In Vitro Characterization of a Purified NS2/3 Protease Variant of Hepatitis C Virus

Thibeault, Diane; Maurice, Roger; Pilote, Louise; Lamarre, Daniel; Pause, Arnim

doi:10.1074/jbc.m108266200

Cited by 53 publications

(48 citation statements)

References 34 publications

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“…However, the fusion of NS2 to the N terminus of NS3 does not completely abolish the interaction with NS4A. In fact, several groups have reported synthetic 4A peptides as potent inhibitors of NS2/3 autoprocessing (19,48). Nevertheless, we found a decrease in NS3 protease kinetics in an in vitro assay dependent on the addition of a 4A peptide using purified NS2/ 3-(904 -1206).…”

Section: Discussioncontrasting

confidence: 53%

“…No homology between NS2/3 and other proteases has been identified, and the catalytic mechanism of action remains unclear. Although the observation that NS2/3 activity is stimulated by zinc and inhibited by EDTA has led some groups to suggest that NS2/3 is a novel metalloprotease, others have proposed that it may function as a cysteine protease, and studies performed with classical protease inhibitors have not yielded a definite classification (17,19,20). Mutagenesis studies have identified His 952 and Cys 993 within NS2 as being essential for NS2/3 protease activity (15,16), and in addition, mutations thought to perturb the local conformation of the cleavage site also inactivate the enzyme (21).…”

Section: Hepatitis C Virus (Hcv)mentioning

confidence: 99%

“…1D and encompass only the protease domain of NS3. Both the NS2/3 active-site mutants and NS3 proteins were recovered from inclusion bodies utilizing protocols for NS2/3 protease production (19). Following purification on a nickel column, the proteins were subsequently refolded on a gel filtration column.…”

Section: Fig 4 Ns3 Helicase Activity In the Presence Of Uncleaved Ns2mentioning

confidence: 99%

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Hepatitis C Virus NS2/3 Processing Is Required for NS3 Stability and Viral RNA Replication

Welbourn

Green

Gamache

et al. 2005

Journal of Biological Chemistry

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The hepatitis C virus NS2/3 protease is responsible for cleavage of the viral polyprotein between nonstructural proteins NS2 and NS3. We show here that mutation of three highly conserved residues in NS2 (His 952 , Glu 972 , and Cys 993 ) abrogates NS2/3 protease activity and that introduction of any of these mutations into subgenomic NS2-5B replicons results in complete inactivation of NS2/3 processing and RNA replication in both stable and transient replication assays. The effect of uncleaved NS2 on the various activities of NS3 was therefore explored. Unprocessed NS2 had no significant effect on the in vitro ATPase and helicase activities of NS3, whereas immunoprecipitation experiments demonstrated a decreased affinity of NS4A for uncleaved NS2/3 as compared with NS3. This subsequently resulted in reduced kinetics in an in vitro NS3 protease assay with the unprocessed NS2/3 protein. Interestingly, NS3 was still capable of efficient processing of the polyprotein expressed from a subgenomic replicon in Huh-7 cells in the presence of uncleaved NS2. Notably, we show that fusion with NS2 leads to the rapid degradation of NS3, whose activity is essential for RNA replication. Finally, we demonstrate that uncleaved NS2/3 degradation can be prevented by the addition of a proteasome inhibitor. We therefore propose that NS2/3 processing is a critical step in the viral life cycle and is required to permit the accumulation of sufficient NS3 for RNA replication to occur. The regulation of NS2/3 cleavage could constitute a novel mechanism of switching between viral RNA replication and other processes of the hepatitis C virus life cycle.

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Section: Discussioncontrasting

confidence: 53%

Section: Hepatitis C Virus (Hcv)mentioning

confidence: 99%

Section: Fig 4 Ns3 Helicase Activity In the Presence Of Uncleaved Ns2mentioning

confidence: 99%

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Hepatitis C Virus NS2/3 Processing Is Required for NS3 Stability and Viral RNA Replication

Welbourn

Green

Gamache

et al. 2005

Journal of Biological Chemistry

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“…However, the observation that the protease activity is stimulated by metal ions and inhibited by EDTA led to the suggestion that the NS2/3 protease was a metalloprotease (27,31). Furthermore, sequence comparison with other proteases and studies with classical protease inhibitors (64,83) have not resulted in a definitive classification. Additionally, NS2/3 autoprocessing seems to be extremely sensitive to the correct folding of the protease (27,31,66), and a trans-cleavage activity of an active NS2/3 protease containing a mutant cleavage site has been suggested (66).…”

Section: Discussionmentioning

confidence: 99%

Protein-Protein Interactions between Hepatitis C Virus Nonstructural Proteins

Dimitrova

Imbert

Kiény

et al. 2003

J Virol

165

180

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Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirusinfected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo-and heterodimerizations.

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“…7A and B), the bulk of mature NS2 and NS3 should arise from processing of the nascent chain. Previous studies also noted that the posttranslational NS2-3 autocleavage is inefficient, unless special conditions (such as the presence of detergents) are met (56,78).…”

Section: Discussionmentioning

confidence: 99%

Complete Translation of the Hepatitis C Virus Genome In Vitro: Membranes Play a Critical Role in the Maturation of All Virus Proteins except for NS3

Svitkin¹,

Pause

López‐Lastra

et al. 2005

J Virol

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We developed an in vitro translation extract from Krebs-2 cells that translates the entire open reading frame of the hepatitis C virus (HCV) strain H77 and properly processes the viral protein precursors when supplemented with canine microsomal membranes (CMMs). Translation of the C-terminal portion of the viral polyprotein in this system is documented by the synthesis of NS5B. Evidence for posttranslational modification of the viral proteins, the N-terminal glycosylation of E1 and the E2 precursor (E2-p7), and phosphorylation of NS5A is presented. With the exception of NS3, efficient generation of all virus-specific proteins is CMM dependent. A time course of the appearance of HCV products indicates that the viral polyprotein is cleaved cotranslationally. A competitive inhibitor of the NS3 protease inhibited accumulation of NS3, NS4B, NS5A, and NS5B, but not that of NS2 or structural proteins. CMMs also stabilized HCV mRNA during translation. Finally, the formyl-[35 S]methionyl moiety of the initiator tRNA Met was incorporated exclusively into the core protein portion of the polyprotein, demonstrating that translation initiation in this system occurs with high fidelity.Hepatitis C virus (HCV) is an enveloped virus that belongs to the genus Hepacivirus in the family Flaviviridae (which also includes flaviviruses and pestiviruses) and is the leading cause of chronic hepatitis and liver cirrhosis in humans in the developed world (77). The genome of HCV is a ϳ9.6-kb-long positive-strand RNA that is translated into a polyprotein of approximately 3,010 amino acids (58). For some HCV strains, evidence for an alternative open reading frame that overlaps the core protein gene has also been reported (83,88). Translation of the HCV RNA is accomplished by binding of ribosomes to an internal ribosome entry site (IRES) (80). A salient feature of the HCV IRES is its ability to recruit ribosomes with the aid of only two canonical initiation factors (eIF3 and eIF2) (29, 55), in contrast to most other IRESs, e.g., IRESs from picornaviruses, which also require the eIF4 initiation factors (52,54,73).HCV does not replicate in cultured cells. However, viral protein detection and mapping were achieved by using transient expression systems (1,18,20) and, more recently, the replicon system (38, 57). These studies have suggested that the viral polyprotein is cleaved co-and posttranslationally at specific sites into at least 10 polypeptides ordered from the N terminus as follows: C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. Cleavages within the structural region and at the p7/NS2 junction are thought to be mediated by host cell signal peptidase(s), which is located in the lumen of the endoplasmic reticulum (ER) and cleaves behind stretches of hydrophobic amino acids. The first cleavage product (C; core) is highly basic and constitutes the major component of the nucleocapsid. Envelope proteins E1 and E2 are type I transmembrane glycoproteins. Processing of the NS region is mediated by two overlapping virus-specific proteases. The NS2-NS3 zinc-...

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In Vitro Characterization of a Purified NS2/3 Protease Variant of Hepatitis C Virus

Cited by 53 publications

References 34 publications

Hepatitis C Virus NS2/3 Processing Is Required for NS3 Stability and Viral RNA Replication

Hepatitis C Virus NS2/3 Processing Is Required for NS3 Stability and Viral RNA Replication

Protein-Protein Interactions between Hepatitis C Virus Nonstructural Proteins

Complete Translation of the Hepatitis C Virus Genome In Vitro: Membranes Play a Critical Role in the Maturation of All Virus Proteins except for NS3

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