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Non-structural protein-1 is required for West Nile virus replication complex formation and viral RNA synthesis.

Youn S, Ambrose RL, Mackenzie JM, Diamond MS - Virol. J. (2013)

Bottom Line: Despite its transit through the secretory pathway and intracellular localization in the lumen of the endoplasmic reticulum and Golgi vesicles, NS1 is as an essential gene for flavivirus replication.In viral lifecycle experiments, we demonstrate that WNV NS1 was not required for virus attachment or input strand translation of the infectious viral RNA, but was necessary for negative and positive strand RNA synthesis and formation of the endoplasmic reticulum-associated replication complex.These results expand on prior studies with yellow fever and Kunjin viruses to show that flavivirus NS1 has an essential co-factor role in regulating replication complex formation and viral RNA synthesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, Saint Louis, MO 63110, USA. diamond@borcim.wustl.edu.

ABSTRACT

Background: Flavivirus NS1 is a non-structural glycoprotein that is expressed on the cell surface and secreted into the extracellular space, where it acts as an antagonist of complement pathway activation. Despite its transit through the secretory pathway and intracellular localization in the lumen of the endoplasmic reticulum and Golgi vesicles, NS1 is as an essential gene for flavivirus replication. How NS1 modulates infection remains uncertain given that the viral RNA replication complex localizes to the cytosolic face of the endoplasmic reticulum.

Methods and results: Using a trans-complementation assay, we show that viruses deleted for NS1 (∆-NS1) can be rescued by transgenic expression of NS1 from West Nile virus (WNV) or heterologous flaviviruses in the absence of adaptive mutations. In viral lifecycle experiments, we demonstrate that WNV NS1 was not required for virus attachment or input strand translation of the infectious viral RNA, but was necessary for negative and positive strand RNA synthesis and formation of the endoplasmic reticulum-associated replication complex.

Conclusions: WNV RNA lacking intact NS1 genes was efficiently translated but failed to form canonical replication complexes at early times after infection, which resulted in an inability to replicate viral RNA. These results expand on prior studies with yellow fever and Kunjin viruses to show that flavivirus NS1 has an essential co-factor role in regulating replication complex formation and viral RNA synthesis.

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Trans-complementation of ∆-NS1-WNV with ectopically expressed WNV NS1. A. Scheme for construction of ∆-NS1-WNV. Nucleotides 87 to 928 of the NS1 gene were deleted after restriction digest with the BstEII enzyme. B. Recovery of WNV RNA in supernatant after transfection of ∆-NS1-WNV or WNV-WT RNA into BHK21 or BHK21-VEEV-WNV NS1 cells. 12 hours post transfection, supernatant was harvested and assessed for levels of viral RNA by qRT-PCR of the E gene. The results are the average of two independent experiments performed in triplicate. C-D. Immunofluorescence (C) or flow cytometry (D) staining of WNV E and NS5 antigen after mock infection or infection of ∆-NS1-WNV or WNV-WT in BHK21 or BHK21-VEEV-WNV NS1 cells. Cells were infected at an MOI of 5, and at 26 hr post infection, cells were fixed, permeabilized, and stained with anti-E (red), anti-NS5 (green), or a nuclear stain (blue). Results are representative of several independent experiments. E. Single-step growth kinetics of ∆-NS1-WNV or WNV-WT in BHK21 or BHK21-VEEV-WNV NS1 cells. Cells were infected at an MOI of 5 and supernatants were titrated on BHK21-VEEV-WNV NS1 cells by focus-forming assay. The dashed line indicates the limit of detection of the assay. Results are the average of several independent experiments performed in triplicate. Error bards indicate standard deviations.
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Figure 1: Trans-complementation of ∆-NS1-WNV with ectopically expressed WNV NS1. A. Scheme for construction of ∆-NS1-WNV. Nucleotides 87 to 928 of the NS1 gene were deleted after restriction digest with the BstEII enzyme. B. Recovery of WNV RNA in supernatant after transfection of ∆-NS1-WNV or WNV-WT RNA into BHK21 or BHK21-VEEV-WNV NS1 cells. 12 hours post transfection, supernatant was harvested and assessed for levels of viral RNA by qRT-PCR of the E gene. The results are the average of two independent experiments performed in triplicate. C-D. Immunofluorescence (C) or flow cytometry (D) staining of WNV E and NS5 antigen after mock infection or infection of ∆-NS1-WNV or WNV-WT in BHK21 or BHK21-VEEV-WNV NS1 cells. Cells were infected at an MOI of 5, and at 26 hr post infection, cells were fixed, permeabilized, and stained with anti-E (red), anti-NS5 (green), or a nuclear stain (blue). Results are representative of several independent experiments. E. Single-step growth kinetics of ∆-NS1-WNV or WNV-WT in BHK21 or BHK21-VEEV-WNV NS1 cells. Cells were infected at an MOI of 5 and supernatants were titrated on BHK21-VEEV-WNV NS1 cells by focus-forming assay. The dashed line indicates the limit of detection of the assay. Results are the average of several independent experiments performed in triplicate. Error bards indicate standard deviations.

Mentions: Prior studies showed that deletion of NS1 from infectious cDNA clones of Kunjin (KUNV) [25] or yellow fever (YFV) [22] flaviviruses impaired virus replication, which suggested that NS1 was an essential gene for infection. These studies also demonstrated that KUNV and YFV lacking NS1 could be complemented in trans by ectopic expression of the homologous NS1. To assess the role of NS1 in infection of a North American strain of WNV (WNV-New York 1999), we generated an infectious cDNA clone (∆-NS1-WNV) that deleted 840 nucleotides in-frame (from 87 to 928) of the NS1 gene (Figure 1A and see Methods); this left a small fragment of NS1 consisting of the 86 N-terminal and 201 C-terminal nucleotides.


Non-structural protein-1 is required for West Nile virus replication complex formation and viral RNA synthesis.

Youn S, Ambrose RL, Mackenzie JM, Diamond MS - Virol. J. (2013)

Trans-complementation of ∆-NS1-WNV with ectopically expressed WNV NS1. A. Scheme for construction of ∆-NS1-WNV. Nucleotides 87 to 928 of the NS1 gene were deleted after restriction digest with the BstEII enzyme. B. Recovery of WNV RNA in supernatant after transfection of ∆-NS1-WNV or WNV-WT RNA into BHK21 or BHK21-VEEV-WNV NS1 cells. 12 hours post transfection, supernatant was harvested and assessed for levels of viral RNA by qRT-PCR of the E gene. The results are the average of two independent experiments performed in triplicate. C-D. Immunofluorescence (C) or flow cytometry (D) staining of WNV E and NS5 antigen after mock infection or infection of ∆-NS1-WNV or WNV-WT in BHK21 or BHK21-VEEV-WNV NS1 cells. Cells were infected at an MOI of 5, and at 26 hr post infection, cells were fixed, permeabilized, and stained with anti-E (red), anti-NS5 (green), or a nuclear stain (blue). Results are representative of several independent experiments. E. Single-step growth kinetics of ∆-NS1-WNV or WNV-WT in BHK21 or BHK21-VEEV-WNV NS1 cells. Cells were infected at an MOI of 5 and supernatants were titrated on BHK21-VEEV-WNV NS1 cells by focus-forming assay. The dashed line indicates the limit of detection of the assay. Results are the average of several independent experiments performed in triplicate. Error bards indicate standard deviations.
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Figure 1: Trans-complementation of ∆-NS1-WNV with ectopically expressed WNV NS1. A. Scheme for construction of ∆-NS1-WNV. Nucleotides 87 to 928 of the NS1 gene were deleted after restriction digest with the BstEII enzyme. B. Recovery of WNV RNA in supernatant after transfection of ∆-NS1-WNV or WNV-WT RNA into BHK21 or BHK21-VEEV-WNV NS1 cells. 12 hours post transfection, supernatant was harvested and assessed for levels of viral RNA by qRT-PCR of the E gene. The results are the average of two independent experiments performed in triplicate. C-D. Immunofluorescence (C) or flow cytometry (D) staining of WNV E and NS5 antigen after mock infection or infection of ∆-NS1-WNV or WNV-WT in BHK21 or BHK21-VEEV-WNV NS1 cells. Cells were infected at an MOI of 5, and at 26 hr post infection, cells were fixed, permeabilized, and stained with anti-E (red), anti-NS5 (green), or a nuclear stain (blue). Results are representative of several independent experiments. E. Single-step growth kinetics of ∆-NS1-WNV or WNV-WT in BHK21 or BHK21-VEEV-WNV NS1 cells. Cells were infected at an MOI of 5 and supernatants were titrated on BHK21-VEEV-WNV NS1 cells by focus-forming assay. The dashed line indicates the limit of detection of the assay. Results are the average of several independent experiments performed in triplicate. Error bards indicate standard deviations.
Mentions: Prior studies showed that deletion of NS1 from infectious cDNA clones of Kunjin (KUNV) [25] or yellow fever (YFV) [22] flaviviruses impaired virus replication, which suggested that NS1 was an essential gene for infection. These studies also demonstrated that KUNV and YFV lacking NS1 could be complemented in trans by ectopic expression of the homologous NS1. To assess the role of NS1 in infection of a North American strain of WNV (WNV-New York 1999), we generated an infectious cDNA clone (∆-NS1-WNV) that deleted 840 nucleotides in-frame (from 87 to 928) of the NS1 gene (Figure 1A and see Methods); this left a small fragment of NS1 consisting of the 86 N-terminal and 201 C-terminal nucleotides.

Bottom Line: Despite its transit through the secretory pathway and intracellular localization in the lumen of the endoplasmic reticulum and Golgi vesicles, NS1 is as an essential gene for flavivirus replication.In viral lifecycle experiments, we demonstrate that WNV NS1 was not required for virus attachment or input strand translation of the infectious viral RNA, but was necessary for negative and positive strand RNA synthesis and formation of the endoplasmic reticulum-associated replication complex.These results expand on prior studies with yellow fever and Kunjin viruses to show that flavivirus NS1 has an essential co-factor role in regulating replication complex formation and viral RNA synthesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, Saint Louis, MO 63110, USA. diamond@borcim.wustl.edu.

ABSTRACT

Background: Flavivirus NS1 is a non-structural glycoprotein that is expressed on the cell surface and secreted into the extracellular space, where it acts as an antagonist of complement pathway activation. Despite its transit through the secretory pathway and intracellular localization in the lumen of the endoplasmic reticulum and Golgi vesicles, NS1 is as an essential gene for flavivirus replication. How NS1 modulates infection remains uncertain given that the viral RNA replication complex localizes to the cytosolic face of the endoplasmic reticulum.

Methods and results: Using a trans-complementation assay, we show that viruses deleted for NS1 (∆-NS1) can be rescued by transgenic expression of NS1 from West Nile virus (WNV) or heterologous flaviviruses in the absence of adaptive mutations. In viral lifecycle experiments, we demonstrate that WNV NS1 was not required for virus attachment or input strand translation of the infectious viral RNA, but was necessary for negative and positive strand RNA synthesis and formation of the endoplasmic reticulum-associated replication complex.

Conclusions: WNV RNA lacking intact NS1 genes was efficiently translated but failed to form canonical replication complexes at early times after infection, which resulted in an inability to replicate viral RNA. These results expand on prior studies with yellow fever and Kunjin viruses to show that flavivirus NS1 has an essential co-factor role in regulating replication complex formation and viral RNA synthesis.

Show MeSH
Related in: MedlinePlus