Flaviviruses are arthropod-borne pathogens that cause a number of serious human diseases throughout the world. Despite their importance as human pathogens, the molecular mechanisms of flavivirus replication are minimally understood, hindering the development of effective antiviral therapies and vaccines. Yellow fever virus (YFV) is the prototype member of the Flavivirus genus. Like many other flaviviruses, YFV is transmitted by mosquitoes. Symptoms of YFV infection include kidney failure, internal bleeding, high fever, and hepatitis, which leads to the yellow coloring of the skin for which the disease is named. Although vaccination using a live attenuated strain has been successful for decades, yellow fever still causes over 30,000 deaths per year, mostly in Africa and South America.
In addition to YFV, the Flavivirus genus also includes West Nile virus, four serotypes of dengue virus, and Japanese encephalitis virus. Flavivirus is the largest genus in the Flaviviradae family, whose other genera are Hepacivirus and Pestivirus. The only characterized hepacivirus is hepatitis C virus (HCV), which is well studied due to its importance in chronic liver disease and in liver failure leading to transplant. Although many molecular details differ between HCV and the flaviviruses and their protein sequences are less than 20% identical, there are sufficient similarities in replication strategy to allow comparisons between the viral proteins.
The flavivirus genome is a ≈11-kb plus-sense RNA containing a 5′ cap (m7
G5′ppp5′A) but lacking a 3′ poly(A) tail (16
). The genome encodes a 370-kDa polyprotein precursor, which is inserted into the membrane of the endoplasmic reticulum and processed to yield three structural proteins (C, M, and E) and seven replication proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (16
). Host proteases process the polyprotein at sites in the endoplasmic reticulum lumen and a viral protease cleaves at specific sites on the cytoplasmic side of the endoplasmic reticulum membrane. The viral serine protease is formed by the N-terminal ≈175 residues of the NS3 protein and a cofactor peptide within the NS2B protein (2
The C-terminal 440 amino acids of the NS3 protein constitute a helicase region, based on sequence analysis (30
). The precise biological functions of the NS3 helicase region are unknown, but it is thought to separate RNA daughter and template strands and to assist replication initiation by unwinding RNA secondary structure in the 3′ nontranslated region (NTR) (12
Helicases catalyze a large number of nucleoside triphosphate (NTP)-dependent nucleic acid strand separation and remodeling reactions. They are classified into three superfamilies and one family based on several conserved motifs (19
). The flavivirus helicase is a member of the DEAH/D box family within helicase superfamily 2. Seven conserved sequence motifs in superfamily 2 helicases, beginning near position 200 in flavivirus NS3, are associated with NTP hydrolysis and nucleic acid binding (11
). Motifs I and II, also known as Walker A and Walker B, respectively (51
), exist in all helicase superfamilies. Within the DEAH/D-box family of superfamily 2, the DEAH and DEAD subgroups are defined according to the sequence of the Walker B motif, which binds divalent cations. According to these rules of classification, flavivirus NS3 is a member of the DEAH subgroup.
Crystal structures have been reported for several superfamily 2 helicases, including the HCV helicase (13
), bacterial RecQ (7
), bacteriophage T4 UvsW (39
), yeast eIF4A (10
), and bacterial UvrB (47
). The helicase motifs form an NTPase active site at the interface of two domains that are common to all helicases. Many helicases also have a third domain. A cleft between the third domain and the two common domains is the binding site for single-stranded nucleic acid. A single strand of DNA has the same position and orientation with respect to the common domains in the structures of helicase-nucleic acid complexes (26
), suggestive of a common mechanism for coupling NTP hydrolysis and strand unwinding. Among many proposed mechanisms, an inchworm in which the single strand is translocated through the cleft by NTPase-associated hinging of the common domains (41
) seems most plausible. This is based on the structure of a helicase-DNA complex that includes regions of both duplex and single-stranded DNA (49
Among helicases of known structure, HCV helicase is most closely related to Flavivirus helicase. However, the YFV and HCV helicase sequences are only 17% identical overall and have especially weak similarity in the C-terminal region corresponding to the HCV helicase third domain. In contrast, helicases from different members of the Flavivirus genus are much more closely related, with 40 to 90% identical sequences.
NTPase activity was demonstrated for flavivirus NS3 and for constructs of the helicase region (8
). Recently, a conserved Q motif upstream of the Walker A motif was reported to be essential for the ATPase activity of DEAD-box helicases (43
). Flavivirus NS3 has no conserved Q motif, although a glutamine residue upstream of Walker A was reported to be essential for ATPase activity of the Powassan flavivirus (18
Little is known about the unwinding mechanism of flavivirus helicases, including the specificity for DNA and/or RNA duplex and a requirement for a 3′ or 5′ overhang. In most cases, only low levels of helicase activity have been reported for recombinant flavivirus helicases (31
), in contrast to Flaviviridae
helicases from HCV and a pestivirus (25
). Association of the flavivirus helicase with other replicase proteins may influence activity. For example, the flavivirus NS3 helicase domain associates with NS5, which contains the RNA-dependent RNA polymerase (12
), and an NS5 requirement for helicase activity has been described (22
). However, high levels of helicase activity in the absence of NS5 or other replicase proteins were reported recently for a recombinant dengue virus helicase (6
We report the cloning, expression, and purification of ATPase-active recombinant NS3 helicases from YFV and West Nile virus, the 1.8-Å crystal structure of the YFV NS3 helicase, and the 2.5-Å structure of its complex with ADP. The structure differs substantially from that of HCV helicase in some regions and provides a basis for further study of the molecular mechanisms of flavivirus replication and for rational development of antiflaviviral compounds.