The Interdomain Region of Dengue NS5 Protein That Binds to the Viral Helicase NS3 Contains Independently Functional Importin β1 and Importin α/β-Recognized Nuclear Localization Signals

Abstract

Dengue virus NS5 protein is a multifunctional RNAdependent RNA polymerase that is essential for virus replication. We have shown previously that the 37-amino acid interdomain spacer sequence (residues 369 X 2 KKX 14 KKKX 11 RKX 3 405 ) of Dengue2 NS5 contains a functional nuclear localization signal (NLS). In this study, ␤-galactosidase fusion proteins carrying point mutations of the positively charged residues or truncations of the interdomain linker region (residues 369 -389 or residues 386 -405) were analyzed for nuclear import and importin binding activities to show that the N-terminal part of the linker region (residues 369 -389, a/bNLS) is critical for nuclear localization and is recognized with high affinity by the conventional NLS-binding importin ␣/␤ heterodimeric nuclear import receptor. We also show that the importin ␤-binding site (residues 320 -368, bNLS) adjacent to the a/bNLS, previously identified by yeast two-hybrid analysis, is functional as an NLS, recognized with high affinity by importin ␤, and able to target ␤-galactosidase to the nucleus. Intriguingly, the bNLS is highly conserved among Dengue and related flaviviruses, implying a general role for the region and importin ␤ in the infectious cycle.Dengue virus is a member of the genus Flavivirus within the family Flaviviridae that also contains the genera Pestivirus and Hepacivirus. There are around 70 viruses grouped in the Flavivirus genus, which includes yellow fever virus (YFV), 1 Japanese encephalitis virus, Murray Valley encephalitis virus, Kunjin virus, and tick-borne encephalitis virus. Dengue virus causes a benign syndrome known as Dengue fever and a more severe illness, Dengue hemorrhagic fever or in its severest form Dengue shock syndrome (1-3). There are four serologically and phylogenetically distinguishable Dengue viruses (types 1-4), and the disease they cause is of substantial world wide significance to human health but is mostly restricted to tropical and sub-tropical areas because of its transmission by the Aedes aegypti mosquito (4).Flaviviruses possess a single-strand, positive-sense RNA genome of around 11 kb, which is capped but not polyadenylylated, and encodes a single polyprotein including three structural and seven non-structural proteins in the order C-prM-E-NS1-NS2A-NS3-NS2B-NS2A-NS4B-NS5 (5, 6). Replication of flaviviruses occurs at membrane-associated replicase complexes localized in the perinuclear region. The replicase complex has been extensively characterized in several flaviviruses and includes NS1, NS2A, NS3, NS4A, and NS5 (7-12) and possibly some cellular proteins (13-15). Whereas the protein and RNA interactions of the replicase complexes still require detailed characterization, several recent studies (7, 17) have focused on the structure and function of the NS3 and NS5 proteins.NS3 (69 kDa) is a multifunctional protein that has been shown to have protease, helicase, NTPase, and 5Ј-terminal RNA triphosphatase activities (16 -19). NS5 (104 kDa) contains a well characterized RNA-dependent RNA polymerase ac...

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

“…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. Subsequently, based on available crystal structures, subdomain III of NS3 helicase (residue 483-618) was suggested to be involved in the interaction with NS5 because it has a large protein surface area, located away from the region of main catalytic active sites of NS3 (13,22,36). The NS5 residue Lys-330, which is located on the surface of NS5 thumb subdomain, has been identified as a critical residue in interacting with NS3 helicase (37).…”

“…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).…”

“…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).…”

Nuclear Localization of Dengue Virus Nonstructural Protein 5 Does Not Strictly Correlate with Efficient Viral RNA Replication and Inhibition of Type I Interferon Signaling

“…Two functional nuclear localization signals (NLSs), designated the ␤NLS and the ␣␤NLS, have been identified in DENV NS5. The latter plays the major role in nuclear translocation and viral RNA replication (17)(18)(19)(20). However, the crystal structure of the DENV NS5 polymerase domain (12) suggests that the two NLSs might be integral parts of the NS5 polymerase domain, raising the possibility that the reduced replication of viruses containing mutations in the ␣␤NLS might be due to impairment of their RdRp activity.…”

Comparative Analysis of Full-Length Genomic Sequences of 10 Dengue Serotype 1 Viruses Associated with Different Genotypes, Epidemics, and Disease Severity Isolated in Thailand over 22 Years

“…The first NLS (βNLS) is located between residues 320 and 368 and is responsible for binding cellular factor β1-importin and facilitating protein transport to the nucleus. 46,47 The second NLS (α/βNLS) spans residues 369-405 and is responsible for binding cellular factor α/β-importin. 48 The specific role of nuclear-localized NS5 in the viral life cycle appears to be, in part, to antagonize the antiviral response, including up-regulated modulation of interleukin-8 production by infected cells.…”