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Role for the phosphoprotein P subunit of the paramyxovirus polymerase in limiting induction of host cell antiviral responses - PubMed

Role for the phosphoprotein P subunit of the paramyxovirus polymerase in limiting induction of host cell antiviral responses

Patrick J Dillon et al. J Virol. 2007 Oct.

Abstract

Six amino acid substitutions in the shared N-terminal region of the P subunit of the viral polymerase and the accessory V protein convert the noncytopathic paramyxovirus simian virus 5 (SV5), which is a poor inducer of host cell responses, into a P/V mutant (P/V-CPI-) that induces high levels of apoptosis, interferon-beta (IFN-beta), and proinflammatory cytokines. In this study, we addressed the question of whether these new mutant phenotypes are due to the presence of an altered P protein or of an altered V protein or of both proteins. By the use of the P/V-CPI- mutant as a backbone, new mutant viruses were engineered to express the wild-type (WT) V protein (+V-wt) or WT P protein (+P-wt) from an additional gene inserted between the HN and L genes. In human epithelial cell lines, the +V-wt virus showed reduced activation of apoptosis and lower secretion of IFN-beta and proinflammatory cytokines compared to the parental P/V-CPI- virus. The presence of a V protein lacking the C-terminal cysteine-rich domain (corresponding to the SV5 I protein) did not reduce these host cell responses to P/V-CPI- infection. Unexpectedly, the +P-wt virus, which expressed a WT P subunit of the viral polymerase, also induced much lower levels of host cell responses than the parental P/V-CPI- mutant. For both +V-wt and +P-wt viruses, reduced levels of IFN-beta synthesis correlated with reduced IRF-3 dimerization and nuclear localization of IRF-3 and NF-kappaB, suggesting that the WT P and V proteins acted at an early stage in antiviral pathways. Host cell responses induced by the various P/V mutants directly correlated with levels of viral mRNA accumulation but not with steady-state levels of genomic RNA. Our results support the hypothesis that WT P and V proteins limit induction of antiviral responses by controlling the production of key viral inducers. A model is presented for the mechanism by which both the P subunit of the viral polymerase and the V accessory protein contribute to the ability of a paramyxovirus to limit activation of antiviral responses.

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Figures

FIG. 1.
FIG. 1.

(A) Schematic diagram of SV5 viruses used in this study. Genome structures are shown schematically as negative-sense RNA, with an additional gene (encoding GFP, WT V protein, V-Δcys, or WT P protein) inserted between HN and L. Extra 5′-end P or V genes have an altered editing site, as described in Materials and Methods, such that only the P or V protein is expressed. The black box denotes the cys-rich C-terminal region of V protein which is missing in the case of the V-Δcys open reading frame. With the exception of rSV5-GFP, which has a WT P/V gene, all viruses encoded a P/V gene with a substitution of the CPI- shared P/V N-terminal region as described previously (54). le, leader; tr, trailer. (B) Sequence at the SV5 editing site. The nucleotide and amino acid sequence at the SV5 P/V gene-editing site (51) is shown, with underlined nucleotides denoting the sites of four (V mRNA) or six (P mRNA) G residues. Asterisks denote the locations of substitutions used to disrupt RNA editing.

FIG. 2.
FIG. 2.

Protein expression for the +V-wt virus. (A) Analysis of V protein expression. A549 cells were mock infected (M lane) or infected with rSV5-GFP (S lane), P/V-CPI- (C lane), or +V-wt (+V lane) at an MOI of 10. Cell lysates were harvested at 24 h p.i. and analyzed by Western blotting with V5 antibody, which recognizes WT P and V proteins (α−P+V WT; top panel), or a monoclonal antibody which recognizes both WT and CPI- V proteins (α - V; bottom panel). (B) Time course of WT V protein expression. A549 cells were infected with rSV5-GFP or +V-wt at an MOI of 10. Cell lysates were harvested at the indicated h p.i. and analyzed by Western blotting using the V5 antibody. P/V-CPI- infected cell lysates were loaded as an antibody specificity control (lane C).

FIG. 3.
FIG. 3.

The +V-wt virus induces less apoptotic cell death than the parental P/V-CPI- virus. (A) Microscopy of cells infected with +V-wt virus. A549 cells were examined with a ×20 lens at 72 h p.i. with rSV5-GFP, P/V-CPI-, or +V-wt at an MOI of 10. (B) Cell viability. A549 cells were infected with the indicated viruses at an MOI of 10 and analyzed by a cell proliferation assay at 24, 48, and 72 h p.i. Values are expressed as a percentage of mock-infected sample values at the respective times p.i. with error bars denoting standard deviations from the mean. (C) Annexin staining. A549 cells infected with the indicated viruses at an MOI of 5 were harvested at 24, 48, or 72 h p.i. and analyzed by flow cytometry for cell surface annexin V staining. Data are representative of the results of two independent experiments. (D) TUNEL staining. A549 cells were mock infected or infected with the indicated viruses at an MOI of 10. At 48 h p.i., cells were analyzed for DAPI (4′,6′-diamidino-2-phenylindole) staining or TUNEL staining as described in Materials and Methods.

FIG. 4.
FIG. 4.

A V protein which lacks the cys-rich C-terminal domain cannot limit apoptotic cell death induced by the parental P/V-CPI- virus. (A) Analysis of V protein expression. A549 cells were mock infected or infected with rSV5-GFP or +V-Δcys at an MOI of 10. Cell lysates were harvested at 24 h p.i. and analyzed by Western blotting for levels of NP (top panel), WT P and V proteins (α−P+V WT; middle panel), or actin (bottom panel). (B) Microscopy of cells infected with +V-Δcys virus. A549 cells were examined by microscopy at 24, 48, and 72 h after infection with the indicated viruses at an MOI of 10. (C and D) Cell viability and Annexin V staining. A549 cells were infected with the indicated viruses at an MOI of 10 (panel C) or 5 (panel D) and analyzed by an MTS cell proliferation assay (panel C) or for annexin V staining (panel D) as described in the legend to Fig. 3. Data in panel D are representative of the results of two independent experiments.

FIG. 5.
FIG. 5.

The +P-wt virus induces less apoptotic cell death than the parental P/V-CPI- virus. (A and B) Analysis and time course of P protein expression. A549 cells were mock infected (M lane) or infected with rSV5-GFP (S lane), P/V-CPI- (C lane), or +P-wt (+P lane) at an MOI of 10. Cell lysates were harvested at 24 h p.i. (panel A) or at the indicated times p.i. (panel B) and analyzed by Western blotting with V5 antibody, which recognizes WT P and V proteins (α-P+V WT; top panels), or a polyclonal P-specific antibody which recognizes both WT and CPI- P proteins (α - P; bottom panels). (C) Microscopy of cells infected with +P-wt virus. A549 cells were infected with the indicated viruses at an MOI of 10. Cells were examined by microscopy at 24, 48, and 72 h p.i. (D and E) Cell viability and annexin V staining. A549 cells were infected at an MOI of 10 (panel D) or 5 (panel E) with the indicated viruses and analyzed at 24, 48, or 72 h p.i. by an MTS cell proliferation assay (panel D) or for annexin V staining (panel E) as described in the legend to Fig. 3. Data in panel E are representative of the results of two independent experiments.

FIG. 6.
FIG. 6.

The +V-wt and +P-wt viruses induce lower IFN-beta synthesis and IRF-3 activation compared to the parental P/V-CPI- virus. (A) STAT1 levels. A549 cells were infected at an MOI of 10 with the indicated viruses. Cell lysates were prepared at 8 h p.i., and levels of STAT1 and actin were determined by Western blotting. (B) IFN-beta synthesis. A549 cells were mock infected or infected at an MOI of 10 with rSV5-GFP, P/V-CPI-, +V-wt, or +P-wt. At 24 h p.i., media were analyzed by ELISA for levels of IFN-beta. Error bars denote standard deviations from the mean. (C) IRF-3 nuclear translocation. A549 cells were infected at an MOI of 10 with the indicated viruses. At 24 h p.i., cells were permeabilized and stained for IRF-3 by use of a monoclonal antibody and for the nucleus by use of DAPI (4′,6′-diamidino-2-phenylindole) as described in Materials and Methods. (D) Quantitation of nuclear IRF-3. Samples from the experiment displayed in panel C were used to determine the number cells displaying intense nuclear staining as a percentage of the population. For each sample, four random fields were counted and averaged, with error bars denoting standard deviations. (E) Dimerization of IRF-3. A549 cells were infected at an MOI of 10 with the indicated viruses, and cell lysates prepared at either 8 or 24 h p.i. were analyzed on nondenaturing gels followed by Western blotting with an IRF-3-specific antibody.

FIG. 7.
FIG. 7.

The +V-wt and +P-wt viruses induce lower proinflammatory cytokine synthesis and NF-κB activation than the parental P/V-CPI- virus. (A and B) Samples prepared as described for panel B of Fig. 6 were analyzed by ELISA for levels of IL-6 (panel A) and IL-8 (panel B). Error bars denote standard deviations from the mean. (C) Cells infected with the indicated viruses were permeabilized and stained for NF-κB p65. The number of cells displaying intense nuclear staining was determined as a percentage of the population as described for panel D of Fig. 6.

FIG. 8.
FIG. 8.

RNA synthesis in cells infected with the +V-wt and +P-wt viruses. Vero cells were mock infected or infected at an MOI of 10 with the indicated viruses. At 14 h p.i., nucleocapsid RNA (panel A) or total RNA (panel B) was harvested as described in Materials and Methods. (A) RNA was analyzed by RPA using a 32P-labeled riboprobe that annealed to the leader-NP junction in genomic RNA. The position of the le-NP protected fragment is indicated. Data are representative of the results of four independent experiments. (B) Total RNA was analyzed by RPA using 32P-labeled riboprobe that annealed to positive-sense NP or M mRNA. The position of the virus-specific protected fragment is indicated. Data are representative of the results of three independent experiments. For panels A and B, the amount of RNA in the probe lane represents 1/120 of the amount used to hybridize with samples. Numbers below the lanes denote quantitation of the severalfold increase over the level of radioactivity in the WT rSV5-GFP samples set at 1.0.

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