Functional and genetic characterization of dengue virus methyltransferase.

Abstract

Dengue virus (DENV) is a member of the genus Flavivirus within the family Flaviviridae. DENV consists of four distinct serotypes (DENV 1-4) and causes diseases ranging from self-limiting dengue fever to life threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Dengue fever is the most frequent arthropod transmitted infectious disease in humans and is endemic in the tropics and subtropics regions. Currently there is no antiviral therapy or vaccine available for treatment of DENV infections, thus causing significant human mortality and morbidity in epidemic areas. DENV is an envelope virus with a single-stranded positive-sense RNA genome approximately 11 kb in length (reviewed in Chambers et al., 1990). The open reading frame encodes a polyprotein that is post- and co-translationally cleaved by viral and cellular proteases into three structural proteins (capsid, pre-membrane and envelope) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) (reviewed in Chambers et al., 1990). The N- and C-terminal regions of NS5 encode methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) respectively.

DENV MTase harbours both N-7 and 2′-O activities needed for the formation of type 1 (m7GpppNm) cap structure on the newly synthesized viral RNA. This cap structure is essential for mRNA stability and efficient translation during viral infection. Here, we established robust biochemical assays to study both enzymatic activities and performed kinetic studies to understand their mechanisms of action. DENV N-7 and 2′-O methylations occurred in a sequential manner via a random bi bi mechanism. Progression from N-7 to 2′-O methylation did not involve enzyme-RNA dissociation. Analyses of steady state kinetic parameters showed that N-7 precedes 2′-O methylation as the RNA turnover rate was faster (kcat) resulting in 2.4-fold increase in catalytic efficiency. Michaelis constants of S-adenosyl-methionine (AdoMet) for both reactions were about 10-fold lower than for their respective RNA substrates, suggesting that the rate-limiting steps in methyltransferase reactions were associated with RNA templates.