The crystal structure of Zika virus helicase: basis for antiviral drug design
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KeywordsJapanese Encephalitis Helicase Domain ZIKV Infection Murray Valley Encephalitis Virus Shanghai Synchrotron Radiation Facility
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 single-stranded, 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.
ZIKV helicase is evolutionarily close to those from Murray Valley encephalitis virus (MVEV), DENV-4, DENV-2, YFV, JEV, Kunjin virus (KUNV), and Hepatitis C virus (HCV) from the Flaviviradae family, whose structures have already been solved. To gain further structural insight, we generated a structure-based phylogenetic tree for these homologous helicases (Fig. 1D), using the Structure Homology Program (Stuart et al., 1979). Structural superposition of these 8 structures reveals that all of the flavivirus helicases, including the helicase of ZIKV, cluster into one large group (Group 1), while HCV helicase falls into a separate one (Group 2). In Group 1, ZIKV helicase is evolutionarily closer to those of MVEV, DENV-4 and DENV-2 (Group 1a) while the other members cluster into Group 1b. Clustering of viral helicases indicate that they share more structural features, suggesting it might be possible to design wide-spectrum inhibitors against all the group/subgroup members.
It is worthwhile to note that the P-loop, which is critical for NTP binding and catalysis, has a variety of structural conformations among flavivirus helicases (Fig. 2C), even though the amino acid sequences are stringently conserved. This discrepancy highlights the high degree of intrinsic flexibility of the P-loop. Interestingly, the conformation of the P-loop in ZIKV helicase (apo form) is quite similar to that of DENV-4 helicase complexed with AMPPNP-Mn2+, which is however, distinct from the conformation in its own apo form. This implies that ZIKV helicase might not need to undergo as significant local rearrangement of the NTP binding pocket to transition into the active state as in DENV-4 helicase.
In the structure of ZIKV helicase, a positively charged tunnel can be clearly identified along the domain boundary of Domain III, which directly interacts with Domain I and Domain II (Fig. 2E). The tunnel is lined with positively charged residues and remains wide enough to accommodate a single strand (ss) nucleic acid in an extended conformation running through Domain II to Domain I. The positively charged residues, most of which were contributed by Domain I and Domain II, presumably stabilize the sugar-phosphate backbone of the nucleic acid. Superposition of ZIKV helicase to DENV-4 helicase bound with a 12-mer ssRNA (PDB ID 2JLV) (Luo et al., 2008) generated a model to analyze the potential pattern for nucleic acid binding. It seems that rearrangement of the three domains is required to build a non-clashing model of ZIKV helicase for RNA binding, which has been seen in DENV-4 helicase bound to RNA. Interestingly, P363, P233, D409, and T264, which contribute specificity of DENV-4 helicase for RNA, are entirely conserved in ZIKV helicase, thus implying that ZIKV helicase would prefer RNA to DNA.
In summary, the recent outbreak of ZIKV and its association with fetal abnormalities have caused global public health emergency. Here we present a high-resolution structure of ZIKV helicase, which is an important drug target. The structure has revealed critical substrate-binding pockets for antiviral drug design. Pharmaceutical development of inhibitors targeting the RNA binding tunnel and the pivotal regulatory regions would be a plausible strategy for innovative anti-ZIKV therapies.
We would like to thank Zuokun Lu for data collection at beamline BL18U1 of the Shanghai Synchrotron Radiation Facility (SSRF); Erin Weber and Lanfeng Wang for discussion and advice. This work was supported by the National Basic Research Program (973 Program) (Nos. 2015CB859800 and 2014CB542800) and the National Natural Science Foundation of China (Grant No. 31528006).
Haitao Yang and Hongliang Tian conceived and designed the experiments. Hongliang Tian, Xiaoyun Yang, Wei Xie, Heng Chi and Zhongyu Mu performed the experiments. Haitao Yang, Xiaoyun Ji, Cheng Chen, Chen Wu and Zefang Wang analyzed the data. Haitao Yang, Hongliang Tian, Xiaoyun Ji and Kailin Yang wrote the paper. Hongliang Tian, Xiaoyun Ji, Xiaoyun Yang, Wei Xie, Kailin Yang, Cheng Chen, Chen Wu, Heng Chi, Zhongyu Mu, Zefang Wang, and Haitao Yang declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by the any of the authors.
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