1. |
- Johansson, Magnus, et al.
(författare)
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A small region of the dengue virus-encoded RNA-dependent RNA polymerase, NS5, confers interaction with both the nuclear transport receptor importin-beta and the viral helicase, NS3
- 2001
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Ingår i: Journal of General Virology. - : Microbiology Society. - 0022-1317 .- 1465-2099. ; 82:Pt 4, s. 735-745
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Tidskriftsartikel (refereegranskat)abstract
- The dengue virus RNA-dependent RNA polymerase, NS5, and the protease/helicase, NS3, are multidomain proteins that have been shown to interact both in vivo and in vitro. A hyperphosphorylated form of NS5 that does not interact with NS3 has been detected in the nuclei of virus-infected cells, presumably as the result of the action of a functional nuclear localization sequence within the interdomain region of NS5 (residues 369-405). In this study, it is shown by using the yeast two-hybrid system that the C-terminal region of NS3 (residues 303-618) interacts with the N-terminal region of NS5 (residues 320-368). Further, it is shown that this same region of NS5 is also recognized by the cellular nuclear import receptor importin-beta. The interaction between NS5 and importin-beta and competition by NS3 with the latter for the same binding site on NS5 were confirmed by pull-down assays. The direct interaction of importin-beta with NS5 has implications for the mechanism by which this normally cytoplasmic protein may be targetted to the nucleus.
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2. |
- Vasudevan, S G, et al.
(författare)
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Characterisation of inter- and intra-molecular interactions of the dengue virus RNA dependent RNA polymerase as potential drug targets
- 2001
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Ingår i: Il Farmaco. - : Elsevier BV. - 0014-827X .- 1879-0569. ; 56:1-2, s. 33-36
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Tidskriftsartikel (refereegranskat)abstract
- Our research is directed towards enhancing the understanding of the molecular biology of dengue virus replication with the ultimate goal being to develop novel antiviral strategies based on preventing critical inter- or intra-molecular interactions required for the normal virus life cycle. The viral RNA-dependent RNA polymerase (NS5) and the viral helicase (NS3) interaction offers a possible target for inhibitors to bind and prevent replication. In this study the yeast-two hybrid system was used to show that a small region of NS5 interacts with NS3, and also with the cellular nuclear transport receptor importin-beta. Furthermore, intramolecular interaction between the two putative domains of NS5 can also be detected by the yeast two-hybrid assay. We have also modified the colony lift assay for the beta-galactosidase reporter activity in intact yeast cells which reflects the strength of interaction between two proteins to a microtiter plate format. This assay offers a unique opportunity to screen for small molecule compounds that block physiologically important interactions.
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3. |
- Brooks, Andrew J, et al.
(författare)
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The interdomain region of dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin beta 1 and importin alpha/beta-recognized nuclear localization signals
- 2002
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 277:39, s. 36399-36407
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Tidskriftsartikel (refereegranskat)abstract
- Dengue virus NS5 protein is a multifunctional RNA-dependent 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, beta-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 alpha/beta heterodimeric nuclear import receptor. We also show that the importin beta-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 beta, and able to target beta-galactosidase to the nucleus. Intriguingly, the bNLS is highly conserved among Dengue and related flaviviruses, implying a general role for the region and importin beta in the infectious cycle.
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4. |
- Brooks, Andrew J, et al.
(författare)
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The Interdomain Region of Dengue NS5 ProteinInteracts with NS3 and Host Proteins
- 2002
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Ingår i: Dengue Bulletin. - 1020-895X. ; 26, s. 155-161
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Although dengue virus genome replication occurs in the cytoplasm of infected cells, it has been shown that the NS5 protein (RNA-dependent RNA polymerase) is hyperphosphorylated at a late stage in infection and localized to the cell nucleus. A 37 amino acid sequence of NS5 (residues 369-405) was shown to contain a functional nuclear localization signal (NLS) that interacted with the cellular nuclear transport factor, importin α/β heterodimer. Further studies using the yeast two-hybrid system revealed that the NS5 region (residues 320-368) immediately adjacent to the NLS contained an importin β-binding site that abuts or overlaps the binding site for the NS3 protein (protease/helicase). The importin β-binding site has also been shown to be a functional NLS (bNLS). Intriguingly, when both bNLS and NLS (residues 320-405) were present, the fused β -galactosidase protein did not accumulate in the nucleus. Here we provide a review of our studies on the NS5 interdomain region and compare it to other members of the Flavivirus genus in order to highlight the importance of this region as a possible target for developing broad-acting antiviral agent against dengue and other mechanistically-related viruses.
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5. |
- Vasudevan, Subhash G, et al.
(författare)
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Characterisation of inter- and intra-molecular interactions of the dengue virus RNA dependent RNA polymerase as potential drug targets.
- 2001
-
Ingår i: Il Farmaco. - 0014-827X .- 1879-0569. ; 56:1-2, s. 33-36
-
Tidskriftsartikel (refereegranskat)abstract
- Our research is directed towards enhancing the understanding of the molecular biology of dengue virus replication with the ultimate goal being to develop novel antiviral strategies based on preventing critical inter- or intra-molecular interactions required for the normal virus life cycle. The viral RNA-dependent RNA polymerase (NS5) and the viral helicase (NS3) interaction offers a possible target for inhibitors to bind and prevent replication. In this study the yeast-two hybrid system was used to show that a small region of NS5 interacts with NS3, and also with the cellular nuclear transport receptor importin-beta. Furthermore, intramolecular interaction between the two putative domains of NS5 can also be detected by the yeast two-hybrid assay. We have also modified the colony lift assay for the beta-galactosidase reporter activity in intact yeast cells which reflects the strength of interaction between two proteins to a microtiter plate format. This assay offers a unique opportunity to screen for small molecule compounds that block physiologically important interactions.
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