DENGUE VIRUS NS5 PROTEIN TARGETS PML NUCLEAR BODIES INVOLVED IN INTRINSIC IMMUNITY

PETER HEMMERICH; FEDERICO GIOVANNONI; CYBELE C. GARCÍA

Philadelphia

Congreso; ASCB - EMBO 2017 Meeting; 2017

American Society for Cell Biology - European Molecular Biology Origanization

Promyelocytic leukemia (PML) protein is the key organizer of sub nuclear structures called nuclear bodies (PML-NBs). PML-NBs are known to be involved in many basic cellular processes including apoptosis, proliferation, DNA repair and antiviral response. Accumulating reports have revealed that PML-NBs can restrict the replication of many DNA viruses (that must replicate in the nucleus), but also RNA viruses (that replicate in the cytoplasm), such as dengue virus (DENV). A total of 390 million DENV infections occur each year around the world, turning DENV into a major public health problem. DENV can cause dengue fever, which is often a self-limited febrile illness. However, more severe forms of the disease lead to 500,000 hospitalizations and 25,000 deaths worldwide annually. Here, we shed light on the antiviral role of PML in the in-vitro replication of DENV as well as the mechanism underlying this effect. In order to determine if PML has an inhibitory effect on DENV replication, the expression of PML in A549 cells was silenced by using siRNAs. Then, cells were infected with four DENV serotypes (DENV 1-4) and the amount of virus particles produced was quantified. A549 cells silenced for PML produced 3-4 times more DENV particles than control cells. Next, to determine if DENV infection has an impact on PML-NBs distribution, confocal microscopy of DENV-infected cells was performed. Image analysis revealed that the number of PML-NBs was significantly lower in infected cells. Even though flaviviruses, such as DENV, replicate in the cytoplasm of infected cells, DENV proteins C, NS3 and NS5 can localize to the nucleus. This nuclear localization is still considered enigmatic in DENV biology. To determine if the disruption of PML-NBs could be a consequence of the interaction between PML and viral proteins, cells were transfected with vectors encoding for C or NS5. Confocal images showed that quantitative expression of NS5, but not C, was sufficient to reduce the number of PML-NBs. Furthermore, immunoprecipitation studies confirmed the interaction between NS5 and PML. Finally, we observed that overexpression of PML isoforms III and IV, but not the other nuclear PML isoforms induced an accumulation of NS5 at PML-NBs. Overall, we show for the first time a phenotypic as well as functional interplay between PML and DENV. DENV-2 NS5 is found in a complex with PML and its expression alone is sufficient to disrupt PML-NBs. We conclude that NS5 nuclear localization may be important for inhibiting PML-mediated antiviral response.