus (VSV)/HCV pseudotype. In this study, we have investigated the role of glycosylation upon intracellular transport of chimeric E1-G, and in infectivity of the pseudotyped virus. Interestingly, surface expressed E1-G exhibited sensitivity to Endoglycosidase H (Endo H) treatment, which was similar to full-length E1, suggesting that additional complex oligosaccharides were not added while E1-G was in transit from the endoplasmic reticulum (ER) to the mammalian cell surface. As a next step, each of the four potential N-linked glycosylation sites located at amino acid position 196, 209, 234, or 305 of the E1 ectodomain were mutated separately (asparagine --> glutamine), or in some combination. FACS analysis suggested that mutation(s) of the glycosylation sites affect the translocation of E1-G to the cell surface to different extents, with no single site being particularly essential. VSV pseudotype virus generated from glycosylation mutants exhibited a decrease in titer with an increasing number of mutations at the glycosylation sites on chimeric E1-G. In a separate experiment, N-glycosidase F treatment of pseudotype generated from the already synthesized E1-G or its mutants decreased virus titer by approximately 35%, and the neutralization activity of patient sera was not significantly altered with N-glycosidase F-treated pseudotype virus. Taken together, our results suggested that E1-G does not add complex sugar moieties during transport to the cell surface and retain the glycosylation profile of its parental E1 sequence. Additionally, the removal of glycans from the E1-G reduced, but does not completely impair, virus infectivity.
Virology 07/2004; 324(2):273-85. · 3.35 Impact Factor
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ABSTRACT: The hypervariable region 1 (HVR1) of hepatitis C virus (HCV) E2 envelope glycoprotein is a 27-amino-acid sequence located at its N terminus. In this study, we investigated the functional role of HVR1 for interaction with the mammalian cell surface. The C-terminal truncated E2 glycoprotein was appended to a transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein for generation of the chimeric E2-G gene construct. A deletion of the HVR1 sequence from E2 was created for the construction of E2DeltaHVR1-G. Pseudotype virus, generated separately by infection of a stable cell line expressing E2-G or E2DeltaHVR1-G with a temperature-sensitive mutant of VSV (VSVts045), displayed unique functional properties compared to VSVts045 as a negative control. Virus generated from E2DeltaHVR1-G had a reduced plaquing efficiency ( approximately 50%) in HepG2 cells compared to that for the E2-G virus. Cells prior treated with pronase (0.5 U/ml) displayed a complete inhibition of infectivity of the E2DeltaHVR1-G or E2-G pseudotypes, whereas heparinase I treatment (8 U/ml) of cells reduced 40% E2-G pseudotype virus titer only. E2DeltaHVR1-G pseudotypes were not sensitive to heparin (6 to 50 micro g/ml) as an inhibitor of plaque formation compared to the E2-G pseudotype virus. Although the HVR1 sequence itself does not match with the known heparin-binding domain, a synthetic peptide representing 27 amino acids of the E2 HVR1 displayed a strong affinity for heparin in an enzyme-linked immunosorbent assay. This binding was competitively inhibited by a peptide from the V3 loop of a human immunodeficiency virus glycoprotein subunit (gp120) known to bind with cell surface heparin. Taken together, our results suggest that the HVR1 of E2 glycoprotein binds to the cell surface proteoglycans and may facilitate virus-host interaction for replication cycle of HCV.
Journal of Virology 06/2004; 78(9):4478-86. · 5.40 Impact Factor
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ABSTRACT: Ebola virus glycoprotein (EGP) has been implicated for the induction of cytotoxicity and injury in vascular cells. On the other hand, EGP has also been suggested to induce massive cell rounding and detachment from the plastic surface by downregulating cell adhesion molecules without causing cytotoxicity. In this study, we have examined the cytotoxic role of EGP in primary endothelial cells by transduction with a replication-deficient recombinant adenovirus expressing EGP (Ad-EGP). Primary human cardiac microvascular endothelial cells (HCMECs) transduced with Ad-EGP displayed loss of cell adhesion from the plastic surface followed by cell death. Transfer of conditioned medium from EGP-transduced HCMEC into naive cells did not induce loss of adhesion or cell death, suggesting that EGP needs to be expressed intracellularly to exert its cytotoxic effect. Subsequent studies suggested that HCMEC death occurred through apoptosis. Results from this study shed light on the EGP-induced anoikis in primary human cardiac endothelial cells, which may have significant pathological consequences.
Virology 05/2004; 321(2):181-8. · 3.35 Impact Factor
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ABSTRACT: Hepatitis C virus (HCV) is a serious human pathogen and an estimated 170 million people are infected worldwide. Current therapeutic regimens have shown limited efficacy against selected genotypes of the virus. The phenomenon of RNA interference can be used to selectively block homologous genes post-transcriptionally, and has revolutionized approaches to study gene function. In this report, we have demonstrated that small interfering RNAs (siRNAs) targeted against NS5A of HCV genotype 1a specifically inhibit NS5A RNA and protein expression in a human hepatoma (HepG2) cell line. Expression of endogenous alpha-actin and the ds-RNA activated serine/threonine kinase-PKR were unaltered, demonstrating that the inhibitory effect observed from siRNA was specific to the HCV NS5A protein. We next examined whether siRNA directed against NS5A could inhibit core protein expression, the first gene product synthesized in virus infected cells due to its localization at the 5' end of the HCV polyprotein. For this purpose, a full-length cDNA clone from HCV (H77, genotype 1a) was used, and results indicated that the introduction of NS5A targeted siRNA resulted in an inhibition of NS5A and core protein expression. Moreover, we observed that this siRNA effectively inhibited NS5A mediated activation of the IL-8 promoter. Taken together, our results demonstrated that siRNA was effective in inhibiting HCV protein expression, and may have therapeutic potential to limit HCV replication in chronically infected patients.
Virus Research 11/2003; 96(1-2):27-35. · 2.94 Impact Factor
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ABSTRACT: HCV envelope glycoproteins play an important role in the initiation of viral infection. The functional dichotomy of the individual HCV glycoproteins was investigated using VSV/HCV pseudotype virus. Surprisingly, VSV/HCV pseudotype virus generated from either E1 or E2 displayed infectivity of a number of mammalian cells. The use of pseudotyped virus has allowed us to better understand the similar and divergent properties of E1 and E2 glycoproteins decorating the envelope of HCV. The serum pseudotype virus neutralizing activity in patient sera did not exhibit a correlation with the infecting HCV genotype or virus load. HCV E2 glycoprotein induces a weak neutralizing antibody response, however the neutralization function was augmented by complement. Taken together, these observations suggest a role for both the glycoproteins in HCV attachment and entry into susceptible host cells. An understanding of HCV entry and strategies appropriate for mimicking cell surface molecules may help in the development of new therapeutic modalities against HCV infection. Furthermore, incorporation of the HCV glycoproteins in a candidate vaccine may offer protection, although additional work is necessary to enhance their immunogenicity.
Vox Sanguinis 09/2002; 83 Suppl 1:27-32. · 2.86 Impact Factor
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ABSTRACT: Hepatitis C virus (HCV) often causes a prolonged and persistent infection. Sequence divergence in the HCV genome indicates several genotypes and a series of subtypes for this virus. The core protein of HCV has many intriguing functional properties and is implicated as a factor in virus mediated pathogenesis. Nuclear factor kappaB (NF-kappaB), a transcription factor, responds to inflammatory signals, activates the expression of inflammatory mediators, and plays a role in cell proliferation process. In this study, we have investigated NF-kappaB regulation by HCV core protein cloned from three isolates of different genotypes. Our results suggest that core protein from HCV genotype 1a represses NF-kappaB activation, unlike two other core genomic regions from HCV genotype 1b (BK or Taiwan). However, missense mutations in positions (K(9) to R or N(11) to T) of HCV genotype 1a relieve repression of NF-kappaB regulation by core protein. Interestingly, in vitro translation studies suggested that amino acid substitution at position 11 (N-->T) in HCV genotype 1a generated a primary protein product of approximately 17 kDa, smaller than the major approximately 21 kDa protein band apparent in the parental sequence or with one carrying mutation at amino acid position 9 (K-->R). However, the approximately 17 kDa protein did not appear to be involved in NF-kappaB regulation. Taken together, our present data suggest that genomic variation in the core protein determines a distinct functional regulation of NF-kappaB, which may modulate immunnoregulatory molecules early in viral infection.
Virus Research 07/2002; 87(1):21-9. · 2.94 Impact Factor
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ABSTRACT: Hepatitis C virus (HCV) core protein has many intriguing properties and plays an important role in cell growth regulation. We have recently shown that the HCV core protein from genotype 1a promotes primary human hepatocytes to an immortalized phenotype. Here, we investigated whether the presence of core protein is necessary for maintenance of the immortalized hepatocytes and investigated its consequences on cellular gene expression. The introduction of an antisense orientation of the core gene into immortalized hepatocytes led to the onset of cell death. Further analysis suggested that cell death occurred through apoptosis associated with the activation of tumor suppressor pathways. Antisense core gene expression in immortalized hepatocytes increased p53 expression at both the mRNA and the protein levels. A decreased telomere length and reduced c-myc protein expression were also observed in hepatocytes when the antisense core gene was introduced. Results from these studies suggested that modulation of cell cycle regulatory genes by repression of core protein expression is responsible for reversion of the immortalized phenotype of the hepatocytes. Thus, targeted inhibition may contribute to the development of new therapeutic modalities for prevention of HCV core protein function.
Virology 07/2002; 298(1):53-62. · 3.35 Impact Factor
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ABSTRACT: We previously reported a number of features of hepatitis C virus (HCV) chimeric glycoproteins related to pseudotype virus entry into mammalian cells. In this study, pseudotype virus was neutralized by HCV E2 glycoprotein-specific antibodies and infected human sera. Neutralization (50% reduction of pseudotype virus plaque formation) was observed with two human immunoglobulin G1 monoclonal antibodies (MAbs) at concentrations of between 2.5 and 10 microg/ml. A hyperimmune rabbit antiserum to an E2 hypervariable region 1 (HVR1) mimotope also exhibited an HCV E2 pseudotype virus neutralization titer of approximately 1/50. An E1 pseudotype virus used as a negative control was not neutralized to a significant level (<1/10) by these MAbs or rabbit antiserum to E2 HVR1. Since HCV probably has a lipid envelope, the role of complement in antibody-mediated virus neutralization was examined. Significant increases in the neutralization titers of the human MAbs (approximately 60- to 160-fold higher) and rabbit antiserum to HVR1 mimotopes (approximately 10-fold higher) were observed upon addition of guinea pig complement. Further, these studies suggested that complement activation occurred primarily by the classical pathway, since a deficiency in the C4 component led to a significant decrease in the level of virus neutralization. This same decrease was not observed with factor B-deficient complement. We also determined that 9 of 56 HCV-infected patient sera (16%) had detectable pseudotype virus neutralization activity at serum dilutions of between 1/20 and 1/50 and that complement addition enhanced the neutralization activity of some of the HCV-infected human sera. Taken together, these results suggest that during infection, HCV E2 glycoprotein induces a weak neutralizing antibody response, that those antibodies can be measured in vitro by the surrogate pseudotype virus plaque reduction assay, and that neutralization function can be augmented by complement.
Journal of Virology 04/2002; 76(5):2150-8. · 5.40 Impact Factor
