Increased activity of unlinked Zika virus NS2B/NS3 protease compared to linked Zika virus protease

Abstract

Zika virus (ZIKV) is a flavivirus spread by daytime-active Aedes spp. mosquitoes such as A. aegypti and A. albopictus. Previously thought to be a mild infection, the latest ZIKV outbreak in the Americas is causally associated with more severe symptoms as well as severe birth defects, such as microcephaly. Currently no vaccine or antiviral exists. However, recent progress has demonstrated the viral NS2B/NS3 protease may be a suitable target for the development of small-molecule antiviral agents. To better understand the ZIKV protease, we expressed, purified, and characterized unlinked and linked NS2B/NS3 protease corresponding to an isolate from the recent outbreak in Puerto Rico. Unlinked ZIKV protease is more active and binds substrate with greater affinity than linked ZIKV protease. Therefore, we propose that unlinked ZIKV protease be used when evaluating or designing ZIKV protease inhibitors. Additionally, potent inhibitors of related viral proteases, like West Nile Virus and Dengue virus, may serve as advanced starting points to identify and develop ZIKV protease inhibitors.

Cis autocatalytic cleavage of glycine‐linked Zika virus NS2B‐NS3 protease constructs

“…In agreement with previous studies , we also observed cleavage of linked WT ZIKV NS2B CF ‐NS3 pro . We therefore set out to identify the cleavage site, whether cleavage occurs in cis , that is, an individual protease cleaves its own linker or in trans , that is, a protease linker is cleaved by another ZIKV NS2B CF ‐NS3 pro protease in the sample and to compare the melting behaviors of cleaved and uncleaved protease constructs.…”

“…A slight difference in K M values between linked and unlinked ZIKV NS2B CF -NS3 pro protease has been reported previously [16]. In our hands, we did not observe any functional differences between the two protein variants larger than the margin of error.…”

“…A). With or without cleavage, NS3 pro and NS2B CF remain tightly connected as expected from other studies with flaviviral proteases . In addition to the SEC runs, we recorded CD spectra at 15 °C.…”

“…The sizes of the autocatalytic cleavage products led us and others to conclude that there are two possible cleavage sites in ZIKV NS2B CF ‐NS3 pro , one at the NS2B CF C‐terminal R95 NS2B and one at R29 NS3 in the NS3 pro domain. The double mutation simultaneously removing both arginine residues successfully abrogated autocatalytic cleavage . Here, we mutated these sites individually and thus identified the relevant autocatalytic cleavage site to be at position R95 NS2B in the C‐terminus of the NS2B CF .…”

Structural Dynamics of Zika Virus NS2B-NS3 Protease Binding to Dipeptide Inhibitors

“…This construct exhibited a higher k cat value than the conventional single-chain construct (gZiPro) when benzoyl-Nle-Lys-Arg-Arg-aminomethylcoumarin (Bz-nKRR-AMC) was used as a substrate (Phoo et al, 2016). Similar results which showed that the unlinked NS2B-NS3 protease is a more efficient protease construct were also reported recently (Kuiper et al, 2017). We have recently determined the high-resolution crystal structure of free bZiPro enzyme in a closed conformation, which offers new opportunity for the discovery of structure-based inhibitors (Zhang et al, 2016).…”