Functional Analysis of Two Cavities in Flavivirus NS5 Polymerase

Abstract

Flavivirus NS5 protein encodes methyltransferase and RNAdependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.The genus Flavivirus from the family Flaviviridae contains more than 70 viruses, many of which are important human pathogens causing major public health threats worldwide. Dengue virus (DENV) 2 is a mosquito-borne flavivirus responsible for 50 -100 million human infections and ϳ20,000 deaths each year (1). Besides DENV, West Nile virus (WNV), Japanese encephalitis virus, yellow fever virus, and tick-borne encephalitis virus also cause significant human diseases (1). No antiviral therapy is currently available for treatment of flavivirus infections. Therefore, development of antiviral therapy is urgently needed for flaviviruses.The flavivirus genome is a single strand, plus-sense RNA of about 11 kb in length. The genomic RNA contains a 5Ј-untranslated region (UTR), a single open reading frame, and a 3Ј-UTR. The single open reading frame encodes a long polyprotein that is processed by viral and host proteases into 10 mature viral proteins (2). Three structural proteins (capsid, pre-membrane, and envelope) are primarily involved in virus particle formation, and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are mainly responsible for viral replication (2). Among these, NS3 and NS5 possess enzymatic activities and have been targeted for antiviral development. NS3 functions as a protease (with NS2B as a cofactor), helicase, 5Ј-RNA triphosphatase, and nucleoside triphosphatase (3-5). NS5 is the largest and the most conserved viral protein. The N-terminal part of NS5 is a methyltransferase that methylates the N-7 and 2Ј-O positions of the viral RNA cap structure (6 -9);...

The C-terminal 50 Amino Acid Residues of Dengue NS3 Protein Are Important for NS3-NS5 Interaction and Viral Replication

“…1C). The apparent K d values of the NS3-NS5 interaction for NS3(172-618), GST-NS3(482-618), and -(566 -618) were found to be comparable (5.33 Ϯ 0.35, 10.18 Ϯ 0.91, and 10.25 Ϯ 1.14 M (mean Ϯ S.D., n ϭ 3) respectively) and also similar to the K d values for NS3(172-618) as in previous reports (33,37). Next, we also determined the IC 50 value for NS3(172-618), which was 6.07 Ϯ 2.60 M. The IC 50 values for GST-NS3(482-618) and GST-NS3(566 -618) were not determined as we were unable to reach 100% inhibition due to limited availability of truncated protein.…”

“…Because of the potential interdependence of the two proteins in orchestrating viral genome replication in the RC, the interaction between NS3 and NS5 has been proposed as a promising new target (28,29,37,71). It was previously demonstrated by the Y2H study that NS3(303-618) of the helicase domain interacts with NS5(320 -368) of the RdRP domain.…”

“…6B). Interestingly, our detailed analysis suggests that NS5:K330A mutant cannot synthesize negative-strand RNA, although the mutation has been shown to have no effect on the in vitro enzymatic activities (37). These results suggest that the impairment of infectious virus production and viral protein synthesis in the NS3:N570A mutant may be due to the inability of the mutant NS3 to engage with NS5 and use the dsRNA template to produce more positive-strand genomic RNA that can be translated and processed.…”

“…The NS5-binding site appears to be centered at residue Lys-330 because the mutation to alanine disrupted its interaction with NS3 and abolished RNA replication, although the in vitro RdRP activity was unaffected (37). Based on available crystal structures of the RdRP domain of NS5, it has been proposed that the cavity occupied by Lys-330 may be a potential target for antiviral drug design by blocking NS3-NS5 interaction (37). However, the details of the interaction from the NS3 perspective is missing to fully exploit structure-guided drug design.…”

“…There have been several reports of NS3-NS5 interactions that include biochemical pulldown assays from infected cell extracts (28,(32)(33)(34) and two-hybrid (Y2H) studies that mapped the interaction to the C-terminal region of NS3 helicase (residues 303-618) and the N-terminal region of NS5 RdRP (residues 320 -368; known as bNLS (nuclear localization sequence)) (35,36). The NS5-binding site appears to be centered at residue Lys-330 because the mutation to alanine disrupted its interaction with NS3 and abolished RNA replication, although the in vitro RdRP activity was unaffected (37). Based on available crystal structures of the RdRP domain of NS5, it has been proposed that the cavity occupied by Lys-330 may be a potential target for antiviral drug design by blocking NS3-NS5 interaction (37).…”