PMC

Inhibition of NDV-induced nuclear accumulation of GFP-mIRF3 in U3A cells by transient transfection of the influenza virus NS1 protein. Coverslips coated with U3A fibroblasts were transfected with pGFP-mIRF3 plus pCAGGS-NS1(SAM) or an empty vector (pCAGGS). At 24 h posttransfection, cells were infected with NDV at an MOI of 2 for 7 h. Washed cells were then fixed and examined for GFP–mIRF-3 distribution. Fluorescence of GFP–mIRF-3 in NDV infected cells ± NS1 is shown.

Differential induction of IFN-β mRNA in response to either PR8 or delNS1 virus infection. Confluent 100-mm dishes of HEC-1b cells were infected with either NDV, PR8 virus, or delNS1 virus at an MOI of 1 for 14 h. One dish of mock (PBS)-infected cells was included as a negative control (−). At 14 h postinfection, cells were washed twice with cold PBS, and total RNA was subjected to RT-PCR analysis using specific primers for IFN-β or GAPDH. As a control for genomic DNA contamination, PCR was carried out with GAPDH primers using the mock (−RT) reactions as a template. Following PAGE, products were detected by autoradiography.

Nuclear accumulation of hIRF-3 in delNS1 virus-infected cells. (A) Coverslips coated with Hec-1b cells were mock infected or infected with delNS1 or wild-type influenza A virus (PR8) at an MOI of 1. At 8 h postinfection, fixed cells were stained with a monoclonal antibody directed against human IRF-3 (SL-12). (B) HEC-1b cells were infected with delNS1 or PR8 virus for 2, 4, or 8 h. At the given time points, fixed cells were stained with SL-12 and scored according to subcellular distribution of hIRF-3. (C) 293T cells were transfected with pCAGGS-hIRF-3 and infected 24 h later with either Sendai, PR8, or delNS1 virus or were mock infected. At 16 h postinfection, total cell lysates were probed with α-IRF-3 as indicated in Materials and Methods.

Model for the mechanism of inhibition of IFN-α/β induction by the influenza virus NS1 protein. (A) In the absence of the NS1 protein, dsRNA generated during the course of virus infection promotes the phosphorylation and nuclear accumulation of IRF-3 by a mechanism which has not yet been precisely defined. Once inside the nucleus, IRF-3 associates with other transcription factors and initiates the induction of the antiviral state by upregulating the transcription of cellular defense genes, including IFN-β. (B) In the presence of NS1, dsRNA generated during viral infection is sequestered (in the nucleus and/or in the cytoplasm) and is unable to induce the nuclear accumulation of IRF-3. The transcription of IFN-β mRNA is prevented, and the virus is able to avert the antiviral state induced by the IFN cascade.

Effect on delNS1 virus-induced nuclear accumulation of hIRF-3 in HEC-1b cells by transient transfection of influenza virus NS1 proteins. (A) Coverslips coated with HEC-1b cells were transfected with a plasmid expressing the wild-type influenza virus NS1 protein [pCAGGS-NS1(SAM)] or an NS1 RNA binding mutant [pCAGGS-NS1-R38AK41A(SAM)] (mut) or with an empty vector [pCAGGS]. Both NS1 open reading frames contain a splice acceptor mutation (SAM) to ensure that spliced NEP mRNA is not produced. (The NEP is a second protein normally produced by alternative splicing from the influenza A virus NS gene.) Except with PR8-infected cells and with the no-DNA control, the amount of DNA in all transfected cells was standardized to 0.5 μg per coverslip using pCAGGS. Coverslips were transfected with either 0, 0.02, 0.1, or 0.5 μg of pCAGGS-NS1(SAM) or with 0.5 μg of pCAGGS-NS1-R38AK41A(SAM) and infected with delNS1 virus at an MOI of 1 for 8 h. Fixed cells were stained with antibodies directed against the NS1 protein and endogenous hIRF-3. Cells were scored according to NS1 expression and hIRF-3 distribution. Bars represent the mean cell number from between 5 and 13 random fields per experimental condition. Asterisks indicate that there was no NS1 expression. (B) HEC-1b cells were transfected with 0.1 μg of pCAGGS-NS1(SAM) and infected with delNS1 virus as indicated for panel A. Cells were stained with a rabbit polyclonal antibody against NS1 and a mouse monoclonal antibody against hIRF-3. Anti-rabbit IgG (fluorescein isothiocyanate) and anti-mouse IgG (Texas Red) were used as secondary antibodies. In the top panel, arrows indicate two cells expressing the NS1 protein (green). The surrounding cells do not express detectable levels of NS1. In the bottom panel, arrows indicate the same two cells, demonstrating a lack of nuclear accumulation of hIRF-3. Surrounding cells which did not express the NS1 protein show a bright, nuclear accumulation of endogenous IRF-3. (C) Cells were transfected with 0.1 μg of pCAGGS-NS1-R38AK41A(SAM) and infected with delNS1 for 8 h as described for panel A. Fixed cells were stained as described above. In the top panel, arrows indicate cells which show expression of the NS1-R38AK41A protein (green). The immunofluorescence staining pattern of NS1-R38AK41A was essentially the same as that of wild-type NS1, with bright nuclear staining. In addition to the nuclear signal, a diffuse cytoplasmic signal could also be detected in some cells expressing NS1-R38AK41A. The bottom panel shows NS1-R38AK41A-expressing cells with bright, nuclear accumulation of hIRF-3 (red). (D) HEC-1b cells were infected for 8 h with PR8 virus at an MOI of 1. Fixed cells were stained as described for panel B. The top panel shows virally expressed NS1 protein (green). The arrows indicate a cell which, when examined for IRF-3 localization (bottom panel, red), showed nuclear accumulation of IRF-3. Most cells are infected and are expressing viral NS1. The majority of PR8-infected cells do not, however, show nuclear accumulation of IRF-3. As indicated by the arrow (bottom panel), only one out of six infected cells in this field showed nuclear accumulation of IRF-3.