# A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

## TL;DR

This study maps how orthoflaviviruses use a human protein pathway called UFMylation to replicate, suggesting a new antiviral strategy.

## Contribution

The study reveals a conserved role of UFMylation in orthoflavivirus replication through systematic analysis of NS4B interactions.

## Key findings

- NS4B interacts with UBA5, a key enzyme in the UFMylation pathway, across multiple orthoflaviviruses.
- Pharmacological inhibition of UFMylation significantly reduces viral replication in vitro and in vivo.
- UFMylation pathway components are recruited to viral replication sites to support infectious particle production.

## Abstract

Orthoflavivirus infections represent an increasing public health burden, with several members of the genus emerging or re-emerging globally. Several pre-clinical studies identified the non-structural protein 4B (NS4B), as the most promising target for the development of potent direct-acting antivirals. However, its functional roles in viral replication are still elusive. Here, we employ an integrated proteomic approach to systematically identify cellular targets of NS4B across eight prototypic orthoflaviviruses and characterize their influence on the human proteome. Using this approach, we mapped high-confidence NS4B-interacting human proteins across the genus, underlying potentially divergent and convergent mechanisms of host adaptation across orthoflaviviruses spanning diverse pathologies and vector preferences. Among these, we unveil a novel function for UBA5, the E1-activating enzyme of the UFMylation pathway, in orthoflavivirus replication. Mechanistically, we map associations of distinct viral proteins with multiple members of the UFMylation pathway, which are selectively recruited to sites of viral replication to promote infectious particle production. Finally, we demonstrate that pharmacological inhibition of UFMylation exerts potent antiviral activity in vitro and in vivo. This integrative study provides a rational framework for a system-level understanding of orthoflavivirus NS4B effector functions and sheds light on a conserved and unconventional role for UFMylation in orthoflavivirus replication.

Here, Rajasekharan et al. identify human targets of the viral NS4B protein across orthoflaviviruses, showing how these viruses hijack the UFMylation pathway to replicate. Blocking this pathway reduced infection in lab and animal models, highlighting a promising antiviral strategy.