Evolution of Zika virus in <i>Rag1</i>-deficient mice selects for unique envelope glycosylation motif mutants that show enhanced replication fitness

N-linked glycosylation of flavivirus envelope proteins is widely viewed as being required for optimal folding, processing and/or transit of envelope proteins, and the assembling virons, through the endoplasmic reticulum (ER) and the Golgi. Zika virus (ZIKV) has a single N-linked envelope glycan located adjacent to the fusion loop. Herein we show that independent serial passage of ZIKV_Natal in <i>Rag1</i> ^-/- mice for 223 or 386 days generated two unique envelope glycan-deficient mutants, ZIKV-V153D and ZIKV-N154D, respectively. Surprisingly, these mutants grew to titres ∼1 to 2.6 logs higher than the glycosylated parental ZIKV_Natal in Vero E6 cells and human brain organoids. RNA-Seq of infected organoids suggested that this increased replication fitness was associated with upregulation of the unfolded protein response (UPR). Cell death, cellular viral RNA, and viral protein levels were not significantly affected, arguing that these glycan mutants enjoyed faster ER/Golgi folding, processing, assembly, transit, and virion egress, assisted by an upregulated UPR. Thus, ZIKV envelope N-linked glycosylation is not essential for promoting envelope folding, assembly, and transit through the ER/Golgi, since aspartic acid (D) substitutions in the glycosylation motif can achieve this with significantly greater efficiency. Instead, the evolution of glycan mutants in <i>Rag1</i> ^-/- mice indicates that such envelope glycosylation can have a fitness cost in an environment devoid of virus-specific antibody responses. The V153D and N154D mutations, generated by natural selection in <i>Rag1</i> ^-/- mice, have to date not been employed in orthoflavivirus envelope glycosylation studies. Instead, genetic engineering has been used to generate mutant viruses that, for instance, contain a N154A substitution. The latter may impart confounding unfavourable properties, such as envelope protein insolubility, that have a detrimental impact on virus replication. The V153D and N154D substitutions may avoid imparting unfavourable properties by preserving the surface negative charge provided by the glycan moiety in the parental ZIKV_Natal envelope protein. In <i>Ifnar1</i> ^-/- mice ZIKV-V153D and -N154D showed faster viremia onsets, but reduced viremic periods, than the parental ZIKV_Natal, consistent with an established contention that such glycans have evolved to delay neutralizing antibody activity.