Flaviviruses, such as Dengue (DENV), West Nile (WNV) and Zika (ZIKV) are major human pathogens that inflict a significant burden on society. Cellular proteins play important roles in all facets of the flavivirus lifecycle. Therefore, understanding viral-host protein interactions essential for the flavivirius lifecycle can lead to development of effective antiviral strategies.
A CRISPR/Cas9 genome-wide KO screen was employed to isolate novel host factors critical for ZIKV replication. Briefly, cells transduced with sgRNAs targeting every gene in the genome were infected with ZIKV and cells which survived ZIKV-induced cytopathic effect were PCR amplified and sequenced using Illumina NextSeq. Bioinformatics analysis identified previously characterised host factors (EMC1/EMC6) as well as a novel candidate, RACK1. RACK1 plays multiple roles in homeostatic cellular processes as a scaffold protein and acts as an indispensable hub for signalling transduction of multiple pathways. Interestingly, RACK1 was previously identified as essential for replication of several viruses (HCV, Pox virus).
siRNA knockdown of RACK1, followed by infection with flaviviruses ZIKV, DENV, WNV, POWV, TBEV confirmed that RACK1 has a critical role in viral replication. Interestingly, YFV doesn’t require RACK1 for replication. RACK1 was shown to interact with multiple non-structural (NS) viral proteins, indicating a multifaceted role in ZIKV replication. More specifically, interaction of NS1 with RACK1 was shown via confocal microscopy to localise to the ER while knockdown of RACK1 prior to infection showed that NS1 localisation is significantly altered compared to infected wildtype cells. Current work is focused on electron microscopy imaging on flavivirus NS1-RACK1 interactions. Mutational studies of RACK1 will also give further insight into the signalling pathways important for RACK1-supported flavivirus replication. Collectively, these experiments suggest RACK1 is important for replication complex formation, a critical step in establishing flavivirus replication.
Further understanding of the intricate steps in viral replication establishment will advance knowledge of the flavivirus lifecycle and may potentially aid development of broadly acting antivirals against multiple flaviviruses.