N6 -Methyladenosine Negatively Regulates Human Respiratory Syncytial Virus Replication.

Fabian Figueroa,Alonso Vega-Gibson,Aracelly Gaete-Argel,Lorena I Tapia,Monica L Acevedo,Fernando Valiente-Echeverría,Joseline Catrileo,Sebastian Riquelme-Barrios,Ricardo Soto-Rifo,Luis Antonio Alonso-Palomares

doi:10.3389/fcell.2021.739445

Fabian Figueroa, Alonso Vega-Gibson + Show 8 more

Open Access

https://doi.org/10.3389/fcell.2021.739445

Copy DOI

Abstract

N6-methyladenosine (m6A) is the most abundant internal modification described in eukaryotic mRNA and several viral RNA including human respiratory syncytial virus (HRSV). Here, we evaluated the impact of m6A writers, erasers and readers on HRSV genomic RNA accumulation and inclusion bodies assembly during viral replication. We observed that the METTL3/METTL14 m6A writer complex plays a negative role in HRSV protein synthesis and viral titers, while m6A erasers FTO and ALKBH5 had the opposite effect. We also observed that m6A readers YTHDF1-3 bind to the viral genomic RNA inducing a decrease in its intracellular levels and thus, inhibiting viral replication. Finally, we observed that overexpression of YTHDFs proteins caused a decrease in the size of inclusion bodies (IBs), accompanied by an increase in their number. METTL3 knockdown cells showed an opposite effect indicating that the dynamics of IBs assembly and coalescence are strongly affected by m6A readers in a mechanism dependent on m6A writers. Taken together, our results demonstrated that the m6A modification negatively affects HRSV replication, possibly through a mechanism involving the assembly of inclusion bodies, the main factories of viral genomic RNA synthesis.

Highlights

Human respiratory syncytial virus (HRSV) infections are the main cause of viral bronchiolitis and pneumonia in infants, elderly, asthmatic and immunocompromised individuals worldwide reaching 33 million cases annually
To evaluate the impact of the host m6A machinery on human respiratory syncytial virus (HRSV) replication, we first analyzed the role of the methyltransferase complex on the replication of HRSV by overexpressing Flagtagged METTL3 and METTL14 in HEK293T cells
Our results suggest that the m6A methyltransferase complex restricts HRSV replication by at least interfering with F protein synthesis and the production of infectious particles

Summary

Introduction

Human respiratory syncytial virus (HRSV) infections are the main cause of viral bronchiolitis and pneumonia in infants, elderly, asthmatic and immunocompromised individuals worldwide reaching 33 million cases annually. The viral replication occurs in inclusion bodies (IBs), defined as membrane-less ribonucleoprotein complexes containing the genomic RNA (gRNA) together with viral proteins: nucleoprotein (N), phosphoprotein (P), matrix (M), nucleocapsid-associated transcription factor (M2-1) and the large polymerase subunit (L) N6-methyladenosine (m6A) is the most abundant internal modification described in eukaryotic mRNA and viral RNA and has been involved in regulating multiple cellular and pathological processes, including viral infections (Lichinchi et al, 2016a; Courtney et al, 2017; Imam et al, 2018; Lu et al, 2018; Hao et al, 2019; Liu et al, 2019; Price et al, 2020; Wu et al, 2020). The cellular machinery responsible of this regulation includes the complex formed by methyltransferases METTL3 and METTL14, which introduce the methyl group at the N6 position of adenosines mainly located in the DRACH context (Liu et al, 2014), FTO (Jia et al, 2011), and ALKBH5 (Zheng et al, 2013), which have been shown to reverse adenosine methylation and YTH proteins that read the modification and are responsible for exerting the functions of m6A (Meyer and Jaffrey, 2014)

Methods

Results

Conclusion