Abstract
Purpose :
Human adenovirus (HAdV) is an important cause of respiratory, ocular, and enteric disease. HAdVs are small non-enveloped viruses classified into seven species, A-G, and 90 types. Ocular infection by select viruses within human adenovirus species D (HAdV-D), in particular, types 8, 37, 53, 54, 56, and 64 causes epidemic keratoconjunctivitis (EKC), otherwise known as “pink eye”. EKC is a severe, and hyperacute ocular surface infection, associated with prolonged and/or recurrent keratitis in up to one-third of cases. To date there is no treatment for this infection. An in depth understanding of the molecular mechanisms during infection can help to design targeted therapy in the future.
Methods :
The viral coat protein pIIIa is involved in genome packaging and maturation, and the majority of the pIIIa monomer is localized to the outer surface of the virion. Using an inducible expression system for pIIIa at near endogenous levels after infection, affinity purification and LC-MS/MS analysis were employed to identify binding partners. CRAPome database analysis and SAINT probability were used to score true protein interactions and remove background contaminants. Targeted knock downs, immunoprecipitation and Western blots assays were performed to confirm and analyze putative interactions.
Results :
We identified new potential interactions of pIIIa with the host proteins, USP9X and RANBP2. This was validated by reciprocal co-immunoprecipitation in induced and natural infections. RANBP2 expression increased with pIIIa induction in a dose dependent manner. Also, pIIIa interaction with RANBP2 was decreased by treatment with Leptomycin B, an inhibitor of the nuclear export receptor, CRM1 that also interacts with RANBP2. These observation was verified by CRM1 siRNA knockdown.
Conclusions :
Upon entry, viruses hijack host cellular machinery for virus-specific functions, and many such virus-host interactions remain poorly characterized. Here we show interaction between the host proteins, USP9X and RANBP2 with pIIIa. We hypothesize that pIIIa recruits USP9X, a deubiquitinase, to prevent proteasomal degradation of RANBP2. Application of our methodology to other viral proteins may lead to identification of previously unknown functions for both viral and host encoded proteins during viral infection.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.