Date Published: August 30, 2016
Publisher: Public Library of Science
Author(s): Federico A. De Maio, Guillermo Risso, Nestor G. Iglesias, Priya Shah, Berta Pozzi, Leopoldo G. Gebhard, Pablo Mammi, Estefania Mancini, Marcelo J. Yanovsky, Raul Andino, Nevan Krogan, Anabella Srebrow, Andrea V. Gamarnik, Ted C. Pierson.
Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication.
Dengue virus (DENV) is currently the most important human viral pathogen transmitted by insects. It is responsible for about 390 million infections worldwide every year . In spite of this great burden, vaccines and specific antivirals remain elusive. In fact, a steady increase in the number of infections is being registered in the last years (http://apps.who.int/iris/bitstream/10665/75303/1/9789241504034_eng.pdf?ua=1). DENV belongs to the Flavivirus genus in the Flaviviridae family, together with a large number of emerging and re-emerging human pathogens that cause fevers and encephalitis, such as West Nile virus, Japanese encephalitis virus and Zika virus [2,3].
Here, we discovered a novel property of the DENV NS5 protein in subverting cellular splicing. Proteomic analysis and functional studies revealed that NS5 binds to active spliceosomes in infected cells and modulates splicing. Mechanistic studies indicate that NS5 interacts with components of the U5 snRNP and reduces the efficiency of pre-mRNA splicing. Our findings support a model in which NS5-mediated regulation of specific spliceosomal components renders an advantageous cellular environment for DENV replication, providing a new function for the viral NS5 polymerase in infected cells.