Abstract
Zika virus (ZIKV) is a membrane enveloped Flavivirus with a positive strand RNA genome, transmitted by Aedes mosquitoes. The geographical range of ZIKV has dramatically expanded in recent decades resulting in increasing numbers of infected individuals, and the spike in ZIKV infections has been linked to significant increases in both Guillain-Barré syndrome and microcephaly. Although a large number of host proteins have been physically and/or functionally linked to other Flaviviruses, very little is known about the virus-host protein interactions established by ZIKV. Here we map host cell protein interaction profiles for each of the ten polypeptides encoded in the ZIKV genome, generating a protein topology network comprising 3033 interactions among 1224 unique human polypeptides. The interactome is enriched in proteins with roles in polypeptide processing and quality control, vesicle trafficking, RNA processing and lipid metabolism. >60% of the network components have been previously implicated in other types of viral infections; the remaining interactors comprise hundreds of new putative ZIKV functional partners. Mining this rich data set, we highlight several examples of how ZIKV may usurp or disrupt the function of host cell organelles, and uncover an important role for peroxisomes in ZIKV infection.
Highlights
We report that: [1] the Zika virus (ZIKV) Capsid protein is targeted to a variety of host endomembrane locations and can “remodel” nuclear/endoplasmic reticulum (ER) membranes; [2] the NS5 helicase protein is targeted to, and partially disrupts, Cajal Bodies; [3] the NS3-NS2B protease interacts with the HOPS/CORVET complex, and modifies lysosome volume, and; [4] NS2A is targeted to peroxisomal membranes
Host interactions, the ten ZIKV proteins were fused in-frame with an N- or C-terminal Flag-BirA R118G tag (constructs based on ZIKV H/PF/2013 ANO46305.1 [28]; individual proteins based on cleavage sites reported in [18]) and expressed in HEK 293 cells, a system that displays characteristics of immature neurons [29] and which can be infected with ZIKV [30]
Our global ZIKV interactome identifies Ͼ1200 putative host interacting protein partners, physically linking the ten individual viral proteins to specific human polypeptides, and revealing a broad array of putative accessory functions for both the structural and nonstructural Zika polypeptides in the virus life cycle (Fig. 5; for detailed enrichment analysis, see supplemental Table S2)
Summary
Experimental Design and Statistical Rationale—Both BioID and anti-Flag IP-MS analyses were performed on each of the ten ZIKV polypeptides, expressed individually in HEK293 T-REx cells, fused either with an N-terminal FlagBirA* or a C-terminal BirA*Flag epitope tag. Flag Affinity Purification—5 ϫ 150 cm dishes of subconfluent (80%) 293 T-REx cells expressing the protein of interest were scraped into PBS, pooled, washed twice in 10 ml PBS, and collected by centrifugation at 1000 ϫ g for 5 min at 4 °C. Immunofluorescence, Image Acquisition, and Processing—Transiently transfected HeLa cells expressing GFP- or FlagBirA*-tagged proteins were grown on coverslips, fixed with 4% paraformaldehyde for 15 min, and washed in PBS with 0.1% Triton X-100. Cells were washed twice with pre-warmed PBS, before incubating in DMEM with no pH indicator (GIBCO, Invitrogen). Co-IP was performed on 293 T-REx cells expressing FlagBirA*-NS3, as described in [4] and proteins analyzed by SDS-PAGE and Western blotting using anti-CEP85 antibody (Abnova, Taipei City, Taiwan H00064793-B01P, 1:500)
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