New Host Factors Important for Respiratory Syncytial Virus (RSV) Replication Revealed by a Novel Microfluidics Screen for Interactors of Matrix (M) Protein

Although human respiratory syncytial virus (RSV)1, from the Pneumovirus genus of the Paramyxoviridae family, is the most common cause of infantile bronchiolitis and pneumonia in the developed world, there is no vaccine or antiviral therapy available to combat it (1–4). The RSV Matrix (M) protein plays key roles in virus life cycle. Early in infection M localizes in the nucleus via the action of the nuclear transport protein Importin β1 (5), serving an apparent dual role of inhibiting host cell transcription (6) as well as preventing inhibition of viral transcription in the cytoplasm (7). Nuclear targets of M have thus far not been reported. Later in infection, M traffics to the cytoplasm through the action of the nuclear export protein CRM-1 (8) to associate with inclusion bodies (IBs), the site of RSV transcription and replication. It was recently suggested that M also serves to sequester cellular proteins involved in the host innate immune response (9). M localization into IBs is dependent on the RSV protein M2–1 and is believed to represent a potential switch between viral transcription and assembly (10), with M helping coordinate the latter in an adaptor role. M association in IBs with the RSV F (fusion) protein triggers immediate filament formation (11). Ultimately, all of the viral proteins localize at the apical cell surface, where M helps coordinate assembly into virus filaments followed by budding (12, 13). The minimal RSV viral protein requirement for filament formation and budding of virus-like particles (VLPs) are F, M, nucleo (N), and phospho (P) protein (14). Little is known regarding the specific roles of P and N in budding, but the cytoplasmic tail of F appears to be critical to filament formation, presumably through recruiting specific host factor(s) required for virus release (14, 15). M's crucial role in viral filament maturation and elongation relates to the transfer of RNP complexes from IBs to the sites of budding (16). We recently showed that ordered oligomerization of M is central to infectious filamentous virus production (17), potentially through providing the framework for filament morphology (18), in conjunction with M2–1, which serves as a bridging protein between the oligomeric M layer and RNP in the mature virus (19). Additional to the crucial role of M in RSV filament morphology and infectivity, M has been suggested to recruit cellular factor(s) during virus assembly (20–23). Proteins involved in apical recycling endosomes (ARE)-mediated protein sorting (e.g. Myosin 5 beta), have been shown to be essential for RSV assembly (24) with budding of released virus believed to be Vps4-independent and to require Rab11a FIP2 protein (25). However, only Importin-β1 (5) and CRM1 (8) (see above) are known to be direct interactors of M. A proteomic screen for cellular interactors of RSV M, N, and F proteins identified only limited numbers of proteins, none of which could be validated to bind directly to M (26). Overall, the network of RSV-cell interactions is still mostly unknown, with limited targets identified. Protein microarrays technology allows the interrogation of protein–protein interactions, which could possibly overcome the obstacles mentioned above (27). Here we use an in vitro protein expression and interaction analysis platform based on a highly parallel and sensitive microfluidics affinity assay (28) to identify new host factors interacting with RSV M. This is the first time microfluidics has been used to screen for host factors interacting with a protein from a negative strand RNA virus. A range of factors were identified for the first time, including proteins involved in host transcription and translation regulation, innate immunity response, plasma membrane remodeling, cytoskeleton regulation, and cellular trafficking, with a number verified by coprecipitation. Of these, we present initial characterization of key caveolae structural component Caveolin (Cav) and the actin-binding protein Cofilin1 (Cof1) as cellular factors that colocalize with M in viral inclusions and filaments, and of the zinc finger protein ZNF502, which appears to interact with RSV M in the nucleus. These and the other host factor-RSV M interactions identified here for the first time may be exciting possibilities as targets for anti-RSV approaches in the future.