Kailin Yang

The crystal structure of Zika virus helicase: basis for antiviral drug design.

The genus of Flavivirus contains important human pathogens, including dengue (DENV), yellow fever (YFV), West Nile (WNV), Japanese encephalitis (JEV), and tick-borne encephalitis (TBEV) viruses, which cause a number of serious human diseases throughout the world (Pierson TC, 2013). Zika virus (ZIKV) is also an arthropod-borne flavivirus, which was initially isolated in 1947 from a febrile sentinel rhesus monkey in the Zika forest in Entebbe, Uganda. ZIKV is transmitted by multiple Aedes mosquitoes (Lazear and Diamond, 2016). Historically, ZIKV infection typically caused a mild and self-limiting illness in human beings, accompanied by fever, headache, arthralgia, myalgia, and maculopapular rash (Ioos et al., 2014). ZIKV caught global attention in April 2007, when it caused a large epidemic of Asian genotype ZIKV in Yap Island and Guam, Micronesia. From 2013 to 2014, the Asian genotype was found responsible for the epidemics among several Pacific Islands, including French Polynesia, New Caledonia, Cook Islands, Tahiti, and Easter Island (Lazear and Diamond, 2016). In 2015, a rampant outbreak of ZIKV infection struck Brazil and other regions of the Americas, causing an estimated 1.3 million cases (Hennessey et al., 2016; Mlakar et al., 2016). Thereafter, ZIKV was found in fetal brain tissue, presumably accounting for the sharp increase of congenital microcephaly in the epidemic areas (Brasil et al., 2016; Mlakar et al., 2016; Rodrigues, 2016). Recent studies have demonstrated the significant cellular death of neural stem cells once infected with ZIKV, which provides direct evidence for the inhibitory role of ZIKV on fetal brain development (Tang et al., 2016). However, as there are currently no effective vaccines or therapies available to contain ZIKV infection, ZIKV remains a significant challenge to the public health of the Western Hemisphere as well as the whole world (Lazear and Diamond, 2016). Similar to other flaviviruses, ZIKV contains a singlestranded, positive sense RNA genome of 10.7 kb. The genome is translated into a single large polypeptide, which undergoes proteolytic cleavage into 3 structural proteins (C, prM/M, and E), and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Pierson TC, 2013). The NS3 protein is a key component for viral polypeptide processing and genomic replication, with a protease domain at its N-terminus and a helicase domain at the C-terminus. Upon stimulation by RNA, the helicase domain exhibits intrinsic nucleoside triphosphatase activity, which then provides the chemical energy to unwind viral RNA replication intermediates to facilitate replication of the viral genome together with RNA-dependent RNA polymerase (NS5) (Lindenbach, 2001). Given its essential role in genome replication, ZIKV helicase could be an attractive target for drug development against ZIKV. Here we report the crystal structure of ZIKV helicase at 1.8-Å resolution. The helicase structure revealed a conserved triphosphate pocket critical for nonspecific hydrolysis of nucleoside triphosphates across multiple flavivirus species. A positive-charged tunnel has been identified in the viral helicase, which is potentially responsible for accommodating the RNA. This crystal structure of ZIKV helicase provides an accurate model for rational drug design against ZIKV infection. We determined the crystal structure of ZIKV helicase at a resolution of 1.8 Å (Table S1) in the space group P21. Distinct from the DENV-2 helicase, whose two crystal forms both contain two molecules per asymmetric unit (Xu et al., 2005), ZIKV helicase has a solo protein molecule in an asymmetric unit in the crystals. No stable oligomer through crystallographic packing was identified in the crystals, consistent with the observation of a monomeric form of the ZIKV helicase in solution by size exclusion chromatography (Fig. 1A). This observation suggests that ZIKV helicase is able to function as a monomer. The refined model is complete and includes the residues 175–617 from ZIKV NS3. Although the overall structure is generally well ordered, the electron densities are less well defined for residues 193–202 and 249–255 with a higher B factor (>50 compared with an overall average B factor of 27). This indicates that these are possible substrate/ligand binding regions due to the increased flexibility. The tertiary structure of ZIKV helicase reveals three domains, of around 130–160 amino acid residues each (Fig. 1B and 1C). Domain I (residues 175–332) and domain II (residues 333–481) share a similar fold with an expanded six-stranded β-sheet stacked between a large number of loops and four helices, though there is little sequence identity between these two domains. Domain III (residues 482–617) is predominantly comprised of a four-α-

Structural basis of Zika virus helicase in recognizing its substrates

The recent explosive outbreak of Zika virus (ZIKV) infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn2+ and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn2+. The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV) apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.

Crystallization and preliminary crystallographic analysis of a native chitinase from the fungal pathogen Aspergillus fumigatus YJ-407.

Chitinase hydrolyzes chitin, a linear polymer of beta-1,4-linked N-acetylglucosamine (NAG), and plays a variety of roles in the biological world. In addition to endo- and exo-hydrolytic activities, transglycosyl activity has also been observed in the extracellular chitinase (afCHI) from the airborne saprophytic fungi Aspergillus fumigatus YJ-407. Crystals of this native chitinase have been grown at 291 K using PEG 3350 as a precipitant. The diffraction data from the crystal extend to 1.7 A resolution at BSRF, China. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 95.7, b = 100.5, c = 134.3 A. The presence of two molecules per asymmetric unit gives a crystal volume per protein mass (V(M)) of 3.6 A(3) Da(-1) and a solvent content of 65% by volume. A full set of X-ray diffraction data was collected to 2.1 A resolution.

Crystal Structure of Feline Infectious Peritonitis Virus Main Protease in Complex with Synergetic Dual Inhibitors

ABSTRACT Coronaviruses (CoVs) can cause highly prevalent diseases in humans and animals. Feline infectious peritonitis virus (FIPV) belongs to the genus Alphacoronavirus, resulting in a lethal systemic granulomatous disease called feline infectious peritonitis (FIP), which is one of the most important fatal infectious diseases of cats worldwide. No specific vaccines or drugs have been approved to treat FIP. CoV main proteases (Mpros) play a pivotal role in viral transcription and replication, making them an ideal target for drug development. Here, we report the crystal structure of FIPV Mpro in complex with dual inhibitors, a zinc ion and a Michael acceptor. The complex structure elaborates a unique mechanism of two distinct inhibitors synergizing to inactivate the protease, providing a structural basis to design novel antivirals and suggesting the potential to take advantage of zinc as an adjunct therapy against CoV-associated diseases. IMPORTANCE Coronaviruses (CoVs) have the largest genome size among all RNA viruses. CoV infection causes various diseases in humans and animals, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). No approved specific drugs or vaccinations are available to treat their infections. Here, we report a novel dual inhibition mechanism targeting CoV main protease (Mpro) from feline infectious peritonitis virus (FIPV), which leads to lethal systemic granulomatous disease in cats. Mpro, conserved across all CoV genomes, is essential for viral replication and transcription. We demonstrated that zinc ion and a Michael acceptor-based peptidomimetic inhibitor synergistically inactivate FIPV Mpro. We also solved the structure of FIPV Mpro complexed with two inhibitors, delineating the structural view of a dual inhibition mechanism. Our study provides new insight into the pharmaceutical strategy against CoV Mpro through using zinc as an adjuvant therapy to enhance the efficacy of an irreversible peptidomimetic inhibitor.

Michael Acceptor-Based Peptidomimetic Inhibitor of Main Protease from Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus (PEDV) causes high mortality in pigs. PEDV main protease (Mpro) plays an essential role in viral replication. We solved the structure of PEDV Mpro complexed with peptidomimetic inhibitor N3 carrying a Michael acceptor warhead, revealing atomic level interactions. We further designed a series of 17 inhibitors with altered side groups. Inhibitors M2 and M17 demonstrated enhanced specificity against PEDV Mpro. These compounds have potential as future therapeutics to combat PEDV infection.