Human respiratory syncytial virus (hRSV) is the most serious cause of lower respiratory tract infection in infants, young children and immunocompromised individuals. There are no licensed vaccines or efficacious therapeutics to combat hRSV.
Innate immune responses are initiated upon host cell detection of viral-specific molecular patterns, such as virus-specific RNA features. This detection system activates the production of immunomodulatory cytokines (induction), which in turn stimulate the production of antiviral protein effectors (signalling), some of which amplify the cytokine induction process. hRSV produces two small non-structural proteins (NS1 and NS2) which dampen the host cell innate immune system by targeting key players in both induction and signalling pathways. Therapeutic and vaccine developments to combat hRSV may emerge by gaining a better understanding of the interactions between hRSV NS1 and NS2 and host cell antiviral pathways.
We have developed a comprehensive suite of proteomic approaches to identify specific targets of hRSV NS1 and NS2. This includes workflows based on in-solution protein IEF of cell lysates with downstream bottom-up mass spectrometric label free quantification of the differences between hRSV-infected and uninfected cells. Another workflow involves bottom-up analyses of digests of unfractionated whole cell lysates combined with label free quantification. Our infectious experiments include hRSV encoding the complete wild-type genome and genotypes lacking NS1 and/or NS2 coding capacities. As a consequence we have identified key pathways that are promoted and/or blocked in order to achieve survival of the infected host cell in a state conducive to hRSV propagation. Although some observations were common to both proteomic workflows, the in-solution IEF workflow uniquely enabled observation of some regulation at a global level due to selective enrichment of low abundance proteins. Furthermore, the in-solution IEF workflow provided information on regulation at a proteoform level that was not evident on a global quantification level. Of particular interest were the observations that NS1 appears to directly suppress the activation of NF-κB1 and to promote the survival of pro-apoptotic proteins within the infected cell. Furthermore, it was evident that hRSV has tactics to suppress the innate antiviral responses of the infected cell independently of NS1 and/or NS2. The strengths and weaknesses of these workflows will be discussed along with how specific observations provide a better understanding of how hRSV subverts host cell antiviral pathways.