Visualization of positive and negative sense viral RNA for probing the mechanism of direct-acting antivirals against hepatitis C virus

RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((-)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (-)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (-)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action

Identification of a lead like inhibitor of the hepatitis C virus non-structural NS2 autoprotease

Hepatitis C virus (HCV) non-structural protein 2 (NS2) encodes a autoprotease activity that is essential for virus replication and thus represents an attractive anti-viral target. Recently, we demonstrated that a series of epoxide-based compounds, previously identified as potent inhibitors of the clotting factor, FXIII, also inhibited NS2-mediated proteolysis in vitro and possessed anti-viral activity in cell culture models. This suggested that a selective small molecule inhibitor of the NS2 autoprotease represents a viable prospect, therefore in this independent study we applied a structure-guided virtual high-throughput screening approach to identify a lead-like small molecule inhibitor of the NS2 autoprotease. This screen identified a candidate lead-like small molecule that was able to inhibit both NS2-mediated proteolysis in vitro and NS2-dependent genome replication in a cell-based assay. Structure-activity relationship analysis shed light on the nature of the active pharmacophore in this compound and may inform further development into a more potent inhibitor of NS2 mediated proteolysis

A comparative analysis of the fluorescence properties of the wild-type and active site mutants of the hepatitis C virus autoprotease NS2-3

Hepatitis C virus encodes an autoprotease, NS2-3, which is required for processing of the viral polyprotein between the non-structural NS2 and NS3 proteins. This protease activity is vital for the replication and assembly of the virus and therefore represents a target for the development of anti-viral drugs. The mechanism of this auto-processing reaction is not yet clear but the protease activity has been shown to map to the C-terminal region of NS2 and the N-terminal serine protease region of NS3. The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage. Here, intrinsic tryptophan fluorescence has been used to assess refolding in the wild-type protein and specific active site mutants. We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage. Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold

The subcellular localization of the hepatitis C virus non-structural protein NS2 is regulated by an ion channel-independent function of the p7 protein

The hepatitis C virus (HCV) p7 ion channel and non-structural protein 2 (NS2) are both required for efficient assembly and release of nascent virions, yet precisely how these proteins are able to influence this process is unclear. Here, we provide both biochemical and cell biological evidence for a functional interaction between p7 and NS2. We demonstrate that in the context of a genotype 1b subgenomic replicon the localization of NS2 is affected by the presence of an upstream p7 with its cognate signal peptide derived from the C terminus of E2 (SPp7). Immunofluorescence analysis revealed that the presence of SPp7 resulted in the targeting of NS2 to sites closely associated with viral replication complexes. In addition, biochemical analysis demonstrated that, in the presence of SPp7, a significant proportion of NS2 was found in a detergent (Triton X-100)-insoluble fraction, which also contained a marker of detergent resistant rafts. In contrast, in replicons lacking p7, NS2 was entirely detergent soluble and the altered localization was lost. Furthermore, we found that serine 168 within NS2 was required for its localization adjacent to replication complexes, but not for its accumulation in the detergent-insoluble fraction. NS2 physically interacted with NS5A and this interaction was dependent on both p7 and serine 168 within NS2. Mutational and pharmacological analyses demonstrated that these effects were not a consequence of p7 ion channel function, suggesting that p7 possesses an alternative function that may influence the coordination of virus genome replication and particle assembly

A Concerted Action of Hepatitis C Virus P7 and Nonstructural Protein 2 Regulates Core Localization at the Endoplasmic Reticulum and Virus Assembly

Hepatitis C virus (HCV) assembly remains a poorly understood process. Lipid droplets (LDs) are thought to act as platforms for the assembly of viral components. The JFH1 HCV strain replicates and assembles in association with LD-associated membranes, around which viral core protein is predominantly detected. In contrast, despite its intrinsic capacity to localize to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of cell culture-grown HCV (HCVcc)-infected cells, but was mainly localized at endoplasmic reticulum (ER) membranes where it colocalized with the HCV envelope glycoproteins. Furthermore, high-titer cell culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1 virus, strengthening the hypothesis that ER localization of core is required for efficient HCV assembly. Our results further indicate that p7 and NS2 are HCV strain-specific factors that govern the recruitment of core protein from LDs to ER assembly sites. Indeed, using expression constructs and HCVcc recombinant genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity. Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same differential core subcellular localization detected in JFH1- vs. Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between the p7 and the first NS2 trans-membrane domains is required to induce core-ER localization and assembly of extra- and intra-cellular infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing the subcellular localization of HCV core to LDs vs. ER and required for initiation of the early steps of virus assembly

The biochemistry and cellular biology of the hepatitis C virus NS2/3 autoprotease

Hepatitis C virus (HCY) causes a chronic infection that afflicts approximately 2.2% of the world's population. It has a positive-sense RNA genome that encodes a single open reading frame,· translated as a single polyprotein, coding structural proteins at the amino-terminus and non-structural proteins at the carboxyl-terminus. Processing at the NS2INS3 junction of the hepatitis C virus polyprotein occurs via an autoprotease comprising the cytoplasmic region of NS2 and the protease domain of NS3. Mutational analysis has been used to demonstrate that the cysteine residues required for zinc co-ordination in NS3 are similarly required for NS2/3 autoprocessing and to identify a new critical residue - cysteine 922. Detailed examination of zinc coordination in NS3 and NS2/3 using zinc chelation also revealed structural information about NS3 in the context of NS2/3 autoprotease activity. Until recently HCY could not be cultured in cells, therefore subgenomic replicons were developed as a tool to study HCY replication and the behaviour of non-structural proteins in a cellular environment. These replicons were originally produced beginning at either NS2 or NS3, those lacking NS2 were found to replicate more efficiently and were used in almost all subsequent studies. In this work replicons beginning at NS2 were produced with additional upstream sequences to examine their role in the behaviour of NS2 and replication. Some HCY-derived upstream sequences had a lethal effect on replicon replication when present in an atypical context, while an upstream sequence present in a non-lethal context regulates both NS2 localisation and the abundance of non-structural proteins.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Global rescue of defects in HIV-1 envelope glycoprotein incorporation: implications for matrix structure.

The matrix (MA) domain of HIV-1 Gag plays key roles in membrane targeting of Gag, and envelope (Env) glycoprotein incorporation into virions. Although a trimeric MA structure has been available since 1996, evidence for functional MA trimers has been elusive. The mechanism of HIV-1 Env recruitment into virions likewise remains unclear. Here, we identify a point mutation in MA that rescues the Env incorporation defects imposed by an extensive panel of MA and Env mutations. Mapping the mutations onto the putative MA trimer reveals that the incorporation-defective mutations cluster at the tips of the trimer, around the perimeter of a putative gap in the MA lattice into which the cytoplasmic tail of gp41 could insert. By contrast, the rescue mutation is located at the trimer interface, suggesting that it may confer rescue of Env incorporation via modification of MA trimer interactions, a hypothesis consistent with additional mutational analysis. These data strongly support the existence of MA trimers in the immature Gag lattice and demonstrate that rescue of Env incorporation defects is mediated by modified interactions at the MA trimer interface. The data support the hypothesis that mutations in MA that block Env incorporation do so by imposing a steric clash with the gp41 cytoplasmic tail, rather than by disrupting a specific MA-gp41 interaction. The importance of the trimer interface in rescuing Env incorporation suggests that the trimeric arrangement of MA may be a critical factor in permitting incorporation of Env into the Gag lattice

Impact of a brief worry-based cognitive therapy group in psychosis: a study of feasibility and acceptability

Previous research suggests that CBT focusing on worry in those with persecutory delusions reduces paranoia, severity of delusions and associated distress. This preliminary case series aimed to see whether it is feasible and acceptable to deliver worry-focused CBT in a group setting to those with psychosis. A secondary aim was to examine possible clinical changes. Two groups totalling 11 participants were run for seven sessions using the Worry Intervention Trial manual. Qualitative and quantitative data about the experience of being in the group was also collected via questionnaires, as was data on number of sessions attended. Measures were delivered pre- and post-group and at 3-month follow-up. These included a worry scale, a measure of delusional belief and associated distress and quality of life measures. Of the 11 participants who started the group, nine completed the group. Qualitative and quantitative feedback indicated that most of the participants found it acceptable and helpful, and that discussing these issues in a group setting was not only tolerable but often beneficial. Reliable Change Index indicated that 6/7 of the group members showed reliable reductions in their levels of worry post-group and 5/7 at follow-up. There were positive changes on other measures, which appeared to be more pronounced at follow-up. Delivering a worry intervention in a group format appears to be acceptable and feasible. Further research with a larger sample and control group is indicated to test the clinical effectiveness of this intervention

Replication of S66 and T69 mutants in Jurkat cells.

<p>Jurkat cells were transfected with the indicated molecular clones. At 2-day intervals the cells were split and samples of media were assayed for RT activity. In each graph of WT pNL4-3, 12LE, 62QR or 12LE/62QR mutations are combined with (A) WT; (B) 66SA; (C) 69TA; (D) 66SR; (E) 69TR. (F) 293T cells were co-transfected with the indicated molecular clones and vectors expressing HIV-1 Env or VSV-G. At 24 h, supernatants were harvested and assayed for infectivity as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. n = 3, +/− SEM.</p