The long-term goal of this project is to decipher how RNA viruses suppress the host's innate immune response. The short-term goal is to glean a biochemical and mechanistic understanding how the two unique nonstructural (NS) proteins of respiratory syncytial virus (RSV) operate to suppress both the induction and function of type I interferon (IFN), which enables the virus to cause severe lower respiratory tract infection and associated mortality. The proposed studies are founded on several novel observations that we have made recently: (i) RSV NS proteins can form NS1-NS2 heteromer. (ii) Between them, NS1 and NS2 degrade multiple members of both IFN induction and response pathways, such as RIG-I, IKK?, IRF3, IRF7, TRAF3 and STAT2. (iii) By fractionating extracts from A549 cells in which either NS1, or NS2, or both were expressed recombinantly, we isolated three distinct complexes of the following major constituents, depending on which NS was expressed: (1) NS1 Complex or NS1C: NS1, Elongin BC, Cullin 2, Rbx1;(2) NS2 Complex or NS2C: NS2, Elongin BC, Cullin 5, Rbx2;(3) the dual NS1/NS2 Complex or NS1/2C: NS1, NS2, Elongin BC, Cullin 2 &5, Rbx1 &2, BAG2, RAD23B. Thus, NS1/2C appears to be a heterodimer of NS1C and NS2C, but contains two additional host proteins, BAG2 and RAD23B. (v) We have developed in vitro assays for all three NS complexes, showing that they possess E3 ligase activity. (vi) The specificity of recruitment of these IFN-related substrates to the NS complexes seems to be determined by ISG15 conjugation. (vii) We made the unexpected discovery that NS1 is a phosphoprotein, likely phosphorylated by an Ataxia telangiectasia mutated (ATM) family protein kinase, which opens a novel avenue of NS1 activation. In this AREA (R15) project, we will pursue two Specific Aims, in which altogether we will: test the role of the E3 ligase components in the NS complexes;validate the putative BC box domain of NS1 and NS2;interrogate the role of ISGylation in NS E3 ligase substrate specificity;explore the role of accessory subunits (BAG2, RAD23B) and the phosphorylations of NS1 and BAG2;and lastly, validate the role of phosphorylation of NS1 in vivo by constructing and using recombinant phosphosite NS1 mutant RSV in the mouse model. Together, our results should provide new molecular insights into how a virally hijacked host cellular ubiquitin-proteasomal degradation system functions. It may also offer a framework for future antiviral regimen targeting these complexes. Finally, it would offer our students at CSU a springboard to learn various aspects of protein-protein interaction, ubiquitination and protein phosphorylation, with special reference to viral suppression of innate immunity.
Respiratory syncytial virus (RSV) is the most severe respiratory pathogen in children and also affects the elderly and the immunocompromised. In an average year, RSV infects 64 million people globally, and causes 50,000 hospitalizations and 10,000 deaths in the US alone. Our goal is to explore novel, previously unexplored mechanisms by which the unique nonstructural proteins of the virus suppress antiviral defense, which may allow a better understanding and management of the infection.
|Ribaudo, Michael; Barik, Sailen (2017) The nonstructural proteins of Pneumoviruses are remarkably distinct in substrate diversity and specificity. Virol J 14:215|
|Rabbani, M A G; Barik, Sailen (2017) 5-Hydroxytryptophan, a major product of tryptophan degradation, is essential for optimal replication of human parainfluenza virus. Virology 503:46-51|
|Rabbani, M A G; Ribaudo, Michael; Guo, Ju-Tao et al. (2016) Identification of Interferon-Stimulated Gene Proteins That Inhibit Human Parainfluenza Virus Type 3. J Virol 90:11145-11156|
|Wang, Xin; Majumdar, Tanmay; Kessler, Patricia et al. (2016) STING Requires the Adaptor TRIF to Trigger Innate Immune Responses to Microbial Infection. Cell Host Microbe 20:329-341|
|Dhar, Jayeeta; Barik, Sailen (2016) Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins. Sci Rep 6:38139|
|Dhar, Jayeeta; Cuevas, Rolando A; Goswami, Ramansu et al. (2015) 2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1. J Virol 89:10115-9|