Full-length Dengue Virus RNA-dependent RNA Polymerase-RNA/DNA Complexes

Abstract

Fundamental aspects of interactions of the Dengue virus type 3 full-length polymerase with the single-stranded and doublestranded RNA and DNA have been quantitatively addressed. The polymerase exists as a monomer with an elongated shape in solution. In the absence of magnesium, the total site size of the polymerase-ssRNA complex is 26 ؎ 2 nucleotides. In the presence of Mg 2؉ , the site size increases to 29 ؎ 2 nucleotides, indicating that magnesium affects the enzyme global conformation. The enzyme shows a preference for the homopyrimidine ssRNAs. Positive cooperativity in the binding to homopurine ssRNAs indicates that the type of nucleic acid base dramatically affects the enzyme orientation in the complex. Both the intrinsic affinity and the cooperative interactions are accompanied by a net ion release. The polymerase binds the dsDNA with an affinity comparable with the ssRNAs affinity, indicating that the binding site has an open conformation in solution. The lack of detectable dsRNA or dsRNA-DNA hybrid affinities indicates that the entry to the binding site is specific for the sugar-phosphate backbone and/or conformation of the duplex. The dengue virus (DENV)2 is a member of the Flaviviridae family and the etiological agent of dengue fever, a disease affecting worldwide ϳ100 million people each year (1-5). The Flaviviridae family includes such human pathogens as tick-borne encephalitis virus, hepatitis C virus, West Nile virus, yellow fever virus, and Japanese encephalitis virus (1-5). The dengue virus exists in four distinct types, DENV1, -2, -3, and -4, which show ϳ60% genomic sequence identity (1). The dengue fever often develops into the dengue hemorrhagic fever, an acute form of the disease, possibly as a result of sequential infection with different types of the virus. It is characterized by a significant mortality, particularly for individuals with a weakened or undeveloped immune system. The small, 10.7-kbp positive strand RNA genome of the dengue virus encodes only 10 proteins: three structural proteins (capsid, premembrane, and envelope proteins) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 proteins) (3, 6 -8). The entire viral genome is translated as a single polyprotein, subsequently cleaved by viral and cellular proteases into functional entities.The NS5 protein is the viral replicative RNA-dependent RNA polymerase (1, 3, 6 -8). The primary structure of the fulllength polymerase encompasses 900 amino acids (i.e. it is the largest of the nonstructural proteins of the dengue virus) (6 -10). The enzyme is built of two functional domains (6 -10). The N-terminal domain includes the first 263 amino acids. It functions as a methyltransferase (MTase), whose activity is necessary for viral RNA recognition by the host cell translational apparatus. Correspondingly, amino acids ϳ273-906 form the polymerase domain, containing the active site of the RNA synthesis (Fig. 1a) (6 -8). The crystal structures of the isolated N-terminal and polymerase domains have been determined up t...

Interactions between the Dengue Virus Polymerase NS5 and Stem-Loop A

“…3B). This result is in contrast to the NS5-ssRNA interaction, where the release of ϳ6 Na ϩ ions during complex formation was observed (29). The weaker effect of NaCl observed for the NS5-SLA interaction indicates that the binding-site size (the numbers of nucleotides and amino acids involved in interactions) for SLA is smaller than that for ssRNA (Fig.…”

“…The dependence of the logarithm of the intrinsic binding constant on the logarithm of [NaCl] (log-log plot) is shown in Fig. 3B (29,31). This plot is linear in the studied salt concentration range, and the slope, ѨlogK/Ѩlog[NaCl], is Ϫ1.3, which indicates that the release of ϳ1 Na ϩ ion accompanies the intrinsic interactions between NS5 and SLA 80 (31).…”

“…Additionally, the NS5-SLA 80 interaction is not influenced by temperature (Fig. 4), while the binding of NS5 and ssRNA is affected (29). The fact that the association reaction between NS5 and SLA 80 is driven by entropy (Fig.…”

“…2, blue circles). Next, we tested how the magnesium concentration affects the formation of the NS5-SLA 80 complex, since previous binding studies of NS5 and ssRNA showed that magnesium affects the affinity of NS5 for ssRNA (29). Fluorescence titrations of SLA 80 -F with NS5 in buffer B1 containing 0, 0.4, 1.0, 1.3, and 2.0 mM MgCl 2 at 10°C are shown in Fig.…”

“…Despite the importance of SLA in initiating dengue virus RNA replication, interactions between NS5 and SLA have not been quantitatively analyzed, and thus, fundamental aspects of the NS5-SLA interaction, such as stoichiometry and energetics of intrinsic affinities, are unknown. We previously analyzed NS5 interactions with ssRNA and double-stranded RNA (dsRNA), as well as ssDNA and dsDNA, using fluorescence titration techniques (29). Here, we report studies of the interaction between NS5 and SLA and describe the effects of salt, magnesium, and temperature on NS5-SLA binding affinity.…”