Serotype-Specific Structural Differences in the Protease-Cofactor Complexes of the Dengue Virus Family

With an estimated 40% of the world population at risk, dengue poses a significant threat to human health, especially in tropical and subtropical regions. Preventative and curative efforts, such as vaccine development and drug discovery, face additional challenges due to the occurrence of four antigenically distinct serotypes of the causative dengue virus (DEN1 to -4). Complex immune responses resulting from repeat assaults by the different serotypes necessitate simultaneous targeting of all forms of the virus. One of the promising targets for drug development is the highly conserved two-component viral protease NS2B-NS3, which plays an essential role in viral replication by processing the viral precursor polyprotein into functional proteins. In this paper, we report the 2.1-Å crystal structure of the DEN1 NS2B hydrophilic core (residues 49 to 95) in complex with the NS3 protease domain (residues 1 to 186) carrying an internal deletion in the N terminus (residues 11 to 20). While the overall folds within the protease core are similar to those of DEN2 and DEN4 proteases, the conformation of the cofactor NS2B is dramatically different from those of other flaviviral apoprotease structures. The differences are especially apparent within its C-terminal region, implicated in substrate binding. The structure reveals for the first time serotype-specific structural elements in the dengue virus family, with the reported alternate conformation resulting from a unique metal-binding site within the DEN1 sequence. We also report the identification of a 10-residue stretch within NS3pro that separates the substrate-binding function from the catalytic turnover rate of the enzyme. Implications for broad-spectrum drug discovery are discussed.Dengue fever (DF) affects tens of millions of people each year, with an average mortality rate of 5%, comprising mostly young children. The increasing spread and frequency of global epidemics of this disease have heightened the urgency for developing effective strategies for prevention, diagnosis, and treatment. Though several groups are involved in vaccine development (17, 23, 25), dengue presents a unique, complex challenge. Four antigenically distinct serotypes of the causative dengue virus (DENV) (DEN1 to -4) occur in nature, with differing pathogenicities in partially overlapping geographic locations. In individuals previously exposed to a certain serotype, repeat assault by a different serotype can lead to potentially fatal complications of the disease, dengue hemorrhagic fever and dengue shock syndrome. The presence of subneutralizing levels of antibodies against the first serotype, resulting in antibody-dependent enhancement (ADE), is believed to be a causative mechanism underlying this complication. Forty percent of the world population lives in dengue risk areas, mainly in tropical and subtropical regions, which necessitates the development of vaccines and therapeutics that can simultaneously protect against all four serotypes (24).DEN1 to -4 belong to the family Flaviviridae (genus Flavivirus) of positive-stranded RNA viruses transmitted by Aedes aegypti mosquitoes. Upon infection, the genomic RNA is translated by the host cell machinery into a 370-kDa polyprotein, which is subsequently cleaved and processed into 10 distinct structural (C, E, and prM) and nonstructural (NS1, 2A, 2B, 3, 4A, 4B, and 5) proteins. A majority of this processing (junctions of NS2A/2B, 2B/3, 3/4A, and 4B/5, as well as internal sites within C, 2A, 3, and 4A) is carried out by a virus-encoded two-component protease, NS2B-NS3. Additional processing, especially of sites toward the N terminus (junctions of C/prM, prM/E, E/NS1, NS1/NS2A, and NS4A/4B), employs cellular proteases, such as signalase. The NS2B-NS3-mediated cleavage forms an essential step in the viral replicative cycle, as evidenced by the lack of production of infectious virions in mutants carrying inactivating substitutions in the protease. Of the duo, the N terminus of NS3 encodes the enzymatic core, while a hydrophilic core within NS2B provides an essential cofactor function (9).Owing to its essential nature in viral replication and the promise and success of drugs targeting the proteases in the treatment of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections (4), several recent studies have concentrated on identifying inhibitors of the flaviviral protease (5, 10, 21, 26-28). An added advantage of targeting the protease is that it is highly conserved between the serotypes (63 to 74%). For such structure-based drug design efforts, the availability of three-dimensional (3D) structures is an essential prerequisite, and given the differing pathogenicities and immune complexity generated by the multiple serotypes, it is a prudent first step to determine the structures of all four DENV proteases. The crystal structures of the DEN2 and DEN4 proteases in complex with various lengths of NS2B are available (7, 15). In this paper, we present the structures of an optimal construct of DEN1pro in complex with the essential hydrophilic core of NS2B (NS2Bc), as well as its active-site mutant. These structures reveal a novel and unexpected serotype-specific structural element embedded in the DENV genome, with implications for the discovery of broad-spectrum drugs. We also report the identification of a 10-residue stretch within the NS3 sequence that allows the separation of the substrate-binding function of the enzyme from its catalytic efficiency.