Transposon #mutagenesis of the #Zika virus #genome highlights regions essential for #RNA replication and restricted for immune e

(Source: Journal of Virology, full page: (LINK). Abstract, edited.)

Transposon mutagenesis of the Zika virus genome highlights regions essential for RNA replication and restricted for immune evasion

Benjamin O. Fulton1, David Sachs2, Megan C. Schwarz1, Peter Palese1,3# and Matthew J. Evans1#

Author Affiliations: 1 Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA; 2 Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA; 3 Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

ABSTRACT

The molecular constraints affecting Zika virus (ZIKV) evolution are not well understood. To investigate ZIKV genetic flexibility, we used transposon mutagenesis to add 15-nucleotide insertions throughout the MR766 ZIKV genome and subsequently deep sequenced the viable mutants. Few ZIKV insertion mutants replicated, which likely reflects a high degree of functional constraints on the genome. The NS1 gene exhibited distinct mutational tolerances at different stages of the screen. This result may define regions of the NS1 protein that are required for the different stages of the viral life cycle. The ZIKV structural genes showed the highest degree of insertional tolerance. Although the envelope (E) protein exhibited particular flexibility, the highly conserved envelope domain II (EDII) fusion loop of the E protein was highly intolerant to transposon insertions. The fusion loop is also a target of pan-flavivirus antibodies that are generated against other flaviviruses and neutralize a broad range of dengue virus and ZIKV isolates. The genetic restrictions identified within the epitopes in the EDII fusion loop likely explain the sequence and antigenic conservation of these regions in ZIKV and among multiple flaviviruses. Thus, our results provide insights into the genetic restrictions on ZIKV that may affect the evolution of this virus.

IMPORTANCE

Zika virus has recently emerged as a significant human pathogen. Determining the genetic constraints on Zika virus is important for understanding the factors affecting viral evolution. We used a genome-wide transposon mutagenesis screen to identify where mutations were tolerated in replicating viruses. We found that the genetic regions involved in RNA replication were mostly intolerant of mutations. The genes coding for structural proteins were more permissive to mutations. Despite the flexibility observed in these regions, we found that epitopes bound by broadly reactive antibodies were genetically constrained. This finding may explain the genetic conservation of these epitopes among flaviviruses.

FOOTNOTES

#Corresponding authors: Matthew J. Evans, Department of Microbiology, Icahn School of Medicine at Mount Sinai, 1 Gustave L. Levy Place, Box 1124, New York, NY 10029, Tel: 212-241-5576, Email: [email protected]

Peter Palese, Department of Microbiology, Icahn School of Medicine at Mount Sinai, 1 Gustave L. Levy Place, Box 1124, New York, NY 10029, Tel: 212-241-7318, Email: [email protected]

Copyright © 2017 American Society for Microbiology. All Rights Reserved.

–

Keywords: Zika Virus; Genetics.

——