Determinants of Zika virus host tropism uncovered by deep mutational scanning

Abstract

Arboviruses cycle between, and replicate in, both invertebrate and vertebrate hosts, which for Zika virus (ZIKV) involves Aedes mosquitoes and primates1. The viral determinants required for replication in such obligate hosts are under strong purifying selection during natural virus evolution, making it challenging to resolve which determinants are optimal for viral fitness in each host. Herein we describe a deep mutational scanning (DMS) strategy2–5 whereby a viral cDNA library was constructed containing all codon substitutions in the C-terminal 204 amino acids of ZIKV envelope (E) protein. The cDNA library was transfected into C6/36 (Aedes) and Vero (primate) cells, with subsequent deep sequencing and computational analyses of recovered viruses showing that K316Q and S461G, or Q350L and T397S substitutions conferred substantial replicative advantages in mosquito and primate cells, respectively. A 316Q/461G virus was constructed and shown to be replication-defective in mammalian cells due to severely compromised virus particle formation and secretion. The 316Q/461G virus was also highly attenuated in human brain organoids, and illustrated utility as a vaccine in mice. This approach can thus imitate evolutionary selection in a matter of days and identify amino acids key to regulating virus replication in specific host environments.

Deep mutational scanning comprehensively maps how Zika envelope protein mutations affect viral growth and antibody escape

“…By using this plasmid and also choosing a viral strain (MR766) that grows to high titer in cell culture, we were able to generate viral libraries that effectively sampled all amino-acid mutations to E protein. This library diversity contrasts to two other recent ZIKV deep mutational scanning studies 21,22 : both of these studies revealed important biological insights about host adaptation, but neither produced a complete map of amino-acid level selection due to an inability to generate and maintain the viral library diversity to sample all amino acids. Of course, our experiments were still subject to noise, probably because even our high-efficiency reversegenetics had some bottlenecking of viral diversity.…”

“…Second, Vero cells are a relatively permissive monkey-derived cell line, whereas actual ZIKV evolves under selection to efficiently replicate in actual humans and mosquitoes. Indeed, prior deep mutational scanning studies of ZIKV 21,22 and other viruses 37,38 have identified numerous mutations with effects that depend on the host species and level of innate-immune function in the cells used to grow the virus. Therefore, our map should be interpreted as providing a baseline estimate of the functional effects of mutations to the E protein in a relatively permissive context.…”

“…This approach has been used to assess the effects of all viral mutations on the function and antigenicity of key proteins from influenza virus 16–18 and HIV 19,20 . Two studies have applied deep mutational scanning to ZIKV 21,22 , but these studies did not systematically map the mutational tolerance or antigenicity of all amino-acid mutations. Instead, they used libraries comprised of a subset of E protein variants to identify mutations that differentially affect viral growth in mammalian and insect cells.…”

A global lipid map defines a network essential for Zika virus replication

“…Our findings present several avenues to fill in the circuitry of this wiring diagram and connect it to other maps of the molecular factors of ZIKV pathogenesis. From a mechanistic standpoint, it is now clear that individually expressed ZIKV proteins can regulate specific cellular processes 27,[72][73][74][75][76][77] , and that minor changes in genome sequence can greatly affect ZIKV infectivity and virulence [78][79][80] . We show here that ectopic expression of ZIKV NS4B, a transmembrane protein integral to the Flaviviridae RC 22 , causes changes in sphingolipid metabolism similar to what we observed during full viral infection, implying a causal relationship between NS4B and dysregulation of sphingolipid homeostasis.…”

A global lipid map defines a network essential for Zika virus replication

“…Our findings present several avenues to fill in the circuitry of this wiring diagram and connect it to other maps of the molecular factors of ZIKV pathogenesis. From a mechanistic standpoint, it is now clear that individually expressed ZIKV proteins can regulate specific cellular processes 27,70–75 , and that minor changes in genome sequence can greatly affect ZIKV infectivity and virulence 76–78 . We show here that ectopic expression of ZIKV NS4B, a transmembrane protein integral to the Flaviviridae replication complex 22 , causes changes in sphingolipid metabolism similar to what we observed during full viral infection, implying a causal relationship between NS4B and dysregulation of sphingolipid homeostasis.…”