Switching the Substrate Specificity of the Two-Component NS2B-NS3 Flavivirus Proteinase by Structure-Based Mutagenesis

Shiryaev, Sergey A.; Ratnikov, Boris I.; Aleshin, Alexander E.; Kozlov, I. A.; Nelson, Nicholas A.; Lebl, Michal; Smith, Jeffrey W.; Liddington, Robert; Strongin, Alex Y.

doi:10.1128/jvi.02719-06

Cited by 48 publications

(41 citation statements)

References 36 publications

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“…The structures are consistent with the data presented herein that substrate sequences flanking the cleavage site play a role in specificity (70). Our data are also consistent with other observations that NS3 positions 131 and 132 contribute to specificity of the P2= position, as mutation of WNV NS3 P131 and NS3 T132 to match the DENV sequence (P131K plus T132P) shifted the specificity of WNV NS2B/3 to that of DENV NS2B/3 (71). Interactions between protease and substrate on the non-prime site of the scissile bond involve the S2 and S3 pockets of the active site and are occupied by the P2 and P3 positions of the substrate, respectively.…”

Section: Discussionsupporting

confidence: 92%

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

VanBlargan

Davis

Dowd

et al. 2015

J Virol

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The molecular mechanisms that define the specificity of flavivirus RNA encapsulation are poorly understood. Virions composed of the structural proteins of one flavivirus and the genomic RNA of a heterologous strain can be assembled and have been developed as live attenuated vaccine candidates for several flaviviruses. In this study, we discovered that not all combinations of flavivirus components are possible. While a West Nile virus (WNV) subgenomic RNA could readily be packaged by structural proteins of the DENV2 strain 16681, production of infectious virions with DENV2 strain New Guinea C (NGC) structural proteins was not possible, despite the very high amino acid identity between these viruses. Mutagenesis studies identified a single residue (position 101) of the DENV capsid (C) protein as the determinant for heterologous virus production. C101 is located at the P1= position of the NS2B/3 protease cleavage site at the carboxy terminus of the C protein. WNV NS2B/3 cleavage of the DENV structural polyprotein was possible when a threonine (Thr101 in strain 16681) but not a serine (Ser101 in strain NGC) occupied the P1= position, a finding not predicted by in vitro protease specificity studies. Critically, both serine and threonine were tolerated at the P1= position of WNV capsid. More extensive mutagenesis revealed the importance of flanking residues within the polyprotein in defining the cleavage specificity of the WNV protease. A more detailed understanding of the context dependence of viral protease specificity may aid the development of new protease inhibitors and provide insight into associated patterns of drug resistance. West Nile virus (WNV) and the four serotypes of dengue virus (DENV1 to -4) are mosquito-borne viruses of the Flavivirus genus that significantly impact public health (1, 2). Despite a clear need, neither vaccines nor therapeutics for WNV or DENV have been licensed for use in humans. The flavivirus genome is an ϳ11-kb, single-stranded, positive-sense RNA that encodes a single open reading frame flanked by 5= and 3= untranslated regions. The viral genome is translated on endoplasmic reticulum (ER)-derived membranes into a single polyprotein that undergoes co-and posttranslational cleavage by the viral protease NS2B/3 and host proteases into 10 functionally distinct proteins, including the structural proteins capsid (C), premembrane (prM), and envelope (E) that form the virus particle. During assembly, membrane-anchored prM and E glycoproteins are incorporated into virions as they bud into the ER lumen. The C protein associates with the viral genome in the cytoplasm to form an unstructured nucleocapsid that is incorporated into the budding particle via unknown mechanisms (3). The carboxy terminus (C terminus) of the C protein includes a signal sequence, flanked by protease cleavage sites, that directs the translocation of prM into the ER lumen and tethers C to the cytosolic face of the ER membrane.Cleavage at both sites is essential for virion morphogenesis and occurs in a sequenti...

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Section: Discussionsupporting

confidence: 92%

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

VanBlargan

Davis

Dowd

et al. 2015

J Virol

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“…These targets represent human proteins and human pathogens of bacterial and viral origin. Earlier studies have proved the efficiency and the accuracy of the custom-built centrifugal synthesizer, and the synthetic scheme we used and the high quality of the synthesized peptides (22,(35)(36)(37). These synthetic peptides were used in the cleavage reactions to identify the cleavage preferences of furin, PC2, PC4, PC5/6, PC7, and PACE4.…”

Section: Resultsmentioning

confidence: 99%

Substrate Cleavage Analysis of Furin and Related Proprotein Convertases

Remacle

Shiryaev

et al. 2008

Journal of Biological Chemistry

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130

137

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We present the data and the technology, a combination of which allows us to determine the identity of proprotein convertases (PCs) related to the processing of specific protein targets including viral and bacterial pathogens. Our results, which support and extend the data of other laboratories, are required for the design of effective inhibitors of PCs because, in general, an inhibitor design starts with a specific substrate. Seven proteinases of the human PC family cleave the multibasic motifs R-X-(R/K/X)-R2 and, as a result, transform proproteins, including those from pathogens, into biologically active proteins and peptides. The precise cleavage preferences of PCs have not been known in sufficient detail; hence we were unable to determine the relative importance of the individual PCs in infectious diseases, thus making the design of specific inhibitors exceedingly difficult. To determine the cleavage preferences of PCs in more detail, we evaluated the relative efficiency of furin, PC2, PC4, PC5/6, PC7, and PACE4 in cleaving over 100 decapeptide sequences representing the R-X-(R/K/X)-R2 motifs of human, bacterial, and viral proteins. Our computer analysis of the data and the follow-on cleavage analysis of the selected full-length proteins corroborated our initial results thus allowing us to determine the cleavage preferences of the PCs and to suggest which PCs are promising drug targets in infectious diseases. Our results also suggest that pathogens, including anthrax PA83 and the avian influenza A H5N1 (bird flu) hemagglutinin precursor, evolved to be as sensitive to PC proteolysis as the most sensitive normal human proteins.

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“…The specificity could derive from the flexibility and conformation of the peptide that is not immediately obvious from the structure. For an example, mutation of WNV P131 and T132 to the DENV sequence K and P, respectively, changes the P1= and P2= specificity to be more like those of DENV (29). T132 forms a hydrogen bond with the P1= carbonyl in the WNV-aprotinin structure, and this interaction is lost in DENV, where the Thr is replaced by Pro.…”

Section: Discussionmentioning

confidence: 99%

Ligand-Bound Structures of the Dengue Virus Protease Reveal the Active Conformation

Noble

Seh

Chao

et al. 2012

J Virol

257

385

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Dengue is a mosquito-borne viral hemorrhagic disease that is a major threat to human health in tropical and subtropical regions. Here we report crystal structures of a peptide covalently bound to dengue virus serotype 3 (DENV-3) protease as well as the serine-protease inhibitor aprotinin bound to the same enzyme. These structures reveal, for the first time, a catalytically active, closed conformation of the DENV protease. In the presence of the peptide, the DENV-3 protease forms the closed conformation in which the hydrophilic ␤-hairpin region of NS2B wraps around the NS3 protease core, in a manner analogous to the structure of West Nile virus (WNV) protease. Our results confirm that flavivirus proteases form the closed conformation during proteolysis, as previously proposed for WNV. The current DENV-3 protease structures reveal the detailed interactions at the P4= to P3 sites of the substrate. The new structural information explains the sequence preference, particularly for long basic residues in the nonprime side, as well as the difference in substrate specificity between the WNV and DENV proteases at the prime side. Structural analysis of the DENV-3 protease-peptide complex revealed a pocket that is formed by residues from NS2B and NS3; this pocket also exists in the WNV NS2B/NS3 protease structure and could be targeted for potential antivirus development. The structural information presented in the current study is invaluable for the design of specific inhibitors of DENV protease. Dengue virus (DENV) is a member of the flavivirus genus, which includes several viruses that are important human pathogens, including yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV). The four serotypes of DENV are estimated to cause 50 to 100 million human infections worldwide every year in tropical and subtropical regions (16). There are currently no clinically approved vaccines or therapeutics for DENV. Understanding the molecular details of DENV infection is essential for vaccine and antiviral development.Flaviviruses contain a single strand of positive-sense RNA that encodes three structural and seven nonstructural (NS) proteins that are translated as a single polypeptide chain. NS3 contains two functions, an N-terminal serine protease and a C-terminal RNA helicase. The catalytic triad (His51, Asp75, and Ser135) is within the NS3 protease domain, but a region of NS2B is also required for catalytic activity (15). NS2B contains three predicted transmembrane helices, ensuring that the NS2B-NS3 protease is bound to the endoplasmic reticulum. The viral polyprotein is cleaved into the individual proteins by a combination of host proteases and the viral NS2B-NS3 protease (6, 30). The proteolytic activity of the viral protease is essential for viral replication, making it an excellent antiviral target (18).Ligand-free structures of flavivirus proteases have been solved for DENV-1 and -2 and a WNV active-site mutant (1, 7, 13). In addition, the structure of Mur...

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Switching the Substrate Specificity of the Two-Component NS2B-NS3 Flavivirus Proteinase by Structure-Based Mutagenesis

Cited by 48 publications

References 36 publications

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

Substrate Cleavage Analysis of Furin and Related Proprotein Convertases

Ligand-Bound Structures of the Dengue Virus Protease Reveal the Active Conformation

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