Probing Zika Virus Neutralization Determinants with Glycoprotein Mutants Bearing Linear Epitope Insertions.

Zika virus (ZIKV) glycoproteins are the primary target of the humoral immune response. In this study, we explored the capacity of these glycoproteins to tolerate insertion of linear epitope sequences and the potential of antibodies that bind these epitopes to inhibit infection. We first created a panel of ZIKV mutants with the FLAG epitope inserted in the premembrane (prM) and envelope (E) glycoprotein regions. The insertion locations were based on the results of our recent transposon insertional mutagenesis screen. Although FLAG insertions in prM greatly impaired viral fitness, this sequence was tolerated in numerous surface-exposed E protein sites. We observed that mutants bearing FLAG epitopes in E domains I and II and the E domain I-II hinge region were all neutralized by FLAG antibody; however, the neutralization sensitivity varied highly. We measured the antibody binding efficiency and found that this closely matched the pattern of neutralization sensitivity. We determined that E glycosylation did not affect antibody binding to a nearby epitope or its capacity to serve as a neutralization target. Although we could not generate infectious viruses with FLAG epitope insertions in a buried region of E protein domain III, we found that the V5 epitope could be inserted at this site without greatly impacting fitness. Furthermore, this virus was efficiently neutralized by V5 antibodies, highlighting that even buried epitopes can function as neutralization targets. Finally, we analyzed the timing of antibody neutralization activity during cell entry and found that all antibodies blocked a step after cell attachment.