Kinetic and structural analyses of hepatitis C virus polyprotein processing

Bartenschlager, Ralf; Ahlborn-Laake, L; Mous, Jan; Jacobsen, Helmut

doi:10.1128/jvi.68.8.5045-5055.1994

Cited by 301 publications

(184 citation statements)

References 38 publications

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“…The 9.6-kb HCV RNA genome consists of one large open reading frame flanked on the 5= and 3= ends by untranslated regions important for efficient replication and translation of the polyprotein. The HCV polyprotein is processed into proteins that are essential for the virus replication cycle (2). The bifunctional NS3 protein consists of an N-terminal catalytic subunit that, when complexed with its activating cofactor NS4A, is required for cleavage of the polyprotein and subsequent viral replication (3,41).…”

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confidence: 99%

“…The bifunctional NS3 protein consists of an N-terminal catalytic subunit that, when complexed with its activating cofactor NS4A, is required for cleavage of the polyprotein and subsequent viral replication (3,41). Inhibition of proteolytic cleavage has been an effective approach for anti-HCV therapy since its identification (2). Efforts were aided early on by the availability of a crystal structure of the enzyme's NS3/4A protease active site (1).…”

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Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032)

McPhee

Sheaffer

Friborg

et al. 2012

Antimicrob Agents Chemother

177

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Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032)

McPhee

Sheaffer

Friborg

et al. 2012

Antimicrob Agents Chemother

177

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“…The processing of the core (nucleocapsid protein), E1, and E2 (envelope proteins) involves host signal peptidases, 5 while the HCV NS3 protein is required for maturation of the NS4a, NS4b, NS5a, and NS5b proteins. [6][7][8][9] The NS3 serine protease activity has been found to be associated with the amino-terminal portion of the NS3 protein. The NS2/3 boundary is cleaved by a second protease that includes the carboxy-terminus of NS2 and overlaps with the serine protease.…”

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confidence: 99%

“…8,10 The enzymatic activity of the NS3 protease is partly dependent on formation of a complex with NS4a. 9,[11][12][13][14][15] Chraracterization of the functional properties of the carboxy-terminal portion of NS3 of flaviviruses and HCV have revealed adenosine phosphatase (ATPase) and helicase activities that are involved in viral replication. 16,17 A portion of the NS3 sequence expressed as a fusion protein with superoxide dismutase (C33) appears to be serologically reactive during the early phase of HCV infections, and for this reason, C33 is routinely incorporated in clinical diagnostic HCV antibody immunoassays.…”

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confidence: 99%

Human and murine antibody recognition is focused on the ATPase/Helicase, but not the protease domain of the hepatitis C virus nonstructural 3 protein

et al. 1998

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The hepatitis C virus (HCV) nonstructural (NS) 3 protein has been shown to possess at least two enzymatic domains. The amino terminal third contains a serine-protease domain, whereas the carboxy terminal two thirds is comprised of an adenosine triphosphatase (ATPase)/helicase domain. These domains are essential for the maturation of the carboxy-terminal portion of the HCV polyprotein and catalyze the cap synthesis of the RNA genome. In this report, human and murine antibody responses induced by NS3 were characterized using a recombinant full-length NS3 (NS3-FL) protein, or the isolated protease or ATPase/ helicase domains, expressed and purified from Escherichia coli. Sera from 40 patients with chronic HCV infection were assayed in enzyme-linked immunoassays (EIAs) for antibody binding to the panel of NS3 proteins. Virtually all patient sera contained antibodies specific for NS3-FL and the ATPase/helicase domain, whereas only 10% of sera reacted with the protease domain of NS3. Human antibodies reactive with NS3-FL were highly restricted to the immunoglobulin G1 (IgG1) isotype and were inhibited by soluble ATPase/helicase, but not by the protease domain. The anti-NS3 (ATPase/helicase) reactivity decreased on denaturation by sodium dodecyl sulfate (SDS) and ␤-mercaptoethanol (2ME), suggesting the recognition of nonlinear or conformational B-cell determinants. Similar to infected humans, mice immunized with NS3-FL developed high-titered primary antibody responses to the NS3 ATPase/ helicase domain, whereas an anti-NS3 protease response was not observed after primary or secondary immunizations. Thus, the human and murine humoral immune responses to the HCV NS3 protein are focused on the ATPase/helicase domain, are restricted to the IgG1 isotype in humans, and are conformationally dependent. Unexpectedly, in both species, the NS3 protease domain, present in the context of the full-length NS3, appears to possess low intrinsic immunogenicity in terms of antibody production. (HEPATOLOGY 1998;28:219-224.)The hepatitis C virus (HCV) is a recent member in the virus family Flaviviridae, and has been classified as a separate genus from the pestiviruses and the flaviviruses. 1,2 The protein sequences of the three genera share short stretches of homologies that, upon comparison at the molecular level, led to the identification of the putative structural organization of the HCV polyprotein. 3,4 This was soon followed by in vitro transcriptional and translational analysis with the characterization of functional properties of the predicted viral genes. Data available today indicate that the HCV polyprotein-NH2-core (C)-envelope 1 (E1)-E2-nonstructural protein 2 (NS2)-NS3-NS4a-NS4b-NS5a-NS5b-COOH is encoded by a single positive-stranded RNA genome of approximately 9,400 nucleotides. Like other members in the family Flaviviridae, posttranslational processing of this precursor protein is required to generate the HCV viral particles and the individual viral proteins needed for its replicative cycle. The processing of the core (nucle...

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“…The NS3-NS4A PCS protease contains a typical dual ␤-barrel core observed in the chymotrypsin-like serine proteases, and the PCS participates in the folding of both the N-and C-terminal barrels (N-barrel and C-barrel) (22). The primary pestivirus NS3 cleavage sites are Leu/Ser, Leu/Ala, and Leu/Asn (23), which differ from the Cys/X sites in HCV (24) and the dibasic sites in flaviviruses (25). Recently, two minor autocleavage sites (Leu 192 /Met 193 and Leu 159 /Lys 160 ) within the NS3 protease module were identified, and altering either of the cleavage sites by Leu deletion or mutation greatly inhibited RNA replication in a CSFV replicon system and resulted in a loss of genome RNA infectivity (10).…”

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Uncoupling of Protease trans -Cleavage and Helicase Activities in Pestivirus NS3

Zheng

et al. 2017

J Virol

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The nonstructural protein NS3 from the family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A -cleavage event. Although this conformation is incompatible with protease-cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities and virus production Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease-cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different conformational states.

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Kinetic and structural analyses of hepatitis C virus polyprotein processing

Cited by 301 publications

References 38 publications

Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032)

Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032)

Human and murine antibody recognition is focused on the ATPase/Helicase, but not the protease domain of the hepatitis C virus nonstructural 3 protein

Uncoupling of Protease trans -Cleavage and Helicase Activities in Pestivirus NS3

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