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The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with Molecular Dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5’ UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in the stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel antiviral agents.

Hepatitis B virus (HBV) is an enveloped DNA virus that contains a partially double-stranded relaxed circular (rc) DNA. Upon infection, rcDNA is delivered to the nucleus where it is repaired to covalently closed circular (ccc) DNA that serves as the transcription template for all viral RNAs. Our understanding of HBV particle entry dynamics and host pathways regulating intracellular virus trafficking and cccDNA formation is limited. The discovery of sodium taurocholate co-transporting peptide (NTCP) as the primary receptor allows studies on these early steps in viral life cycle. We employed a synchronised infection protocol to quantify HBV entry kinetics. HBV attachment to cells at 4 degrees C is independent of NTCP, however, subsequent particle uptake is NTCP-dependent and reaches saturation at 12 h post-infection. HBV uptake is clathrin- and dynamin dependent with actin and tubulin playing a role in the first 6 h of infection. Cellular fractionation studies demonstrate HBV DNA in the nucleus within 6 h of infection and cccDNA was first detected at 24 h post-infection. Our studies show the majority (83%) of cell bound particles enter HepG2-NTCP cells, however, only a minority (

Type 1 interferon (IFN) continues to be the foundation for the current standard of care combination therapy for chronic hepatitis C virus (HCV) infection, yet the component interferon-stimulated genes (ISGs) that mediate the antiviral actions of IFN are not fully defined. Interferon-induced transmembrane protein 1 (IFITM1) is an ISG product that suppresses early stage infection by a number of viruses through an unknown mechanism of action. Moreover, the actions of IFITM1 on HCV infection are not fully elucidated. Here we identify IFITM1 as a hepatocyte tight junction protein and a potent anti-HCV effector molecule. IFITM1 expression is induced early during IFN treatment of hepatocytes and accumulates at hepatic tight junctions in HCV-infected human patient liver during IFN therapy. Additionally, we found that IFITM1 interacts with HCV coreceptors, including CD81 and occludin, to disrupt the process of viral entry. Thus, IFITM1 is an anti-HCV ISG whose actions impart control of HCV infection through interruption of viral coreceptor function. Conclusion: This study defines IFITM1 as an ISG effector with action against HCV entry. Design of therapy regimens to enhance IFITM1 expression should improve the virologic response among HCV patients undergoing treatment with type I IFN. (HEPATOLOGY 2013)

Hepatitis C virus (HCV) is an enveloped, positive-strand RNA virus of the family Flaviviridae that primarily infects hepatocytes, causing acute and chronic liver disease. HCV is also associated with a variety of extrahepatic symptoms including central nervous system (CNS) abnormalities, cognitive dysfunction, fatigue and depression. These symptoms do not correlate with the severity of liver disease and are independent of hepatic encephalopathy. HCV RNA has been associated with CNS tissue, and reports of viral sequence diversity between brain and liver tissue suggest independent viral evolution in the CNS and liver. This review will explore the data supporting HCV infection of the CNS and how this fits into our current understanding of HCV pathogenesis.

HCV is a blood-borne pathogen that affects approximately 3% of the global population and leads to progressive liver disease. Recent advances have identified an essential role for host cell molecules: tetraspanin CD81, scavenger receptor B1 and the tight junction proteins claudin-1 and occludin in HCV entry, suggesting a complex multi-step process. The conserved nature of this receptor-dependent step in the viral life cycle offers an attractive target for therapeutic intervention. Evidence is emerging that additional factors other than classical receptors, such as inflammatory mediators regulate the ability of hepatocytes to support HCV entry, and as such may provide potential avenues for drug design and development. In this review, we summarise the recent literature on HCV entry mechanisms with a view to realising the future potential of therapeutically targeting this process.

Much of our current understanding of hepatitis C virus (HCV) replication has hailed from the use of a small number of cloned viral genomes and transformed hepatoma cell lines. Recent evidence suggests that lipoproteins play a key role in the HCV life cycle and virus particles derived from the sera of infected patients exist in association with host lipoproteins. This report will review the literature on HCV replication in primary hepatocytes and transformed cell lines, focusing largely on host factors defining particle entry.

1. We investigated the effects of a number of naturally occurring chemokines (MIP-1alpha, MIP-1beta, RANTES, MCP-2, MCP-3, MCP-4) on different processes linked to the chemokine receptor CCR5 in recombinant CHO cells expressing the receptor at different levels. 2. Internalization of CCR5 following chemokine treatment was studied and MIP-1alpha, MIP-1beta and RANTES (50 nM) were able to induce internalization (similar50%) of the receptor. Internalization due to MCP-2, MCP-3 and MCP-4 was less (similar20%). 3. Phosphorylation of CCR5 following chemokine treatment was studied and MIP-1alpha, MIP-1beta and RANTES (50 nM) were able to induce phosphorylation of CCR5 whereas the other chemokines did not induce CCR5 phosphorylation. 4. MIP-1alpha, MIP-1beta, RANTES and MCP-2 were able to stimulate [(35)S]-GTPgammaS binding, an index of receptor/G protein activation, whereas MCP-3 and MCP-4 had no effect in this assay. MCP-2 was a partial agonist (similar80%) compared to MIP-1alpha, MIP-1beta and RANTES, which gave similar maximal stimulations in this assay. 5. MIP-1alpha, MIP-1beta, RANTES, MCP-2 and MCP-4 were able to stimulate increases in intracellular calcium ions via activation of CCR5 whereas MCP-3 was without effect. 6. It is concluded that different chemokines interacting with CCR5 mediate different patterns of cellular responses.

HCV encodes two glycoproteins, E1 and E2, that are believed to be exposed on the surface of virions. These molecules are likely to be involved in viral interactions with the host immune response and responsible for mediating viral entry into target cells. They are obvious major components for prototype vaccine studies. Recently, E2 has been reported to bind to the tetraspan molecule CD81, which represents a putative receptor for HCV. Here, we discuss the role the HCV gps may play during infection, the contribution of E2 gp variation to HCV evasion from the immune response and possible implications of the E2-CD81 interaction for HCV pathogenesis.

During development of an enzyme immunoassay for the detection of cytomegalovirus (CMV) we previously discovered that virus found naturally in urine specimens could not be captured onto the solid phase by CMV-specific monoclonal antibodies, whereas these same antibodies could capture CMV from cell culture supernatants. We now report that urine from normal CMV-seronegative individuals contains a substance of molecular weight 11-12,000 daltons that inhibits the ELISA detection of cell culture-grown CMV. The addition of a known urinary protein of this molecular weight, beta 2 microglobulin (beta 2m; 11,700 daltons), inhibited the detection of cell culture-grown CMV in the ELISA over the concentration range found in clinical urine samples. In contrast, another low molecular weight urinary protein, lysozyme, had no inhibitory effect. beta 2m caused inhibition only when added to the virus preparation and not to the antibody-capture stage. We conclude that beta 2m in urine prevents the detection of CMV by ELISA by binding to the virus and masking its antigenic determinants and we calculate that of the order of 10(5) molecules of beta 2m bind to each particle of cell culture-grown CMV. We postulate that CMV in fresh urine specimens is similarly coated with beta 2m, accounting for the failure to detect it by ELISA.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytomegalovirus (CMV) in urine using monoclonal antibodies directed against CMV as a capture for viral antigen. The assay was capable of detecting virus at 10(2.3)TCID50/ml as determined by titration of stock virus, strain Ad169. The assay was found to have a sensitivity of 65% and a specificity of 100% when 73 coded stored urine specimens were examined. Assuming that the poor sensitivity was due to loss of antigen following storage, we proceeded to analyse fresh urine specimens. Surprisingly, the assay gave negative results with 46 fresh urines known to contain CMV; however, following storage at +4 degrees C for two weeks, 35 (76%) of these samples gave ELISA results in the positive range. This detection of CMV, after storage at +4 degrees C, could be due to degradation of virus particles leading to release of soluble glycoproteins into the medium or to the presence of an inhibitory substance in fresh urine that is destroyed during storage.

The susceptibility of common marmosets and cotton-top tamarins to infection by HIV-2 in vivo was tested. One year and 19 months, respectively, post-inoculation, sera taken from three of four animals from each species are reactive for HIV-2 antibodies and HIV-specific nucleotide sequences were demonstrated in short-term cultures of PBL from two cotton-top tamarins. The animals remain in good health.