Genome-Wide CRISPR Screen Identifies RACK1 as a Critical Host Factor for Flavivirus Replication

Abstract

Cellular factors have important roles in all facets of the flavivirus replication cycle. Deciphering viral-host protein interactions is essential for understanding the flavivirus lifecycle as well as development of effective antiviral strategies. To uncover novel host factors that are co-opted by multiple flaviviruses, a CRISPR/Cas9 genome wide knockout (KO) screen was employed to identify genes required for replication of Zika virus (ZIKV). Receptor for Activated Protein C Kinase 1 (RACK1) was identified as a novel host factor required for ZIKV replication, which was confirmed via complementary experiments. Depletion of RACK1 via siRNA demonstrated that RACK1 is important for replication of a wide range of mosquito- and tick-borne flaviviruses, including West Nile Virus (WNV), Dengue Virus (DENV), Powassan Virus (POWV) and Langat Virus (LGTV) as well as the coronavirus SARS-CoV-2, but not for YFV, EBOV, VSV or HSV. Notably, flavivirus replication was only abrogated when RACK1 expression was dampened prior to infection. Utilising a non-replicative flavivirus model, we show altered morphology of viral replication factories and reduced formation of vesicle packets (VPs) in cells lacking RACK1 expression. In addition, RACK1 interacted with NS1 protein from multiple flaviviruses; a key protein for replication complex formation. Overall, these findings reveal RACK1’s crucial role to the biogenesis of pan-flavivirus replication organelles. Importance Cellular factors are critical in all facets of viral lifecycles, where overlapping interactions between the virus and host can be exploited as possible avenues for the development of antiviral therapeutics. Using a genome-wide CRISPR knock-out screening approach to identify novel cellular factors important for flavivirus replication we identified RACK1 as a pro-viral host factor for both mosquito- and tick-borne flaviviruses in addition to SARS-CoV-2. Using an innovative flavivirus protein expression system, we demonstrate for the first time the impact of the loss of RACK1 on the formation of viral replication factories known as 'vesicle packets' (VPs). In addition, we show that RACK1 can interact with numerous flavivirus NS1 proteins as a potential mechanism by which VP formation can be induced by the former.

RACK1 associates with RNA-binding proteins Vigilin and SERBP1 to control dengue virus replication

“…Our study provided an unprecedented mapping of the DENV RC host interactome and identified cellular modules exploited by DENV during active replication. By combining these proteomics data with gene silencing experiments, we identified a set of Host Dependency Factors (HDFs) that critically impact DENV infection and established an important role for Receptor for Activated C-Kinase 1 (RACK1) in DENV vRNA amplification 12 , which was recently confirmed by others 13 .…”

Let’s Get Physical: Flavivirus-Host Protein–Protein Interactions in Replication and Pathogenesis

“…The Receptor for Activated C Kinase 1 (RACK1) protein is a known scaffolding protein with roles in protein shuttling, anchoring, and stabilization, as well as mediating cellular pathways through protein interactions ( Adams et al, 2011 ). The interaction between DENV NS1 and host RACK1 was first identified in a DENV NS1 specific proteomics screen ( Hafirassou et al, 2017 ) but the role of this interaction was not fully explored until recently by Shue et al (2021) . They performed a genome-wide CRISPR knockout screen in Huh7 cells to identify host genes involved in ZIKV replication.…”

“…Using a Renilla luciferase DENV replicon they determined that RACK1 specifically played a role in viral genome replication, rather than viral entry or translation. Using replication-independent expression system that induces the formation of replication compartments in the ER without virus infection they found that RACK1 silencing led to reduced formation of these compartments in the ER ( Shue et al, 2021 ). These studies are a great example of the power of integrating proteomic and genetic screens to identify mechanisms of virus replication.…”

Flavivirus–host interactions: an expanding network of proviral and antiviral factors

“…In addition, recent CRISPR KO screens have been published including a genome-wide screen with ZIKV performed in human stem cells differentiated into neural progenitor cells [ 37 ], a ZIKV screen in Huh-7.5 cells [ 38 • ], and screens with ZIKV and YFV in HAP1 cells [ 21 • ]. Highlights from these reports include mechanistic follow-up where Shue et al show that RACK1 is required for multiple mosquito-borne and tick-borne flaviviruses as well as the coronavirus, SARS-CoV-2 [ 38 • ]. The results indicate that RACK1 may be involved in establishing ROs on the ER membrane.…”