Emerging infections of mosquito-borne Zika virus (ZIKV) pose an increasing threat to human health, as documented over the recent years in South Pacific islands and the Americas in recent years. To better understand molecular mechanisms underlying the increase in human cases with severe pathologies, we recently demonstrated the functional roles of structural proteins capsid (C), pre-membrane (prM), and envelop (E) of ZIKV epidemic strains with the initiation of viral infection in human cells. Specifically, we found that the C-prM region contributes to permissiveness of human host cells to ZIKV infection and ZIKV-induced cytopathic effects, whereas the E protein is associated with viral attachment and early infection. In the present study, we further characterize ZIKV E proteins by investigating the roles of residues isoleucine 152 (Ile152), threonine 156 (Thr156), and histidine 158 (His158) (i.e., the E-152/156/158 residues), which surround a unique N-glycosylation site (E-154), in permissiveness of human host cells to epidemic ZIKV infection. For comparison purpose, we generated mutant molecular clones of epidemic BeH819015 (BR15) and historical MR766-NIID (MR766) strains that carry each others E-152/156/158 residues, respectively. We observed that the BR15 mutant containing the E-152/156/158 residues from MR766 was less infectious in A549-Dual™ cells than parental virus. In contrast, the MR766 mutant containing E-152/156/158 residues from BR15 displayed increased infectivity. The observed differences in infectivity were, however, not correlated with changes in viral binding onto host-cells or cellular responses to viral infection. Instead, the E-152/156/158 residues from BR15 were associated with an increased efficiency of viral membrane fusion inside infected cells due to conformational changes of E protein that enhance exposure of the fusion loop. Our data highlight an important contribution of E-152/156/158 residues to the early steps of ZIKV infection in human cells.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus which is of major public health concern. ZIKV infection is recognized as the cause of congenital Zika disease and other neurological defects, with no specific prophylactic or therapeutic treatments. As the humoral immune response is an essential component of protective immunity, there is an urgent need for effective vaccines that confer protection against ZIKV infection. In the present study, we evaluate the immunogenicity of chimeric viral clone ZIKBeHMR-2, in which the region encoding the structural proteins of the African strain MR766 backbone was replaced with its counterpart from the epidemic strain BeH819015. Three amino-acid substitutions I152T, T156I, and H158Y were introduced in the glycan loop of the E protein (E-GL) making ZIKBeHMR-2 a non-glycosylated virus. Adult BALB/c mice inoculated intraperitoneally with ZIKBeHMR-2 developed anti-ZIKV antibodies directed against viral proteins E and NS1 and a booster dose increased antibody titers. Immunization with ZIKBeHMR-2 resulted in a rapid production of neutralizing anti-ZIKV antibodies. Antibody-mediated ZIKV neutralization was effective against viral strain MR766, whereas epidemic ZIKV strains were poorly sensitive to neutralization by anti-ZIKBeHMR-2 immune sera. From our data, we propose that the three E-GL residues at positions E-152, E-156, and E-158 greatly influence the accessibility of neutralizing antibody epitopes on ZIKV.

Heme oxygenase-1 (HO-1), a rate-limiting enzyme involved in the degradation of heme, is induced in response to a wide range of stress conditions. HO-1 exerts antiviral activity against a broad range of viruses, including the Hepatitis C virus, the human immunodeficiency virus, and the dengue virus by inhibiting viral growth. It has been reported that HO-1 displays antiviral activity against the Zika virus (ZIKV) but the mechanisms of viral inhibition remain largely unknown. Using a ZIKV RNA replicon with the Green Fluorescent Protein (GFP) as a reporter protein, we were able to show that HO-1 expression resulted in the inhibition of viral RNA replication. Conversely, we observed a decrease in HO-1 expression in cells replicating the ZIKV RNA replicon. The study of human cells infected with ZIKV showed that the HO-1 expression level was significantly lower once viral replication was established, thereby limiting the antiviral effect of HO-1. Our work highlights the capacity of ZIKV to thwart the anti-replicative activity of HO-1 in human cells. Therefore, the modulation of HO-1 as a novel therapeutic strategy against ZIKV infection may display limited effect.