Structural and functional characterization of the replication and transcription activities of Hantaan virus polymerase

Abstract

The Bunyavirales order is a large and worldwide distributed group of segmented negative sense single-stranded RNA viruses (sNSV) that includes more than 350 species (nine families). Arthropods and rodents are their natural reservoirs and humans are occasionally infected resulting in severe diseases. Particularly, the pathogenic prototype viruses Hantaan virus (HTNV, family Hantaviridae) and Crimean-Congo Haemorrhagic fever virus (CCHFV, family Nairoviridae) have increased their geographical expansion and as a consequence, also cases of human diseases. Thus, it is necessary to (i) understand their mechanism of infection and (ii) to develop effective drugs to counteract them. In this perspective, we are working on two critical steps of bunyavirus viral cycle : replication and transcription. These processes are carried out by the multifunctional viral polymerase (L). In order to decipher the molecular mechanisms of bunyavirus replication, we present the study of interactions between hantavirus L proteins and their genomic RNA and replication assays of the full-length Hantaan polymerase. By electrophoresis mobility shifts assays and fluoresce anisotropy we determined the viral sequences specifically binding the L proteins anddefined their length and measured their affinity. We could see differences in the interactions between bunyaviral families L proteins suggesting differences on their mechanisms of replication and the way they are regulated. On the other hand, we performed replication assays for HNTV and LACV L and we have obtained a different result patron. These studies on replication and transcription reactions have shown that Hantaan L is more active than LACV L in the presence or absence of N terminal TAG. Also, the expected replication products are different and we observed some reproducible abortive products.We observed an unexpected UTP transferase activity by the Hantaan L protein that seems related to the processing of its genomic RNA for preventing recognition by the cell innate immune system and maintaining genome integrity. TheseRNA-protein interactions studies, along with the replication assays, will provide the basis for subsequent biochemical and structural studies to understand the molecular mechanism uncovering these reactions. This will be crutial for the development of effective antiviral drugs.