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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 09 October 2012, Vol.109(41), pp.16534-9
    Description: Alphaviruses, a group of positive-sense RNA viruses, are globally distributed arboviruses capable of causing rash, arthritis, encephalitis, and death in humans. The viral replication machinery consists of four nonstructural proteins (nsP1-4) produced as a single polyprotein. Processing of the polyprotein occurs in a highly regulated manner, with cleavage at the P2/3 junction influencing RNA template use during genome replication. Here, we report the structure of P23 in a precleavage form. The proteins form an extensive interface and nsP3 creates a ring structure that encircles nsP2. The P2/3 cleavage site is located at the base of a narrow cleft and is not readily accessible, suggesting a highly regulated cleavage. The nsP2 protease active site is over 40 Å away from the P2/3 cleavage site, supporting a trans cleavage mechanism. nsP3 contains a previously uncharacterized protein fold with a zinc-coordination site. Known mutations in nsP2 that result in formation of noncytopathic viruses or a temperature sensitive phenotype cluster at the nsP2/nsP3 interface. Structure-based mutations in nsP3 opposite the location of the nsP2 noncytopathic mutations prevent efficient cleavage of P23, affect RNA infectivity, and alter viral RNA production levels, highlighting the importance of the nsP2/nsP3 interaction in pathogenesis. A potential RNA-binding surface, spanning both nsP2 and nsP3, is proposed based on the location of ion-binding sites and adaptive mutations. These results offer unexpected insights into viral protein processing and pathogenesis that may be applicable to other polyprotein-encoding viruses such as HIV, hepatitis C virus (HCV), and Dengue virus.
    Keywords: Alphavirus -- Metabolism ; Polyproteins -- Metabolism ; Viral Nonstructural Proteins -- Metabolism ; Viral Proteins -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 2012, Vol.109(41), pp.16534-16539
    Description: Alphaviruses, a group of positive-sense RNA viruses, are globally distributed arboviruses capable of causing rash, arthritis, encephalitis, and death in humans. The viral replication machinery consists of four nonstructural proteins (nsP1–4) produced as a single polyprotein. Processing of the polyprotein occurs in a highly regulated manner, with cleavage at the P2/3 junction influencing RNA template use during genome replication. Here, we report the structure of P23 in a precleavage form. The proteins form an extensive interface and nsP3 creates a ring structure that encircles nsP2. The P2/3 cleavage site is located at the base of a narrow cleft and is not readily accessible, suggesting a highly regulated cleavage. The nsP2 protease active site is over 40 Å away from the P2/3 cleavage site, supporting a trans cleavage mechanism. nsP3 contains a previously uncharacterized protein fold with a zinc-coordination site. Known mutations in nsP2 that result in formation of noncytopathic viruses or a temperature sensitive phenotype cluster at the nsP2/nsP3 interface. Structure-based mutations in nsP3 opposite the location of the nsP2 noncytopathic mutations prevent efficient cleavage of P23, affect RNA infectivity, and alter viral RNA production levels, highlighting the importance of the nsP2/nsP3 interaction in pathogenesis. A potential RNA-binding surface, spanning both nsP2 and nsP3, is proposed based on the location of ion-binding sites and adaptive mutations. These results offer unexpected insights into viral protein processing and pathogenesis that may be applicable to other polyprotein-encoding viruses such as HIV, hepatitis C virus (HCV), and Dengue virus. ; p. 16534-16539.
    Keywords: Dengue Virus ; Polyproteins ; Proteinases ; Death ; Active Sites ; Arthritis ; Mutation ; Pathogenicity ; Pathogenesis ; Phenotype ; Humans ; Genome ; Temperature ; Virus Replication ; Hepatitis C Virus ; Rna ; Arboviruses ; Human Immunodeficiency Virus ; Viral Nonstructural Proteins ; Encephalitis
    ISSN: 0027-8424
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  • 3
    In: Nature, 2011, Vol.479(7373), p.423
    Description: Retinoic-acid-inducible gene-I (RIG-I; also known as DDX58) is a cytoplasmic pathogen recognition receptor that recognizes pathogen-associated molecular pattern (PAMP) motifs to differentiate between viral and cellular RNAs. RIG-I is activated by blunt-ended double-stranded (ds)RNA with or without a 5' -triphosphate (ppp), by single-stranded RNA marked by a 5'-ppp (1) and by polyuridine sequences (2,3). Upon binding to such PAMP motifs, RIG-I initiates a signalling cascade that induces innate immune defences and inflammatory cytokines to establish an antiviral state. The RIG-I pathway is highly regulated and aberrant signalling leads to apoptosis, altered cell differentiation, inflammation, autoimmune diseases and cancer (4,5). The helicase and repressor domains (RD) of RIG-I recognize dsRNA and 5'-ppp RNA to activate the two amino-terminal caspase recruitment domains (CARDs) for signalling. Here, to understand the synergy between the helicase and the RD for RNA binding, and the contribution of ATP hydrolysis to RIG-I activation, we determined the structure of human RIG-I helicase-RD in complex with dsRNA and an ATP analogue. The helicase-RD organizes into a ring around dsRNA, capping one end, while contacting both strands using previously uncharacterized motifs to recognize dsRNA. Small-angle X-ray scattering, limited proteolysis and differential scanning fluorimetry indicate that RIG-I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full-length RIG-I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG-I helicase-RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including RNA interference and DNA repair, which utilize homologous helicase domains within DICER and FANCM.
    Keywords: Immune Recognition -- Genetic Aspects ; Tretinoin -- Genetic Aspects ; Tretinoin -- Health Aspects ; Rna -- Health Aspects ; Rna -- Structure;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 4
    In: Nature, 2014, Vol.509(7500), p.381
    Description: Hepatitis C virus (HCV) is a significant public health concern with approximately 160 million people infected worldwide. HCV infection often results in chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. No vaccine is available and current therapies are effective against some, but not all, genotypes. HCV is an enveloped virus with two surface glycoproteins (E1 and E2). E2 binds to the host cell through interactions with scavenger receptor class B type I (SR-BI) and CD81, and serves as a target for neutralizing antibodies. Little is known about the molecular mechanism that mediates cell entry and membrane fusion, although E2 is predicted to be a class II viral fusion protein. Here we describe the structure of the E2 core domain in complex with an antigen-binding fragment (Fab) at 2.4 Å resolution. The E2 core has a compact, globular domain structure, consisting mostly of β-strands and random coil with two small α-helices. The strands are arranged in two, perpendicular sheets (A and B), which are held together by an extensive hydrophobic core and disulphide bonds. Sheet A has an IgG-like fold that is commonly found in viral and cellular proteins, whereas sheet B represents a novel fold. Solution-based studies demonstrate that the full-length E2 ectodomain has a similar globular architecture and does not undergo significant conformational or oligomeric rearrangements on exposure to low pH. Thus, the IgG-like fold is the only feature that E2 shares with class II membrane fusion proteins. These results provide unprecedented insights into HCV entry and will assist in developing an HCV vaccine and new inhibitors.
    Keywords: Sciences (General) ; Physics;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 5
    In: Nature, 2005, Vol.435(7040), p.374
    Description: Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The non-structural protein NS5A is an active component of HCV replicase, as well as a pivotal regulator of replication and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth. NS5A is a large phosphoprotein (56-58 kDa) with an amphipathic alpha -helix at its amino terminus that promotes membrane association. After this helix region, NS5A is organized into three domains. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule. Mutations disrupting either the membrane anchor or zinc binding of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-Aa resolution, which contains a novel fold, a new zinc-coordination motif and a disulphide bond. We use molecular surface analysis to suggest the location of protein-, RNA-and membrane-interaction sites.
    Keywords: Protein Structure ; Replication ; Ns5a Protein ; Replicase ; Hepatitis C Virus ; Viral Proteins;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 6
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 05 January 2016, Vol.113(3)
    Description: The cytosolic innate immune receptor Retinoic Acid Inducible Gene-I (RIG-I) is the principal detector of pathogenic RNAs carrying a 5'-triphosphate (5'ppp). Self RNAs like mRNAs evade recognition by RIG-I due to posttranscriptional modifications like 5'-end capping with 7-methyl guanosine (m7G) and 2'-O-methylation of 5'-end nucleotides. Viruses have also evolved mechanisms to mimic these modifications, which in part is believed to aid in immune evasion. Currently, it is unclear how these modifications modulate RIG-I recognition. This paper provides structural and mechanistic insights into the roles of the m7G cap and 2'-O-methylation in RIG-I evasion. We show that RIG-I accommodates the m7G base while maintaining the 5'ppp contacts and can recognize Cap-0 RNAs but not Cap-1.
    Keywords: Sciences (General)
    ISSN: 0027-8424
    E-ISSN: 1091-6490
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  • 7
    In: Nature, 2006, Vol.442(7104), p.831
    Description: Hepatitis C virus is a major global health problem affecting an estimated 170 million people worldwide. Chronic infection is common and can lead to cirrhosis and liver cancer. There is no vaccine available and current therapies have met with limited success. The viral RNA genome encodes a polyprotein that includes two proteases essential for virus replication. The NS2-3 protease mediates a single cleavage at the NS2/NS3 junction, whereas the NS3-4A protease cleaves at four downstream sites in the polyprotein. NS3-4A is characterized as a serine protease with a chymotrypsin-like fold, but the enzymatic mechanism of the NS2-3 protease remains unresolved. Here we report the crystal structure of the catalytic domain of the NS2-3 protease at 2.3 Angstroms resolution. The structure reveals a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer, and the nucleophilic cysteine by the other. The carboxy-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. Proteolysis through formation of a composite active site occurs in the context of the viral polyprotein expressed in mammalian cells. These features offer unexpected insights into polyprotein processing by hepatitis C virus and new opportunities for antiviral drug design. Journal Article.
    Keywords: Materials Sciencecleavage ; Crystal Structure ; Cysteine ; Design ; Hepatitis ; Histidine ; Liver ; Neoplasms ; Processing ; Proteolysis ; Residues ; Resolution ; Rna ; Serine ; Vaccines ; National Synchrotron Light Source;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 8
    In: Nature, 2008, Vol.454(7203), p.523
    Description: Innate immune defences are essential for the control of virus infection and are triggered through host recognition of viral macromolecular motifs known as pathogen-associated molecular patterns (PAMPs). Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people worldwide. Infection is regulated by hepatic immune defences triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals interferon regulatory factor 3 activation to induce the expression of interferon- alpha/beta and antiviral/interferon-stimulated genes (ISGs) that limit infection. Here we identify the polyuridine motif of the HCV genome 3' non- translated region and its replication intermediate as the PAMP substrate of RIG-I, and show that this and similar homopolyuridine or homopolyriboadenine motifs present in the genomes of RNA viruses are the chief feature of RIG-I recognition and immune triggering in human and murine cells. 5' terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent on homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I- dependent signalling to induce a hepatic innate immune response in vivo, and triggered interferon and ISG expression to suppress HCV infection in vitro. These results provide a conceptual advance by defining specific homopolymeric RNA motifs within the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and demonstrate immunogenic features of the PAMP-RIG-I interaction that could be used as an immune adjuvant for vaccine and immunotherapy approaches.
    Keywords: Genomes ; Macromolecules ; Replication ; Immunotherapy ; Interferon Regulatory Factor 3 ; RNA Viruses ; Immunity ; Adjuvants ; Infection ; B-Interferon ; Immunogenicity ; Ribonucleotides ; Liver ; Immune Response ; Vaccines ; DNA Helicase ; a-Interferon ; Signal Transduction ; Hepatitis C Virus ; Microorganisms & Parasites ; RNA ; Ecosystem and Ecology Studies ; Immunology;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 9
    Language: English
    In: PLoS ONE, 30 October 2014, Vol.9(10)
    Description: We report that Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2’s interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421–645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. We used surface plasmon resonance detection to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50’s ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment.
    Keywords: 60 Applied LIFE Sciences ; Sciences (General)
    ISSN: 1932-6203
    E-ISSN: 1932-6203
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  • 10
    Language: English
    In: Biophysical Journal, 31 January 2012, Vol.102(3), pp.601a-601a
    Keywords: Biology
    ISSN: 0006-3495
    E-ISSN: 1542-0086
    Source: ScienceDirect Journals (Elsevier)
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