Cellular splicing is altered by the Dengue Virus 2 NS5 protein during infection

DE MAIO, F.A.; RISSO, G.; IGLESIAS, N.G.; SHAH, P.; KROGAN N; ANDINO R; SREBROA W A; GAMARNIK, A.V.

Mar del Plata

Congreso; SAIB 51 Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2015

The plusstrand dengue virus RNA genome is translated into a single polyprotein which isthen cleaved into 10 different viral proteins. NS5 contains an RNA-dependentRNA polymerase domain and is essential for viral replication. In order toidentify cellular proteins that interact with NS5 during infection, we generatedrecombinant viruses with a purification tag fused to this viral protein andobtained replication competent viruses. Using an affinity purification and massspectrometry strategy about 50 cellular proteins were identified as specificNS5 binders. Among these binders, spliceosome components were highly abundantwith a clear enrichment of U5 components. Different studies including co-IP andRIP analysis confirm the presence of the viral protein in active splicingcomplexes. Studies of endogenous alternative splicing events and splicing oftransfected minigenes, indicated that viral infection or NS5 overexpression,respectively, modulates cellular splicing.Toexplore the relevance of NS5-spliceosome interaction on viral infection, differentspliceosomal components were silenced. Interestingly, interfering components ofthe U5 complex results in a significant increase in viral replication. Theseresults support a model in which  denguevirus manipulates the cellular splicing during infection, likely to create amore favorable cell environment for viral replication.