Localization and Functions of Japanese Encephalitis Virus Nonstructural Proteins NS3 and NS5 for Viral RNA Synthesis in the Infected Cells

Edward, Zulkarnain; Takegami, Tsutomu

doi:10.1111/j.1348-0421.1993.tb03206.x

Cited by 37 publications

(27 citation statements)

References 27 publications

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“…The flavivirus NS5 gene is thought to encode a protein with RNA-dependent RNA polymerase and methyltransferase activity (Bartholomeusz & Wright, 1993;Edward & Takegami, 1993;Koonin, 1993). Antibodies *Author for correspondence.…”

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confidence: 99%

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Nucleotide sequence of the NS5 gene of Banzi virus: comparison with other flaviviruses

Fulop

Barrett

Titball

1995

Journal of General Virology

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confidence: 99%

“…against the NS5 protein inhibit RNA synthesis /n vitro (Edward & Takegami, 1993). The NS5 protein is therefore thought to be important in virus replication.…”

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confidence: 99%

Nucleotide sequence of the NS5 gene of Banzi virus: comparison with other flaviviruses

Fulop

Barrett

Titball

1995

Journal of General Virology

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confidence: 99%

“…Phosphorylation of flavivirus NS5/NS5A proteins occurs at serine/threonine residues (26, 27). YFV NS5 is also phosphorylated and localized in the nucleus (28), whereas NS5 of other flaviviruses such as Japanese encephalitis virus and Kunjin virus have not been detected in the nucleus (8,29).Transport to the nucleus of proteins that are larger than 45 kDa requires intrinsic targeting signals called nuclear localization sequences (NLSs) (30 -33). There are two main classes of basic type NLSs as follows: the simian virus 40 (SV40) large tumor antigen (T-ag) monopartite NLS type, consisting of a single cluster of basic amino acids (PKKKRKV) (34); and bipartite NLSs comprising two clusters of basic amino acids separated by a spacer of 10 -12 amino acids (35).…”

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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

Brooks¹,

Johansson²,

John³

et al. 2002

Journal of Biological Chemistry

128

130

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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...

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“…Therefore, its role in transcription and viral RNA replication is not clear. The majority of the flavivirus RNA-dependent RNA polymerase activity has been shown to be associated with the endoplasmic reticulum membrane fraction (22,32). It has been suggested that EF-1␣ attaches to the endoplasmic reticulum membrane via a posttranslational modification.…”

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confidence: 99%

Mov34 Protein from Mouse Brain Interacts with the 3′ Noncoding Region of Japanese Encephalitis Virus

Vrati

2000

J Virol

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The plus-sense RNA genome of Japanese encephalitis virus (JEV) contains noncoding regions (NCRs) of 95 and 585 bases at its 5 and 3 ends, respectively. The last 83 nucleotides of the 3-NCR are predicted to form stable stem-loop (SL) structures. The shape of this 3-SL structure is highly conserved among divergent flaviviruses even though only small stretches of nucleotide sequence contained within these structures are conserved. These SL structures have been predicted to function as cis-acting signals for RNA replication and as such may bind to viral and cellular proteins that may be involved in viral replication. We have studied the interaction of the JEV 3-NCR RNA with host proteins using gel retardation assays. We show that the JEV 3-SL structure RNA forms three complexes with proteins from the S100 cytoplasmic extract prepared from the neonatal mouse brain. These complexes could be obtained Japanese encephalitis virus (JEV) is a flavivirus with a plussense, single-stranded RNA genome of ϳ11 kb. The genomic RNA contains a single open reading frame capable of encoding a polyprotein of ϳ3,400 amino acids which is subsequently cleaved into three structural (capsid, pre-M, and envelope) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. The coding region in the genome is flanked by 5Ј and 3Ј noncoding regions (NCRs) of 95 and 585 bases (63). The size and sequence of the 3Ј-NCR vary among different flaviviruses although its secondary structure comprising stable stem-loop (SL) formations is predicted to be highly conserved. The last 80 to 90 bases at the extreme 3Ј end of the genome have been predicted to form stable SL structures in different flaviviruses (10,11,63,65). There are only small stretches of nucleotide sequences, mostly located in the loop regions of these SL structures, that are conserved among flaviviruses (10). Conservation of the location and the shape of these SL structures in the 3Ј-NCR of flaviviruses indicates that they may have some functional significance.During the course of flavivirus replication, a minus-sense RNA is synthesized from the plus-sense genomic RNA. This intermediate form of RNA serves as template for the synthesis of the plus-sense genomic RNA which is synthesized to 10-to 100-fold-higher levels than the minus-sense RNA (12, 62). Both the nucleotide sequence and the structural elements of the 3Ј-NCR such as the 3Ј-SL structure are predicted to contain cis-acting signals for the initiation of the viral RNA transcription, and thus, they may specifically interact with viral or cellular proteins during viral RNA replication (12,71).A number of host proteins have been shown to be associated with the RNA-dependent RNA polymerase of phage Q␤ (9), brome mosaic virus (53), cucumber mosaic virus (34), tobacco mosaic virus (48), vesicular stomatitis virus (19), measles virus (44), influenza virus (46), and poliovirus (33, 43). Moreover, a variety of host proteins has been shown to interact with putative cis-acting elements of Sindbis virus (50), mouse hepatitis ...

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Localization and Functions of Japanese Encephalitis Virus Nonstructural Proteins NS3 and NS5 for Viral RNA Synthesis in the Infected Cells

Cited by 37 publications

References 27 publications

Nucleotide sequence of the NS5 gene of Banzi virus: comparison with other flaviviruses

Nucleotide sequence of the NS5 gene of Banzi virus: comparison with other flaviviruses

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

Mov34 Protein from Mouse Brain Interacts with the 3′ Noncoding Region of Japanese Encephalitis Virus

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