Functional characterization of the NS2B/NS3 protease complex from seven viruses belonging to different groups inside the genus Flavivirus

Bessaud, Maël; Pastorino, Boris; Peyrefitte, Christophe N.; Rolland, Dominique; Grandadam, Marc; Tolou, Hugues

doi:10.1016/j.virusres.2006.01.021

Cited by 41 publications

(53 citation statements)

References 56 publications

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“…9B) showed no autoproteolysis at the NS2B-NS3 junction, since the canonical NS2B-NS3 recognition site was not incorporated in the polypeptide sequence. In harmony with observations from other studies (5,19), our recombinant proteases accumulated to levels representing about 30% of total bacterial protein, with Ͼ90% being partitioned to the insoluble fraction. Nevertheless, sufficient soluble proteases were obtained for the in vitro assays in previous studies and this present work.…”

Section: Resultssupporting

confidence: 89%

Mechanism of NS2B-Mediated Activation of NS3pro in Dengue Virus: Molecular Dynamics Simulations and Bioassays

Zuo

Liew

Gang

et al. 2009

J Virol

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The NS2B cofactor is critical for proteolytic activation of the flavivirus NS3 protease. To elucidate the mechanism involved in NS2B-mediated activation of NS3 protease, molecular dynamic simulation, principal component analysis, molecular docking, mutagenesis, and bioassay studies were carried out on both the dengue virus NS3pro and NS2B-NS3pro systems. The results revealed that the NS2B-NS3pro complex is more rigid than NS3pro alone due to its robust hydrogen bond and hydrophobic interaction networks within the complex. These potent networks lead to remodeling of the secondary and tertiary structures of the protease that facilitates cleavage sequence recognition and binding of substrates. The cofactor is also essential for proper domain motion that contributes to substrate binding. Hence, the NS2B cofactor plays a dual role in enzyme activation by facilitating the refolding of the NS3pro domain as well as being directly involved in substrate binding/interactions. Kinetic analyses indicated for the first time that Glu92 and Asp50 in NS2B and Gln27, Gln35, and Arg54 in NS3pro may provide secondary interaction points for substrate binding. These new insights on the mechanistic contributions of the NS2B cofactor to NS3 activation may be utilized to refine current computer-based search strategies to raise the quality of candidate molecules identified as potent inhibitors against flaviviruses.The spectrum of clinical manifestations caused by the dengue virus, namely, dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, are increasing in severity and present major threats to global health. In the tropics and subtropical regions, the fatality rate ranges between 1 and 10% of more than 1 million cases of dengue hemorrhagic fever every year. With exacerbation of disease infection rates aided by global warming, growing urbanization, and rapid expansion of international trade and travel, the virus is now endemic in more than 100 countries and threatens more than a quarter of the world's population. Currently, there is no vaccine or effective therapeutic agent available to protect against or cure acute dengue viral infections.Dengue viruses are members of the Flaviviridae family. They are small, enveloped positive-sense RNA viruses transmitted by Aedes aegypti and Aedes albopictus mosquitoes (6). Dengue virus type 2 (DEN2), the most prevalent of the four serotypes, contains a single-stranded RNA and encodes a large single polyprotein precursor of 3,391 amino acid residues which consists of three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (16). Processing of the polyprotein precursor to release mature viral proteins is mediated co-and posttranslationally by host proteases and the virus-encoded two-component protease NS2B-NS3pro (8). Thus, the essential role of NS2B-NS3pro for viral replication makes it an attractive target for the development of effective antiviral drug inhibitors with therapeutic applications against dengue hemorrhagic feve...

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Section: Resultssupporting

confidence: 89%

Mechanism of NS2B-Mediated Activation of NS3pro in Dengue Virus: Molecular Dynamics Simulations and Bioassays

Zuo

Liew

Gang

et al. 2009

J Virol

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“…These variable results can be explained by the different methods used to predict protein structure in the different software [27,28]. The CFAV V 50 -S 96 region was found aligned with the DNS2B sequences designed in our previous works [9,10] (data not shown), and was cloned as CFAV DNS2B in this work.…”

Section: Resultsmentioning

confidence: 81%

“…The CFAV NS3 protein sequence analysis showed the four boxes conserved amongst the serine protease [10,13,29] and particularly the H, D and S residues that form the catalytic triad of serine proteases (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

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Enzymatic characterization of a trypsin-like serine protease encoded by the genome of Cell fusing agent virus

et al. 2006

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Cell fusing agent virus (CFAV) is a positive strand RNA insect virus first isolated from a mosquito cell line. Based on viral morphology, phenotypic and phylogenetic studies, CFAV had been tentatively assigned to the genus Flavivirus (family Flaviviridae). The determination of the CFAV polyprotein complete sequence showed a putative serine protease domain analogue to the flaviviral NS2B/NS3 complex. This complex had been extensively studied, because it represented one of the main targets for antiflavivirus therapy development. We report herein the biochemical characterization of CFAV DeltaNS2B-NS3pro protease complex. CFAV polyprotein sequence was computationally analysed to identify the amino-acid regions involved in protease activity. We designed, expressed and purified a catalytically active protease whose enzymatic properties were determined using fluorogenic substrates. Our results showed that, despite the low level of conservation of its amino-acid sequence, CFAV protease exhibited physico-chemical properties of other flaviviruses (high pH value requirement for optimal activity, inhibition by salt and preference for substrates featuring a basic residue at P(1) position).

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“…For example, NS2B-NS3 serine proteases from flaviviruses (e.g. West Nile, dengue, yellow fever) (37)(38)(39)(40)(41)(42) become substantially active in vitro only at alkaline pH (Յ10.5). Those enzymes, like factor B, are thought to interact with other protein domains and to deposit on membrane surfaces under native conditions for proteolytic processing.…”

Section: Discussionmentioning

confidence: 99%

Profiling the Enzymatic Properties and Inhibition of Human Complement Factor B

Abbenante

Fairlie

2007

Journal of Biological Chemistry

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Human complement factor B is the crucial catalytic component of the C3 convertase enzyme that activates the alternative pathway of complement-mediated immunity. Although a serine protease in its own right, factor B circulates in human serum as an inactive zymogen and there is a crystal structure only for the inactive state of factor B and various fragments. To provide greater insight to the catalytic function and properties of factor B, we have used short para-nitroanilide derivatives of 4-to 15-residue peptides as substrates to profile the catalytic properties of factor B. Among factors found to influence catalytic activity of factor B was an unusual dependence on pH. Non-physiological alkaline conditions strongly promoted substrate cleavage by factor B, consistent with a pH-accessible conformation of the enzyme that may be critical for catalytic function. Small N-terminal extensions to conventional hexapeptide para-nitroanilide substrates significantly increased catalytic activity of factor B, which was more selective for its cleavage site than trypsin. The new chromogenic assay enabled optimization of catalysis conditions, the profiling of different substrate sequences, and the development of the first reversible and competitive substrate-based inhibitor of factor B. The inhibitor was also shown to prevent in vitro formation of C3a from C3 by factor B, by synthetic and by natural C3 convertase of the alternative complement activation pathway, and to block formation of membrane attack complex. The availability of a reversible substrate-based inhibitor that could stabilize the active conformation of factor B, in conjunction with a pH-promoted higher processing activity, may offer a new avenue to obtain crystal structures of factor B and C3 convertase in an active conformation.

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Functional characterization of the NS2B/NS3 protease complex from seven viruses belonging to different groups inside the genus Flavivirus

Cited by 41 publications

References 56 publications

Mechanism of NS2B-Mediated Activation of NS3pro in Dengue Virus: Molecular Dynamics Simulations and Bioassays

Mechanism of NS2B-Mediated Activation of NS3pro in Dengue Virus: Molecular Dynamics Simulations and Bioassays

Enzymatic characterization of a trypsin-like serine protease encoded by the genome of Cell fusing agent virus

Profiling the Enzymatic Properties and Inhibition of Human Complement Factor B

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