Molecular Basis for Nucleotide Conservation at the Ends of the Dengue Virus Genome

The dengue virus (DV) is an important human pathogen from the Flavivirus genus, whose genome- and antigenome RNAs start with the strictly conserved sequence pppAG. The RNA-dependent RNA polymerase (RdRp), a product of the NS5 gene, initiates RNA synthesis de novo, i.e., without the use of a pre-existing primer. Very little is known about the mechanism of this de novo initiation and how conservation of the starting adenosine is achieved. The polymerase domain NS5Pol_DV of NS5, upon initiation on viral RNA templates, synthesizes mainly dinucleotide primers that are then elongated in a processive manner. We show here that NS5Pol_DV contains a specific priming site for adenosine 5′-triphosphate as the first transcribed nucleotide. Remarkably, in the absence of any RNA template the enzyme is able to selectively synthesize the dinucleotide pppAG when Mn²⁺ is present as catalytic ion. The T794 to A799 priming loop is essential for initiation and provides at least part of the ATP-specific priming site. The H798 loop residue is of central importance for the ATP-specific initiation step. In addition to ATP selection, NS5Pol_DV ensures the conservation of the 5′-adenosine by strongly discriminating against viral templates containing an erroneous 3′-end nucleotide in the presence of Mg²⁺. In the presence of Mn²⁺, NS5Pol_DV is remarkably able to generate and elongate the correct pppAG primer on these erroneous templates. This can be regarded as a genomic/antigenomic RNA end repair mechanism. These conservational mechanisms, mediated by the polymerase alone, may extend to other RNA virus families having RdRps initiating RNA synthesis de novo.