Identification of Cytoplasmic Chaperone Networks Relevant for Respiratory Syncytial Virus Replication

Abstract

RNA viruses have limited coding capacity and must therefore successfully subvert cellular processes to facilitate their replication. A fundamental challenge faced by both viruses and their hosts is the ability to achieve the correct folding and assembly of their proteome while avoiding misfolding and aggregation. In cells, this process is facilitated by numerous chaperone systems together with a large number of co-chaperones. In this work, we set out to define the chaperones and co-chaperones involved in the replication of respiratory syncytial virus (RSV). Using an RNAi screen, we identify multiple members of cellular protein folding networks whose knockdown alters RSV replication. The reduced number of chaperones and co-chaperones identified in this work can facilitate the unmasking of specific chaperone subnetworks required for distinct steps of the RSV life cycle and identifies new potential targets for antiviral therapy. Indeed, we show that the pharmacological inhibition of one of the genes identified in the RNAi screen, valosin-containing protein (VCP/p97), can impede the replication of RSV by interfering with the infection cycle at multiple steps.

The Respiratory Syncytial Virus M2-2 protein is targeted for proteasome degradation and inhibits translation and stress granules assembly

“…Because many of the proteins identified by co-IP were mainly associated with translation elongation, we chose to investigate potential paths connecting it with viral infection, raising a possible association of M2-2 with ribosome quality control (RQC), among other mechanisms involved with this step of translation [41, 42, 43]. M2-2 potential partners encompass, beyond ribosomal proteins, numerous chaperones, including VCP, recently described as necessary for proper RSV infection [44]. Moreover, besides the already described RACK1, we also detected the proteasome subunit PSMD11, all known proteins whose role in RQC has been for long described [45, 46, 47, 48].…”

Polarised human alveolar epithelia enable identification of dichloroacetate as an effective drug against respiratory viruses

“…The human coronavirus SARS-CoV-2 strain MAD6 was kindly provided by Dr. Luis Enjuanes (CNB-CSIC, Madrid, Spain), while Omicron was isolated at CBMSO. Respiratory syncytial virus A expressing the fluorescent protein mKate-2 (RSV-mKate2) was generated as previously described [ 55 ]. Viral stocks were prepared in DMEM supplemented with 2% HIFBS for HCoV-229E-GFP and SARS-CoV-2 in DMEM supplemented with 2% HIFBS and 5% DMSO for RSV-mKate2.…”

Cetylpyridinium chloride and chlorhexidine show antiviral activity against Influenza A virus and Respiratory Syncytial virus in vitro

“…For the Respiratory syncytial virus (RSV) assays we use a variant that expresses the fluorescent protein mKate2 (RSV-mKate2) [27]. To generate this virus the modified RSV infectious clone system (BEI Resources, Manassas, USA) was transfected into HEK-293 cells together with a codon-optimized T7 polymerase (Addgene, Watertown, USA, 65974), and an RSV N, P, M2-1, and L expression plasmids at a ratio of 4:2:2:2:1 respectively.…”