Direct interaction between RSV polymerase L and active Rab11a mediates viral ribonucleoprotein transport to assembly sites

Abstract

Respiratory syncytial virus (RSV) is an enveloped, negative-sense, single-stranded RNA virus whose ribonucleoproteins (vRNPs) must be transported from cytoplasmic viral factories to the plasma membrane for efficient virion assembly. Viral vRNPs comprise genomic RNA encapsidated by nucleoprotein N and associated with the polymerase complex (L, P, and M2-1). It was previously demonstrated that newly synthesized vRNPs are transported along microtubules by hijacking Rab11a, a small GTPase involved in the regulation of recycling endosomes. In our previous study, we showed an interaction between Rab11a and vRNPs in infected cells by immunoprecipitation assays, nevertheless the molecular mechanisms underlying Rab11a viral hijacking remained unknown. Here, we provide the first comprehensive characterization of the interaction between RSV vRNPs and Rab11a using immunoprecipitation, immunofluorescence colocalization, GST pull-down assays, and biolayer interferometry. We demonstrate that the viral polymerase L is the sole vRNPs component responsible for Rab11a recognition: immunoprecipitation of L specifically co-precipitates HA-tagged Rab11a, whereas other vRNPs proteins show no interaction. In vitro binding studies confirm that L interacts directly and specifically with the active, GTP-bound form of Rab11a with sub-micromolar affinity. Domain mapping using truncated constructs reveals that this interaction requires the C-terminal methyltransferase and CTD domains of L (residues 1756-2165) and depends on Rab11as Switch I region, known to mediate interactions with cellular Rab11a partners. Mutagenesis further highlights leucine 1860 in the L polymerase as critical for Rab11a binding. Competitive inhibition of Rab11a-L interaction using the minimal Rab11a-binding domain reveals its involvement in vRNPs transport as it significantly impairs vRNP dynamics during infection. Together, these findings establish RSV polymerase L as the key mediator of Rab11a engagement, define the molecular interface of their interaction, and reveal a potentially conserved viral strategy for genome transport. Targeting the L-Rab11a interaction could therefore be a promising strategy for the development of RSV-specific or broad-spectrum antiviral therapies. Author SummaryRespiratory syncytial virus is the leading cause of severe lower respiratory infection in children worldwide and is increasingly recognized as a major respiratory pathogen in the elderly and immunocompromised. Although vaccines have recently become available, treatment remains largely supportive in the absence of virus-specific antivirals, highlighting the urgent need for new therapeutic strategies. In the infected cell, the viral genome is encapsidated and associated to the viral polymerase complex, to form viral ribonucleoproteins (vRNP). The vRNPs are produced in cytoplasmic viral factories and transported to the plasma membrane for assembly of new viral particles. Previous work has shown that RSV exploits Rab11a, a host GTPase that regulates recycling endosome trafficking, to mediate this transport. However, exactly how the virus connects to this transport system awaits to be precisely defined. In this study, we identify the viral polymerase L as the key RSV protein that directly binds the active form of Rab11a. We mapped the domains of L and Rab11a that interact and showed that this connection is essential for efficient vRNPs trafficking. These findings reveal a critical step in the RSV life cycle and suggest that disrupting the L-Rab11a interface could be a novel target for broad-spectrum antiviral development.