# Tree Shrew Genome-Wide CRISPR Screen Identifies RNF6 as a Proviral Host Factor for Zika Virus Replication in Brain Microvascular Endothelial Cells

## TL;DR

Researchers used a tree shrew CRISPR screen to find RNF6, a protein that helps Zika virus replicate in brain cells, offering new insights for antiviral strategies.

## Contribution

The first tree shrew genome-wide CRISPR screen identifies RNF6 as a proviral host factor for Zika virus in brain microvascular endothelial cells.

## Key findings

- RNF6 knockout or knockdown significantly reduces Zika virus infection in brain microvascular endothelial cells.
- RNF6 interacts with the Zika virus NS5 protein and regulates interferon and MAPK signaling pathways.
- RNF6 is highly conserved between humans and tree shrews with shared NS5-binding residues.

## Abstract

Zika virus (ZIKV), a unique flavivirus with neurotropic and teratogenic potential, can cross the blood–brain barrier and persist in human brain microvascular endothelial cells (BMECs); however, no approved vaccines or specific antivirals exist, and its barrier-crossing and neuroinvasive mechanisms remain elusive. Innovative strategies to identify additional host factors mediating ZIKV infection could yield key insights and help address these challenges. To uncover novel host factors, we established the first tree shrew (Tupaia belangeri) genome-wide CRISPR/Cas9 knockout (GeCKO) library and performed a screen in BMECs, identifying ring finger protein 6 (RNF6) as a novel proviral factor for ZIKV. ZIKV infection in BMECs was significantly reduced following RNF6 knockout or knockdown but enhanced upon RNF6 overexpression or rescue. Mechanistically, RNF6 interacts with the ZIKV NS5 protein and acts as a potential negative regulator of the type I interferon and MAPK signaling pathways. Evolutionary and structural analyses revealed that RNF6 is highly conserved between humans and tree shrews; molecular docking further identified shared NS5-binding residues (Gln-59, Arg-140), supporting the conserved proviral role of human RNF6 in ZIKV infection. Our findings highlight tree shrew GeCKO screening as an efficient approach for identifying novel host factors and establish RNF6 as a critical proviral factor for ZIKV replication in BMECs, providing new insights into ZIKV neurotropic pathogenesis and informing potential antiviral strategies.