# N6-methyladenosine modification of the subgroup J avian leukosis viral RNAs attenuates host innate immunity via MDA5 signaling

## TL;DR

This study shows how a virus called ALV-J uses a chemical modification called m6A to avoid triggering the chicken's immune system, allowing it to cause long-term infections.

## Contribution

The study reveals for the first time that m6A modifications in ALV-J RNA help the virus evade the host's innate immune response via the MDA5 signaling pathway.

## Key findings

- ALV-J genomic RNA has m6A modifications mainly in the Env and 3′UTR regions.
- m6A-deficient ALV-J increases IFN-β production, indicating a role in immune evasion.
- MDA5 signaling is central to how m6A modifications help ALV-J avoid immune detection.

## Abstract

Subgroup J avian leukosis virus (ALV-J), a retrovirus, elicits immunosuppression and persistent infections in chickens. Although it is widely acknowledged that ALV-J can evade the host’s innate immune defenses, the mechanisms behind this immune evasion remain elusive. N6-methyladenosine (m6A), the most prevalent internal RNA modification, plays a role in innate immune evasion. Our research identified ALV-J as an inefficient stimulator of innate immunity in vitro and in vivo, with its genomic RNA featuring m6A modifications predominantly in the envelope protein (Env) region and 3′ untranslated region (3′UTR). To elucidate the functional consequences of m6A modification, we subsequently generated m6A-deficient ALV-J through its culturing in the DF-1 overexpressing fat mass and obesity-associated protein (FTO) cells. The m6A-deficient ALV-J virus, or its RNAs significantly enhanced IFN-β production compared to the wild-type (wt) ALV-J, suggesting a pivotal regulatory function of m6A modifications in modulating innate immune response. Mechanistically, the m6A modification of the ALV-J genomic RNA directly impacted its recognition by MDA5, weakening its binding and ubiquitination and attenuating IFN-β activation. Moreover, m6A-deficient ALV-J, created by inducing mutations in m6A sites within Env and 3′UTR, exhibited reduced replication capacity and elevated IFN-β expression in host cells. Importantly, this phenomenon was abolished in MDA5-knockout DF-1 cells, further demonstrating the core role of MDA5. These data demonstrate that m6A modification of ALV-J genomic RNA dampens the host’s innate immune response through MDA5 signaling pathway.

ALV-J has a long latency and can cause persistent infections with continuous viral shedding, leading to severe immunosuppression and tumor induction in chickens and consequently significant economic losses to the poultry industry. However, the mechanisms by which ALV-J escapes the host’s innate immune recognition and clearance and achieves persistent infection remain elusive. In this study, we systematically identified, for the first time, that the m6A modifications of ALV-J genomic RNA were primarily concentrated in the Env and 3′UTR regions and that this modification is crucial in the viral evasion of the host’s innate immunity. Furthermore, we parsed the in-depth mechanism that the m6A modifications of ALV-J RNA mediate the evasion of RNA recognition by the host’s innate immunity through the MDA5 signaling pathway to attenuate IFN-β expression. Our findings reveal the significance of m6A modifications in the immune evasion strategies of ALV-J and offer a novel perspective on the complex interactions between the virus and the host’s innate immune response.