posted on 2020-03-09, 18:34authored byShayli Varasteh Moradi, Dejan Gagoski, Sergey Mureev, Patricia Walden, Kerrie-Ann McMahon, Robert G. Parton, Wayne A. Johnston, Kirill Alexandrov
The rapid spread of arthropod-borne
Zika virus poses a serious
public health threat that calls for effective ways of controlling
and treating viral infection. This in turn necessitates better understanding
of the mechanisms of virus assembly and its interaction with the host
cells. In order to facilitate such efforts, we developed a new multihost
expression vector pmCellFree that allows rapid and multiplexed production
of ZIKV proteins in any in vitro translation system as well as in
mammalian cells. Using a combination of in vitro expression in Leishmania cell-free system and AlphaLISA interaction
assay, pairwise protein interactions of all ZIKV proteins were systematically
tested. We identified thirty-three intraviral binary protein interactions,
of which 13 interactions are novel. These findings were further validated
by expressing selected protein pairs in mammalian HEK293T cell line
and assessing their interactions in the cellular lysate. The results
of these interaction assays were identical to those obtained with
in vitro expressed proteins. The observed novel protein–protein
interactions were further validated using a pulldown assay. The unrevealed
novel protein interactions may point to the previously unappreciated
complexity of the ZIKV assembly process and may play an important
role in the infection process. These interactions may represent new
targets for antiviral drug development.