Virus-host interactions during hepatitis C virus entry — implications for pathogenesis and novel treatment approaches

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.      

Fibronectin is essential for hepatitis B virus propagation in vitro: may be a potential cellular target?

Previous studies in our laboratory strongly suggested that fibronectin was upregulated by hepatitis B virus (HBV) in HepG2.2.15 cells. Report by Budkowska A also indicated that human liver fibronectin could bind HBV in a species-restricted manner. Therefore, it is reasonable to ask whether inhibiting fibronectin expression might have anti-HBV activity and whether fibronectin might be developed as a new potential cellular target for anti-HBV drugs. By using fibronectin antisense oligonucleotide (ASODN), fibronectin antibody, and Protocatechuic aldehyde (PA), we were able to show that HBV productions in HepG2.2.15 cell culture were reduced in a dose-dependent manner by fibronectin inhibition. In addition, we found that treatment with ASODNs, fibronectin antibody, and PA did not affect HepG2.2.15 cell viability. Furthermore, we observed that fibronectin inhibition sensitized HBV to anti-HBV drugs. In summary, this study demonstrates that fibronectin is essential for HBV propagation and also provides some evidences for the potential of fibronectin as a new cellular target for HBV infection therapy.     

Adipose tissue: a key target for diabetes pathophysiology and treatment?

Excess adipose tissue brings with it a number of adverse consequences, many of which may stem from the development of insulin resistance. An emerging view is that inflammatory changes occurring in expanding adipose tissue are associated with the secretion of peptide and other factors that can adversely affect metabolic processes in other key insulin-target tissues, especially liver and skeletal muscle. However, there is still a commonly-expressed view that the adverse changes in other tissues are ultimately due to an excess of fatty acids, liberated by a metabolically-challenged adipose tissue. Our own studies of adipose tissue metabolism and physiological function (especially blood flow) IN VIVO suggest that these two views of adipose tissue function may be closely linked. Enlarged adipocytes are less dynamic in their responses, just as 'enlarged adipose tissue' is less dynamic in blood flow regulation. Adipocytes seem to be able to sense the appropriate level of fat storage. If the normal mechanisms regulating adipocyte fat storage are interfered with (either in genetically-modified animals or by increasing the size of the adipocytes), then perhaps some sort of cellular stress sets in, leading to the inflammatory and endocrine changes. Some evidence for this comes from the effects of the thiazolidinediones, which improve adipose tissue function and in parallel reduce inflammatory changes.     

Development and evaluation of a new detection tool—visual DNA microarray for simultaneous and specific detection of human immunodeficiency virus type-1 and hepatitis C virus

Human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health. Based on multiplex asymmetrical PCR and coupled with gold labelled silver stain (GLSS), we developed the visual DNA microarray for sensitive and specific detection of these two viruses. Capturing probes of 5′-end-amino-modified oligonucleotides were immobilized on glass surface to bind the complement biotinylated target DNA. The Au–streptavidin probe was introduced to the microarray for specific binding to biotin. Black images of microarray spots which result from the precipitation of silver onto Au–streptavidin probes, were visualized by naked eyes. In order to improve the efficiency of microarray hybridization, triplex asymmetrical PCR of HIV-1, HCV and Human enterovirus 71 (EV-71, used as positive control) were performed to prepare abundant biotinylated single-stranded target DNA. The sensitivity of visual DNA microarray (10³ copies/ml) was higher than conventional PCR (10⁴ copies/ml) and was identical to FQ-PCR (10³ copies/ml). Total 152 blood samples containing the two viruses were tested using the DNA microarray and fluorescence quantitative real-time PCR (FQ-PCR). The results were identical (P > 0.05). So this system has high sensitivity and may have potential in clinical applications.     

NFκB signaling in carcinogenesis and as a potential molecular target for cancer therapy

It has become increasingly clear that deregulation of the NFκB signaling cascade is a common underlying feature of many human ailments including cancers. The past two decades of intensive research on NFκB has identified the basic mechanisms that govern the functioning of this pathway but uncovering the details of why this pathway works differently in different cellular contexts or how it interacts with other signaling pathways remains a challenge. A thorough understanding of these processes is needed to design better and more efficient therapeutic approaches to treat complex diseases like cancer. In this review, we summarize the literature documenting the involvement of NFκB in cancer, and then focus on the approaches that are being undertaken to develop NFκB inhibitors towards treatment of human cancers.     

Viral interference: A potential therapeutic method for chronic viral hepatitis?

Viral interference exists between different viral hepatitis. Acute Hepatitis C virus (HCV) super-infection on Hepatitis B virus (HBV) chronic carriers showed an inhibition of the HBV genome. And acute HBV super-infection on HCV chronic carriers indicated a similar interaction. In these cases, if the acute liver viral super-infection presents a self-limited course, the patients may be free from both viral infections or at least with undetectable underlying chronic viremia. The mechanism of viral interference is still undefined. Anyway this still leads to the hypothesis of using one hepatitis virus (live attenuated vaccine) to treat another hepatitis virus.     

Can modulation of viral fitness represent a target for anti-HIV-1 strategies?

Prolonged use of anti-retroviral compounds in human immunodeficiency virus type 1 (HIV-1) infection selects for drug-resistant and often mutidrug-resistant viral variants. Drug-resistance mutations may also affect viral fitness. Interestingly, recent research has indicated that some of the unfit drug-resistant variants may be less pathogenic, suggesting that decreased viral fitness is beneficial for the host and may be driven by specific treatments during anti-HIV-1 infection. A second potential antiviral strategy starting with profound inteference with viral fitness aims at forcing viruses towards lethal mutagenesis (the so-called "error catastrophe"). This review summarizes the methods for addressing HIV-1 fitness in vitro and ex vivo, the current understanding of clinical implications of reduced HIV-1 fitness, and the potential use of anti-HIV-1 strategies aiming at modulating viral fitness. Finally, it is emphasized how the peculiar features of HIV-1 quasispecies (displaying two different forms of memory, a replicative and a non-replicative form) may sharply influence the design of future diagnostic methodologies for fitness analysis.     

sFRP-mediated Wnt sequestration as a potential therapeutic target for Alzheimer’s disease

The extracellular ligand, Wnt, and its receptors are involved in sign al transduction and play an important role in axis formation and neural development. In neurodegenerative disorders such as Alzheimer’s disease (AD), a decrease of the intracellular Wnt effector, β-catenin, has been linked to amyloid-β-peptide-induced neurotoxicity. Despite this knowledge, targeting Wnt inhibitors as potential biomarkers has not been explored, and harnessing Wnt activators as therapeutic candidates remains largely not investigated. A wide acting family of Wnt mediators, secreted frizzled-related proteins (sFRPs), has not been probed so far as molecular indicators of disease occurrence and progression of Alzheimer’s. Unlike the effect of the Dickkopf (DKK) family of Wnt antagonists on AD, the sFRP molecules have a more pleiotropic impact on the Wnt signaling cascade and probably have a far-reaching involvement in neurodegeneration. The role of sFRPs has been poorly described in AD, and in this review, we analyze the present status of the role of sFRPs on neurodegeneration, their likely involvement, and potential implications in treatment modalities of AD. This information would provide valuable clues for the development of potential therapeutic targets for aberrant neurodegenerative disorders.     

A quantitative assay for the detection of hepatitis B virus DNA employing a biotin-labeled DNA probe and the avidin-β-galactosidase complex

We have developed a procedure for the quantitation of specific DNA which employs nonradioisotopic probes and β-galactosidase as a detector. The sample DNA was immobilized on a nitrocellulose filter paper. After the filter paper had been processed to hybridization with a biotinylated probe DNA, the paper was incubated with avidin-β-galactosidase complex. The optimum ratio of avidin to biotinylated β-galactosidase for preparation of a complex between the two was determined. The filter paper was punched. Each punched piece was put into a microtiter well and β-galactosidase activity was measured using 4-methylumbelliferyl β-d-galactosidase as a substrate. By this method, we were able to quantify as little as a few picograms of specific DNA. The application of this method for the quantitative assay of hepatitis B virus DNA in serum sample is also described. The sensitivity for the detection of the DNA by our method was practically comparable to that of the conventional radioisotopic method. The validity of our method for detection of the virus DNA was further supported by comparison with the serological data.     

The ribonuclease l-dependent antiviral roles of human 2′,5′-oligoadenylate synthetase family members against hepatitis C virus

The latent ribonuclease RNase L and the interferon-inducible 2′,5′-oligoadenylate synthetase (OAS) have been implicated in the antiviral response against hepatitis C virus (HCV). However, the specific roles of these enzymes against HCV have not been fully elucidated. In this study, a scarce endogenous expression and RNA degrading activity of RNase L in human hepatoma Huh7 cells enabled us to demonstrate the antiviral activity of RNase L against HCV replication through the transient expression of the enzyme. The antiviral potential of specific members of the OAS family was further examined through overexpression and RNA interference approaches. Our data suggested that among the members of the OAS family, OAS1 p46 and OAS3 p100 mediate the RNase l-dependent antiviral activity against HCV.     

Evolution of Pinnipedia lactation strategies: a potential role for α-lactalbumin?

Despite the considerable variation in milk composition found among mammals, a constituent common across all groups is lactose, the main sugar and osmole in most eutherians milk. Exceptions to this are the families Otariidae (fur seals and sea lions) and Odobenidae (walruses), where lactose has not been detected. We investigated the molecular basis for this by cloning alpha-lactalbumin, the modifier protein of the lactose synthase complex. A mutation was observed which, in addition to preventing lactose production, may enable otariids to maintain lactation despite the extremely long inter-suckling intervals during the mother's time at sea foraging (more than 23 days in some species).     

Herpes B virus utilizes human nectin-1 but not HVEM or PILRα for cell-cell fusion and virus entry

To investigate the requirements of herpesvirus entry and fusion, the four homologous glycoproteins necessary for herpes simplex virus (HSV) fusion were cloned from herpes B virus (BV) (or macacine herpesvirus 1, previously known as cercopithecine herpesvirus 1) and cercopithecine herpesvirus 2 (CeHV-2), both related simian simplexviruses belonging to the alphaherpesvirus subfamily. Western blots and cell-based enzyme-linked immunosorbent assay (ELISA) showed that glycoproteins gB, gD, and gH/gL were expressed in whole-cell lysates and on the cell surface. Cell-cell fusion assays indicated that nectin-1, an HSV-1 gD receptor, mediated fusion of cells expressing glycoproteins from both BV and CeHV-2. However, herpesvirus entry mediator (HVEM), another HSV-1 gD receptor, did not facilitate BV- and CeHV-2-induced cell-cell fusion. Paired immunoglobulin-like type 2 receptor alpha (PILRα), an HSV-1 gB fusion receptor, did not mediate fusion of cells expressing glycoproteins from either simian virus. Productive infection with BV was possible only with nectin-1-expressing cells, indicating that nectin-1 mediated entry while HVEM and PILRα did not function as entry receptors. These results indicate that these alphaherpesviruses have differing preferences for entry receptors. The usage of the HSV-1 gD receptor nectin-1 may explain interspecies transfer of the viruses, and altered receptor usage may result in altered virulence, tropism, or pathogenesis in the new host. A heterotypic cell fusion assay resulting in productive fusion may provide insight into interactions that occur to trigger fusion. These findings may be of therapeutic significance for control of deadly BV infections.     

Inhibition of hepatitis C virus NS5A by fluoro-olefin based γ-turn mimetics

The HCV non-structural protein NS5A has been established as a viable target for the development of direct acting antiviral therapy. From computational modeling studies strong intra-molecular hydrogen bonds were found to be a common structural moiety within known NS5A inhibitors that have low pico-molar replicon potency. Efforts to reproduce these γ-turn-like substructures provided a novel NS5A inhibitor based on a fluoro-olefin isostere. This fluoro-olefin containing inhibitor exhibited picomolar activity (EC(50)=79 pM) against HCV genotype 1b replicon without measurable cytotoxicity. This level of activity is comparable to the natural peptide-based inhibitors currently under clinic evaluation, and demonstrates that a peptidomimetic approach can serve as a useful strategy to produce potent and structurally unique inhibitors of HCV NS5A.     

Evaluation of immunological paradigms in a virus model: are dendritic cells critical for antiviral immunity and viral clearance?

We have examined the role of dendritic cells (DCs) in the antiviral immune response and viral clearance using a transgenic mouse model (CD11c-diphtheria toxin (DT) receptor GFP) that allows for their conditional ablation in vivo. DT administration systemically ablated conventional and IFN-producing plasmacytoid DCs (pDCs) in transgenic, but not nontransgenic littermates, without elimination of splenic macrophages. Unexpectedly, early (12 and 48 h postinfection) viral clearance of vesicular stomatitis virus was normal in DC-depleted mice despite markedly reduced serum titers of type I IFN. DC-depleted mice remained virus-free with the exception of a subset (approximately 30%) that developed overwhelming and fatal brain infections 6 days postinfection. However, DT treatment profoundly inhibited clonal expansion of naive CD8+ vesicular stomatitis virus-specific T cells without altering the primary Th1 and Th2 cytokine response. Optimal clonal expansion required pDCs because selective elimination of these cells in vivo with a depleting Ab also suppressed expansion of tetramer+ cells, although Th1/Th2 cytokine production remained unaltered. Collectively, these data indicate that conventional DCs and to a lesser extent pDCs are critical for proliferation of naive antiviral T cells. However, other components of the primary adaptive immune response (Th1/Th2 cytokines) are essentially normal in the absence of DCs, which may account for the efficient viral clearance seen in DC-depleted mice. Thus, sufficient redundancy exists in the immune system to sustain efficient viral clearance despite loss of an APC considered essential for induction of a primary antiviral immune response.