The interaction of GRP78 and Zika virus E and NS1 proteins occurs in a chaperone-client manner.

Glucose regulated protein 78 (GRP78) is a chaperone protein that is a central mediator of the unfolded protein response, a key cellular stress response pathway. GRP78 has been shown to be critically required for infection and replication of a number of flaviviruses, and to interact with both non-structural (NS) and structural flavivirus proteins. However, the nature of the specific interaction between GRP78 and viral proteins remains largely unknown. This study aimed to characterize the binding domain and critical amino acid residues that mediate the interaction of GRP78 to ZIKV E and NS1 proteins. Recombinant EGFP fused GRP78 and individual subdomains (the nucleotide binding domain (NBD) and the substrate binding domain (SBD)) were used as a bait protein and co-expressed with full length or truncated ZIKV E and NS1 proteins in HEK293T/17 cells. Protein-protein interactions were determined by a co-immunoprecipitation assay. From the results, both the NBD and the SBD of GRP78 were crucial for an effective interaction. Single amino acid substitutions in the SBD showed that R492E and T518A mutants significantly reduced the binding affinity of GRP78 to ZIKV E and NS1 proteins. Notably, the interaction of GRP78 with ZIKV E was stably maintained against various single amino acid substitutions on ZIKV E domain III and with all truncated ZIKV E and NS1 proteins. Collectively, the results suggest that the principal binding between GRP78 and viral proteins is mainly a classic canonical chaperone protein-client interaction. The blocking of GRP78 chaperone function effectively inhibited ZIKV infection and replication in neuronal progenitor cells. Our findings reveal that GRP78 is a potential host target for anti-ZIKV therapeutics.

Structural Transition and Antibody Binding of EBOV GP and ZIKV E Proteins from Pre-Fusion to Fusion-Initiation State.

Membrane fusion proteins are responsible for viral entry into host cells—a crucial first step in viral infection. These proteins undergo large conformational changes from pre-fusion to fusion-initiation structures, and, despite differences in viral genomes and disease etiology, many fusion proteins are arranged as trimers. Structural information for both pre-fusion and fusion-initiation states is critical for understanding virus neutralization by the host immune system. In the case of Ebola virus glycoprotein (EBOV GP) and Zika virus envelope protein (ZIKV E), pre-fusion state structures have been identified experimentally, but only partial structures of fusion-initiation states have been described. While the fusion-initiation structure is in an energetically unfavorable state that is difficult to solve experimentally, the existing structural information combined with computational approaches enabled the modeling of fusion-initiation state structures of both proteins. These structural models provide an improved understanding of four different neutralizing antibodies in the prevention of viral host entry.

Preventative Vaccines for Zika Virus Outbreak: Preliminary Evaluation.

Since it emerged in Brazil in May 2015, the mosquito-borne Zika virus (ZIKV) has raised global concern due to its association with a significant rise in the number of infants born with microcephaly and neurological disorders such as Guillain-Barré syndrome. We developed prototype subunit and adenoviral-based Zika vaccines encoding the extracellular portion of the ZIKV envelope gene (E) fused to the T4 fibritin foldon trimerization domain (Efl). The subunit vaccine was delivered intradermally through carboxymethyl cellulose microneedle array (MNA). The immunogenicity of these two vaccines, named Ad5.ZIKV-Efl and ZIKV-rEfl, was tested in C57BL/6 mice. Prime/boost immunization regimen was associated with induction of a ZIKV-specific antibody response, which provided neutralizing immunity. Moreover, protection was evaluated in seven-day-old pups after virulent ZIKV intraperitoneal challenge. Pups born to mice immunized with Ad5.ZIKV-Efl were all protected against lethal challenge infection without weight loss or neurological signs, while pups born to dams immunized with MNA-ZIKV-rEfl were partially protected (50%). No protection was seen in pups born to phosphate buffered saline-immunized mice. This study illustrates the preliminary efficacy of the E ZIKV antigen vaccination in controlling ZIKV infectivity, providing a promising candidate vaccine and antigen format for the prevention of Zika virus disease.