CymaBay Therapeutics.

The mechanisms by which infectious hepatitis C virus (HCV) particles are assembled and released from infected cells remain poorly characterized. In this regard, many other enveloped viruses, notably human immunodeficiency virus type 1, have been shown to utilize the host vacuolar protein sorting machinery (also known as the endosomal sorting complex required for transport; ESCRT) to traffic through the cell and effect the membrane rearrangements required for the formation of enveloped particles. We postulated that this might also apply to HCV. To test this hypothesis, we established a method of conditional virus-like particle assembly involving transcomplementation of an envelope-deleted JFH-1 genome using plasmid transfection. This system reliably produced virus particles that were infectious and could be enumerated easily by focusforming assay in Huh7 cells. Following co-transfection with plasmids expressing various dominant-negative forms of either components of the ESCRT-III complex or Vps4 (the AAA ATPase that recycles the ESCRT complexes), a reduction in particle production was seen. No significant effect was observed after co-transfection of dominant-negative ESCRT-I or Alix, an ESCRT associated protein. Dominant-negative Vps4 or ESCRT-III components had no effect on either virus genome replication or the accumulation of intracellular infectious particles. These data were confirmed using cell culture infectious HCV and we conclude that HCV requires late components of the ESCRT pathway for release of infectious virus particles. INTRODUCTIONHepatitis C virus (HCV) represents a major global health burden. An estimated 170 million people are chronically infected worldwide and a significant proportion of these will go on to develop end stage liver disease. Current drug treatments are poorly tolerated and are only effective in approximately 50 % of individuals. There is, therefore, a pressing need for a greater understanding of the molecular mechanisms involved in HCV infection in order for novel drug targets to be identified.HCV is the prototype member of the genus Hepacivirus in the family Flaviviridae. It is a single-stranded, positive-sense RNA virus whose genome encodes a single ORF. This is translated in a cap-independent manner from an internal ribosome entry site (IRES) located in the 59 untranslated region, into a single polyprotein of approximately 3000 aa. The polyprotein is proteolytically cleaved by host and viral proteases to form 10 mature proteins; the structural proteins (core, E1 and E2), a viroporin p7 and the non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B).Although recent advances in the development of tissue culture systems for the study of the HCV life cycle (Lindenbach et al., 2005;Wakita et al., 2005;Zhong et al., 2005) have defined a critical role for lipid droplets in virus assembly (Miyanari et al., 2007), the precise mechanism by which infectious HCV particles are assembled and released remains poorly characterized. In particular, it has not been established how nascent...

Polycystic ovary syndrome (PCOS) increases the risk of metabolic syndrome and non-alcoholic-fatty-liver disease (NAFLD). Vitamin D supplementation may exert positive effects on liver biochemistry in patients with NAFLD; however, its effects on PCOS are unknown. This randomized, double-blind, placebo-controlled study explored the effect of vitamin D supplementation on cardiovascular risk factors (high-sensitivity C-reactive protein (hs-CRP), weight, body mass index (BMI), lipid profile, glucose levels, insulin levels, the homeostatic model assessment-insulin resistance (HOMA-IR), hormones (free androgen index (FAI), testosterone, sex hormone binding globulin (SHBG), and liver markers (alanine aminotransferase (ALT), hyaluronic acid (HA), N-terminal pro-peptide of type III procollagen (PIIINP), tissue inhibitor of metallo-proteinases-1 (TIMP-1), and the enhanced liver fibrosis (ELF) score). Forty women with PCOS were recruited and randomized to vitamin D (3200 IU) or placebo daily for 3 months. All outcomes were measured at baseline and 3 months follow-up (FU). Greater increases in vitamin D levels were shown in the supplementation group (vitamin D, baseline: 25.6 ± 11.4 nmol/L, FU: 90.4 ± 19.5 nmol/L vs. placebo, baseline: 30.9 ± 11.1 nmol/L, FU: 47.6 ± 20.5 nmol/L, p < 0.001). Between groups comparisons (% baseline change) revealed significant differences in ALT (p = 0.042) and a weak effect indicating a greater reduction in the HOMA-IR in the vitamin D group (p = 0.051). No further between group differences were seen in other cardiovascular risk factor, liver markers, or hormones. This study supports beneficial effects of vitamin D supplementation on liver markers and modest improvements in insulin sensitivity in vitamin D deficient women with PCOS.

A characteristic of many positive-strand RNA viruses is that, whilst replication of the viral genome is dependent on the expression of the majority of non-structural proteins in cis, virus particle formation can occur when most or all of the structural proteins are co-expressed in trans. Making use of a recently identified hepatitis C virus (HCV) isolate (JFH1) that can be propagated in tissue culture, this study sought to establish whether this is also the case for hepaciviruses. Stable cell lines containing one of two bicistronic replicons derived from the JFH1 isolate were generated that expressed non-structural proteins NS3-5B or NS2-5B. Release and transmission of these replicons to naïve Huh7 cells could then be demonstrated when baculovirus transduction was used to express the HCV proteins absent from the subgenomic replicons. Transmission could be blocked by a neutralizing antibody targeted at the E2 envelope protein, consistent with this phenomenon occurring via trans-encapsidation of replicon RNA into virus-like particles. Transmission was also dependent on expression of NS2, which was most effective at promoting virus particle formation when expressed in cis on the replicon RNA compared with in trans via baculovirus delivery. Density gradient analysis of the particles revealed the presence of a broad infectious peak between 1.06 and 1.11 g ml "1, comparable to that seen when propagating full-length virus in tissue culture. In summary, the trans-encapsidation system described offers a complementary and safer approach to study HCV particle formation and transmission in tissue culture. INTRODUCTIONCurrent estimates are that 170 million individuals are persistently infected with hepatitis C virus (HCV) throughout the world. Although initially many HCVpositive individuals will exhibit few clinical signs of infection, a significant health burden is associated with long-term chronic virus infection, with recent estimates suggesting that there is a three-fold increased risk of death above background levels, primarily due to liver disease, suicide and possibly cardiovascular complications (Guiltinan et al., 2008). Therapeutic intervention is available, but therapies are poorly tolerated and only partially effective (Deutsch & Hadziyannis, 2008). Furthermore, a vaccine for HCV is not available, and whilst it may be possible to develop one, our current understanding of how the immune system eliminates this virus suggests that such a vaccine would need to stimulate a cell-mediated immune response against a broad range of major histocompatibility complex class I and II epitopes (Day et al., 2003;Lauer et al., 2004). Therefore, there is a need for further research into various aspects of the HCV life cycle, both in terms of identification of novel targets for therapeutic intervention and to establish how the virus is able to circumvent both adaptive and innate immunity.HCV is a positive-strand, enveloped RNA virus belonging to the family Flaviviridae and possesses a 9.6 kb genome containing a single open reading fr...