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Biomedical subjects

Matthew J Evans

Publications and source records attributed to Matthew J Evans.

9 recordsLinked to original sources
PubMed2006

Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry.

Hepatitis C virus (HCV) is an important human pathogen associated with chronic liver disease. Recently, based on a genotype 2a isolate, tissue culture systems supporting complete replication and infectious virus production have been developed. In this study, we used cell culture-produced infectious HCV to analyze the viral entry pathway into Huh-7.5 cells. Bafilomycin A1 and concanamycin A, inhibitors of vacuolar ATPases, prevented HCV entry when they were present prior to infection and had minimal effect on downstream replication events. HCV entry therefore appears to be pH dependent, requiring an acidified intracellular compartment. For many other enveloped viruses, acidic pH triggers an irreversible conformational change, which promotes virion-endosomal membrane fusion. Such viruses are often inactivated by low pH. In the case of HCV, exposure of virions to acidic pH followed by return to neutral pH did not affect their infectivity. This parallels the observation made for the related pestivirus bovine viral diarrhea virus. Low pH could activate the entry of cell surface-bound HCV but only after prolonged incubation at 37 degrees C. This suggests that there are rate-limiting, postbinding events that are needed to render HCV competent for low-pH-triggered entry. Such events may involve interaction with a cellular coreceptor or other factors but do not require cathepsins B and L, late endosomal proteases that activate Ebola virus and reovirus for entry.

Cathepsin B↗
PubMed2005

Complete replication of hepatitis C virus in cell culture.

Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 10(5) infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-alpha and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.

Antibodies, Monoclonal↗
PubMed2005

Single point mutations in the zinc finger motifs of the human immunodeficiency virus type 1 nucleocapsid alter RNA binding specificities of the gag protein and enhance packaging and infectivity.

A specific interaction between the nucleocapsid (NC) domain of the Gag polyprotein and the RNA encapsidation signal (Psi) is required for preferential incorporation of the retroviral genomic RNA into the assembled virion. Using the yeast three-hybrid system, we developed a genetic screen to detect human immunodeficiency virus type 1 (HIV-1) Gag mutants with altered RNA binding specificities. Specifically, we randomly mutated full-length HIV-1 Gag or its NC portion and screened the mutants for an increase in affinity for the Harvey murine sarcoma virus encapsidation signal. These screens identified several NC zinc finger mutants with altered RNA binding specificities. Furthermore, additional zinc finger mutants that also demonstrated this phenotype were made by site-directed mutagenesis. The majority of these mutants were able to produce normal virion-like particles; however, when tested in a single-cycle infection assay, some of the mutants demonstrated higher transduction efficiencies than that of wild-type Gag. In particular, the N17K mutant showed a seven- to ninefold increase in transduction, which correlated with enhanced vector RNA packaging. This mutant also packaged larger amounts of foreign RNA. Our results emphasize the importance of the NC zinc fingers, and not other Gag sequences, in achieving specificity in the genome encapsidation process. In addition, the described mutations may contribute to our understanding of HIV diversity resulting from recombination events between copackaged viral genomes and foreign RNA.

Amino Acid Motifs↗
PubMed2004

Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication.

The study of the hepatitis C virus (HCV) has been hindered by the lack of in vitro model systems. The recent development of HCV subgenomic RNA replicons has permitted the study of viral RNA replication in cell culture; however, the requirements for efficient replication of replicons in this system are poorly understood. Many viral isolates do not function as replicons and most require conserved changes, termed adaptive mutations, to replicate efficiently. In this report, we focus on the HCV nonstructural protein 5A (NS5A), a frequent locus for adaptive mutation. We found the interaction between NS5A and human vesicle-associated membrane protein-associated protein A (hVAP-A), a cellular target N-ethylmaleimide-sensitive factor attachment protein receptor, to be required for efficient RNA replication: NS5A mutations that blocked interaction with hVAP-A strongly reduced HCV RNA replication. Further analyses revealed an inverse correlation between NS5A phosphorylation and hVAP-A interaction. A subset of the previously identified adaptive mutations suppressed NS5A hyperphosphorylation and promoted hVAP-A binding. Our results support a model in which NS5A hyperphosphorylation disrupts interaction with hVAP-A and negatively regulates viral RNA replication, suggesting that replicon-adaptive mutations act by preventing the phosphorylation-dependent dissociation of the RNA replication complex.

Genotype↗
PubMed2004

Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

Amino Acid Sequence↗
PubMed2004

RNA sequences in the Moloney murine leukemia virus genome bound by the Gag precursor protein in the yeast three-hybrid system.

Encapsidation of the Moloney murine leukemia virus (MMLV) genome is mediated through a specific interaction between the major viral structural protein, Gag, and an RNA packaging signal, Psi. Many studies have investigated this process in vivo, although the specific examination of the Gag-RNA interaction in this context is difficult due to the variety of other viral functions involved in virion assembly in vivo. The Saccharomyces cerevisiae three-hybrid assay was used to directly examine the interaction between MMLV Gag and Psi. In this system, MMLV RNA regions exhibiting high-affinity Gag binding were mapped. All Gag-binding regions were located 3' to the viral splice donor sequence of the viral RNA transcript. No single short RNA sequence within Psi supported strong Gag interaction. Instead, an RNA comprised of nearly the entire Psi region was necessary to demonstrate an appreciable Gag interaction in the yeast three-hybrid system. These finding support the notion that two stem-loops (C and D) are not sufficient to form a core MMLV encapsidation signal.

Amino Acid Sequence↗
PubMed2004

Genetic interactions between hepatitis C virus replicons.

To investigate interactions between hepatitis C virus (HCV) RNA replication complexes, a system was developed to simultaneously select different HCV subgenomic replicons within the same cell. Transcomplementation of defective replicons was not observed, suggesting an isolated and independent nature of the HCV RNA replication complex. In contrast, a high level of competition between replicons was observed, such that the presence and increased fitness of one replicon reduced the capacity of a second one to stably replicate. These results suggest that at least one factor in Huh7 cells required for HCV RNA replication is limiting and saturable.

Genetic Complementation Test↗
PubMed2004

Goal planning: a retrospective audit of rehabilitation process and outcome.

OBJECTIVE: To consider the effectiveness of a goal planning programme for people with spinal cord injury (SCI) and address some of the current evidence gaps in goal setting. DESIGN: Retrospective audit. SUBJECTS: Consecutive series of 65 newly injured SCI patients. MAIN OUTCOME MEASURES: The Needs Assessment Checklist (NAC) has been specifically developed for the SCI population, and is used to assess patient attainment in core rehabilitation areas. A 'Goal Planning Progress' form was also used to specifically detail the goal planning process. RESULTS: Across the 65 patients, 396 goal planning meetings were held with 6176 goals set in total. Seventy-two per cent of the goals set at the first goal planning meeting were achieved by the second meeting. The rate of achievement at subsequent meetings was 68%. Significant differences in the number of planned rehabilitation days, number of goal planning meetings, and goals set were identified between injury categories. Significant positive correlations were found between the number of goals set and achievement, as measured by the NAC, in certain rehabilitation domains. CONCLUSIONS: The findings of this study demonstrate that the Needs Assessment and Goal Planning framework is effective in planning SCI rehabilitation. The capacity of this goal planning system to reflect individual need has also been established. Further systematic analyses of this process could potentially lead to more efficient rehabilitation and the identification of care pathways within clinical areas.

Adolescent↗