Zika virus (ZIKV) is a member of the flaviviridae family and infection of pregnant females can result in placental insufficiency, spontaneous abortion and microcephaly. Host factors play important roles in all facets of the flavivirus life cycle and while some have been identified, many remain to be identified and characterised. Thus, a CRISPR Cas9 genome-wide KO screen approach (LentiCRISPRv2: GeCKO) was utilised to identify novel ZIKV essential host factors. This library consists of 122,471 sgRNAs (single guide RNA) which targets every gene in the genome.
Huh7 cells were transduced with lentivirus derived from the LentiCRISPRv2 library at a M.O.I of 0.3 to ensure knockout of a single gene per cell. Following puromycin selection of transduced cells with sgRNA library integration, cells were infected with a cytopathic strain of ZIKV (MR766, MOI 5). A higher proportion of cells transduced with the LentiCRISPRv2 library survived compared to control, suggesting these cells carry a sgRNA which knocked out critical host factors for ZIKV replication. Following specific PCR amplification of the sgRNA integrated region, NGS (Illumina MiSeq) was performed on the isolated PCR products and bioinformatics analysis (University of Adelaide Bioinformatics Hub) identified enrichment of numerous sgRNA sequences, representing putative ZIKV essential cellular factors. One factor identified across multiple screens was RACK1.
Apart from the multiple roles RACK1 plays in cellular processes, it is a dispensable subunit of the 40S ribosome shown to be essential for replication of both HCV and poxvirus vaccinia virus. Current work involves verification of RACK1 as a host factor for ZIKV through single knockouts and establishing the mechanism by which RACK1 aids ZIKV replication. Application of the CRISPR Cas9 genome-wide KO screen to identify indispensable host factors for ZIKV replication furthers understanding of the virus lifecycle and may also aid development of broadly acting antivirals against multiple flaviviridae.