Identification and characterization of key residues in Zika virus envelope protein for virus assembly and entry

Abstract

Zika virus (ZIKV), a family member in the Flavivirus genus, has re-emerged as a global public health concern. The envelope (E) proteins of flaviviruses play a dual role in viral assembly and entry. To identify the key residues of E in virus entry, we generated a ZIKV trans -complemented particle (ZIKV TCP ) system, in which a subgenomic reporter replicon was packaged by trans -complementation with expression of CprME. We performed mutagenesis studies of the loop regions that protrude from the surface of the virion in the E ectodomains (DI, DII, DIII). Most mutated ZIKV TCPs exhibited deficient egress. Mutations in DII and in the hinge region of DI and DIII affected prM expression. With a bioorthogonal system, photocrosslinking experiments identified crosslinked intracellular E trimers and demonstrated that egress-deficient mutants in DIII impaired E trimerization. Of these mutants, an E-trimerization-dead mutation D389A that nears the E-E interface between two neighbouring spikes in the immature virion completely abolished viral egress. Several mutations abolished ZIKV TCPs ’ entry, without severely affecting viral egress. Further virus binding experiments demonstrated a deficiency of the mutated ZIKV TCPs in virus attachment. Strikingly, synthesized peptide containing residues of two mutants (268-273aa in DII) could bind to host cells and significantly compete for viral attachment and interfere with viral infection, suggesting an important role of these resides in virus entry. Our findings uncovered the requirement for DIII mediated-E trimerization in viral egress, and discovered a key residue group in DII that participates in virus entry.

Broad-spectrum activity against mosquito-borne flaviviruses achieved by a targeted protein degradation mechanism

“…These significant structural changes provide the driving force for fusion of the viral lipid bilayer with the endosomal membrane, creating a fusion pore that allows the viral genome to escape to the cytoplasm where it can be expressed. The E protein is also essential at later stages of the replication cycle, in that no new infectious virions can be produced in the absence of E. Mutagenesis studies have identified point mutations with effects on entry or viral particle production as well as mutations that affect both processes [18][19][20][21][22][23] , suggesting that small molecules targeting E might act through one or more modes of action. The development of small molecule inhibitors of DENV E-mediated fusion was facilitated by serendipitous co-crystallization of the detergent n-octyl-β-D-glucoside (βOG) in a conserved pocket (dubbed the "βOG pocket") located between domains I and II 24 (Fig.…”