Structure of the Flavivirus Helicase: Implications for Catalytic Activity, Protein Interactions, and Proteolytic Processing

Abstract

Yellow fever virus (YFV), a member of the Flavivirus genus, has a plus-sense RNA genome encoding a single polyprotein. Viral protein NS3 includes a protease and a helicase that are essential to virus replication and to RNA capping. The 1.8-Å crystal structure of the helicase region of the YFV NS3 protein includes residues 187 to 623. Two familiar helicase domains bind nucleotide in a triphosphate pocket without base recognition, providing a site for nonspecific hydrolysis of nucleoside triphosphates and RNA triphosphate. The third, C-terminal domain has a unique structure and is proposed to function in RNA and protein recognition. The organization of the three domains indicates that cleavage of the viral polyprotein NS3-NS4A junction occurs in trans.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 Ϸ11-kb plus-sense RNA containing a 5Ј cap (m 7 G5Јppp5ЈA) but lacking a 3Ј poly(A) tail (16, 38). 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 Ϸ175 residues of the NS3 protein and a cofactor peptide within the NS2B protein (2,15,16,34,54).The C-terminal 440 amino acids of the NS3 protein constitute a helicase region, based on sequence analysis (30). The precise ...

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

“…1A) was previously shown by Y2H study to interact with NS5(320 -368) (bNLS) (36) of the RdRP domain (residues 273-900) (35,36), the amino acid residues of NS3 helicase that are responsible for binding to NS5 RdRP have not been pinpointed. However, it was hypothesized that helicase subdomain III, NS3(482-618), contains the interaction site (13,22). To test this, we first established that the binding of NS2B 18 NS3 to coated NS5 RdRP increased in a dose-dependent manner as described previously (33) and carried out the NS3-NS5 interaction assay in a competitive ELISA format, with an increasing amount of the following competing proteins, namely NS3(172-618) , GST-NS3(482-618) (subdomain III, or GST-NS3(566 -618) (Fig.…”

“…NS3 contains an N-terminal serine protease domain (residues 1-170) that requires NS2B to be an active protease (4 -8). Its C-terminal domain contains ATPase/helicase activity for unwinding of the double-stranded RNA (dsRNA) intermediate (9 -14) and RNA 5Ј-triphosphatase activity for viral RNA 5Ј-capping reaction that is carried out together with the N-terminal domain of NS5, which has methyltransferase activity (3,9,13,(15)(16)(17)(18)(19)(20)(21)(22). The C-terminal domain of NS5 has RNA-dependent RNA polymerase (RdRP) activity, which is crucial for RNA replication (19,(23)(24)(25)(26).…”

Functional Analysis of Two Cavities in Flavivirus NS5 Polymerase

“…Because the NS3/NS5 interaction was previously mapped to the helicase domain of NS3 (30,31), we performed SPR assays using recombinant helicase domain (without protease domain) and various NS5 proteins. The results showed that the K D value of helicase/NS5 was 4.4-fold higher than that of the full-length NS3/NS5 (Fig.…”

Rapid fixation of a distinctive sequence motif in the 3′ noncoding region of the clade of West Nile virus invading North America

“…This change (I→T at residue 356 of the NS3 protein) may have functional importance. The NS3 protein has RNA helicase activity, and the three-dimensional structure of the homologous helicase from another flavivirus, Yellow fever virus (YFV), has been determined (Wu et al 2005). By analogy with YFV, residue 356 is located near the beginning of a β-strand that includes the initial two residues of a sequence motif (Motif IV) believed to play a role in translocating RNA through the helicase (Wu et al 2005).…”