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. 2003 Feb;77(4):2321-9.
doi: 10.1128/jvi.77.4.2321-2329.2003.

The amino-terminal extensions of the longer Sendai virus C proteins modulate pY701-Stat1 and bulk Stat1 levels independently of interferon signaling

Dominique Garcin  1 , Jean-Baptiste Marq, Stephen Goodbourn, Daniel Kolakofsky
Affiliations

The amino-terminal extensions of the longer Sendai virus C proteins modulate pY701-Stat1 and bulk Stat1 levels independently of interferon signaling

Dominique Garcin et al. J Virol. 2003 Feb.

Abstract

The Sendai virus (SeV) C proteins are known to interact with Stat1 to prevent interferon (IFN)-induced pY701-Stat1 formation and IFN signaling. Nevertheless, pY701-Stat1 levels paradoxically increase during SeV infection. The C proteins also induce bulk Stat1 instability in some cells, similar to rubulavirus V proteins. We have found that SeV infection increases pY701-Stat1 levels even in cells in which bulk Stat1 levels strongly decrease. Remarkably, both the decrease in bulk Stat1 levels and the increase in pY701-Stat1 levels were found to be independent of the IFN signaling system, i.e., these events occur in mutant cells in which various components of the IFN signaling system have been disabled. Consistent with this, the C-induced decrease in Stat1 levels does not require Y701 of Stat1. We present evidence that C interacts with Stat1 in two different ways, one that prevents IFN-induced pY701-Stat1 formation and IFN signaling that has already been documented, and another that induces pY701-Stat1 formation (while decreasing bulk Stat1 levels) in a manner that does not require IFN signaling. These two types of Stat1 interaction are also distinguishable by C gene mutations. In particular, the IFN signaling-independent Stat1 interactions specifically require the amino-terminal extensions of the longer C proteins. The actions of the SeV C proteins in counteracting the cellular antiviral response are clearly more extensive than previously appreciated.

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Figures

FIG. 1.
FIG. 1.
(A) Open reading frame organization and expression of the SeV P gene. The four open reading frames (ORFs) expressed as proteins P, C, V, and W are shown as horizontal boxes, drawn roughly to scale. Several domains of the P protein, notably its tetramerization domain, including the L protein binding site and that which binds to the N: RNA nucleocapsid, are indicated. The double-headed arrow shows the site (codon 317) where G residues are added cotranscriptionally to access the V and W open reading frames. The crenellated beginning of the C open reading frame box indicates the four independently initiated C proteins, which all terminate at codon 205. aa, amino acid. (B) Sendai virus C proteins and their interactions with Stat1. The nested set of four C proteins (C′, C, Y1, and Y2) that initiate from ACG87, AUG114, AUG183, and AUG201, respectively, are shown as a telescoping set of boxes. Their respective lengths in amino acids are indicated; AUG114/C is set at amino acid position 1. The critical Phe170 present in all four C proteins and the deletion of amino acids 10 to 15 (which are present only in C′ and C) are indicated. The IFN-dependent interactions of C and Stat1 require F170 but not the N-terminal extensions found on C′ and C, whereas the IFN-independent interactions of C and Stat1 require the N-terminal extensions found only on C′ but not necessarily F170. These two different C/Stat1 interactions are shown schematically, along with the consequences of these interactions. The IFN-independent properties shared by the SeV C proteins and the rubulavirus V proteins are also indicated.
FIG. 2.
FIG. 2.
Effect of SeV infection on bulk Stat1 and pY702-Stat1 levels of MEFs. (A) Parallel cultures of MEFs were infected or not with 20 PFU of SeV-wt, SeV-CΔ10-15, or SeV-[C′/C-minus] (or Y1/Y2) per cell. Total cell extracts were prepared at 24 h postinfection, and equal amounts were Western blotted with anti-Stat1 antibody (bulk), anti-pY701-Stat1 plus antiactin antibodies (middle panel), or a polyclonal antibody to the viral P, C, V, and W proteins. Each pair of lanes represent duplicate, independent infections. °, *, and #, C′, C, and Y protein bands, respectively. (B) MEFs were infected or not with 20 PFU of SeV-wt per cell. After 24 h, some of the cultures were treated with 0.5 μM staurosporine for either 1 or 2 h. Total cell extracts were prepared, and equal amounts of extract were Western blotted with either anti-Stat1 (bulk) plus antiactin (the latter as a loading control) or anti-pY701-Stat1 antibodies in the ECL system. The results were quantitated in a Bio-Rad light detector and are presented below in graphic form and as their ratios. h., a host band.
FIG. 3.
FIG. 3.
Effect of various SeV infections of U5A cells with and without IFNAR2 on bulk Stat1 levels. Parallel cultures of U5A or U5A+IFNAR2 cells were infected or not with 20 PFU of either SeV-wt, SeV-CΔ10-15, SeV-[C′/C-minus], or SeV-CF170S per cell. After 24 h, total cell extracts were prepared, and equal amounts of extract were Western blotted with anti-Stat1 (bulk) plus antiactin, or polyclonal antibodies that recognize the SeV C, P, V, and W proteins.
FIG. 4.
FIG. 4.
Effect of SeV infection of various cell lines defective in specific components of the IFN signaling system on bulk Stat1 and pY7021-Stat1 levels. Parallel cultures of U6A (Stat2-minus), U5A (IFNAR2-minus), U4A (JAK1-minus), or the parental (wild type) 2fTGH cells were infected or not with 20 PFU of SeV-wt per cell. Total cell extracts were prepared at 24 h postinfection, and equal amounts of extract were Western blotted with either anti-Stat1 (bulk) plus antiactin or anti-pY701-Stat1 antibodies (panel A). Panel B shows the effect of 45 min of IFN treatment on pY701-Stat1 levels of the various cell lines.
FIG. 5.
FIG. 5.
Effect of SeV infection on transfected Stat1 levels in BSR T7 cells. Parallel cultures of BSR T7 cells were transfected with pEBS plasmids expressing either human wild-type Stat1 (1 to 750), Stat1-Y701F, Stat1-Δ694-750, or empty plasmid. After 24 h of transfection, the cultures were infected with 20 PFU of SeV-wt per cell for a further 24 h. Total cell extracts were prepared, equal amounts of extract were precipitated with anti-Stat1, and the precipitates were Western blotted with anti-Stat1.
FIG. 6.
FIG. 6.
Effect of various transfected C genes on cotransfected Stat1 in cell lines defective in known IFN-dependent signaling components. Parallel cultures of U1A and U1A+Tyk2 cells (A) and U4A and U4A+JAK2 cells (B) were cotransfected with pEBS plasmids expressing Stat1 and GFP along with one of the various C gene plasmids listed or an empty plasmid. Total cell extracts were prepared at 24 h posttransfection, and equal amounts of extract were Western blotted with either anti-Stat1 (bulk) plus anti-GFP (top panel) or anti-pY701-Stat1 plus antiactin (middle panel) or anti-C (bottom panel) antibodies. (C) 2fTGH cells were also transfected with the various C gene constructs (as well as the SV5 V gene) along with Stat1 and GFP for 72 h, and the steady-state level of Stat1 remaining was determined as in panels A and B.
FIG. 7.
FIG. 7.
IFN-dependent C/Stat1 interaction requires F170 but not the longer C proteins, whereas IFN-independent C/pY701-Stat1 interaction requires the longer C proteins but not F170. (A) Parallel cultures of 2fTGH cells were cotransfected with pISRE-luc (firefly) and pTK-r.luc (Renilla) as well as one of the various C genes listed or an empty plasmid. Some of the cultures were treated with 1,000 IU of IFN-α for 14 h at 24 h posttransfection. Cell extracts were then prepared, and the relative activities of both luciferases were determined. The bars show the results of duplicate determinations. (B) MEF cell extracts were mixed with those of BSR T7 cells that had been infected or not for 24 h with 20 PFU of either SeV-wt, SeV-CF170S, or SeV-CΔ10-15 per cell. The mixed extracts were then either precipitated with anti-C and Western blotted with anti-Stat1 or anti-pY701-Stat1 or precipitated with anti-Stat1 and Western blotted with anti-C.
FIG. 8.
FIG. 8.
Rubulavirus SV5 V gene induces Stat1 ubiquitination but not pY701-Stat1 formation. 2fTGH cells were cotransfected with plasmids expressing GFP and Stat1 along with the SeV C, SeV CΔ10-15, or SV5 V gene or an empty plasmid. Total cell extracts were prepared at 24 h posttransfection, and equal amounts of extract were Western blotted with either anti-Stat1 (bulk, top panel) or anti-pY701-Stat1 plus antiactin (middle panel). Equal amounts of extract were also precipitated with antiubiquitin (Ub) and then Western blotted with anti-Stat1 (bottom panel). The right-hand lane in the bottom panel has not been immunoprecipitated and shows the electrophoretic position of bulk Stat1. IgG-hc, IgG heavy chain.
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References

    1. Bontron, S., C. Ucla, B. Mach, and V. Steimle. 1997. Efficient repression of endogenous major histocompatibility complex class II expression through dominant negative CIITA mutants isolated by a functional selection strategy. Mol. Cell. Biol. 17:4249-4258. - PMC - PubMed
    1. Buchholz, U. J., S. Finke, and K. K. Conzelmann. 1999. Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J. Virol. 73:251-259. - PMC - PubMed
    1. Cadd, T., D. Garcin, C. Tapparel, M. Itoh, M. Homma, L. Roux, J. Curran, and D. Kolakofsky. 1996. The Sendai paramyxovirus accessory C proteins inhibit viral genome amplification in a promoter-specific fashion. J. Virol. 70:5067-5074. - PMC - PubMed
    1. Chang, Y. E., and L. A. Laimins. 2000. Microarray analysis identifies interferon-inducible genes and Stat-1 as major transcriptional targets of human papillomavirus type 31. J. Virol. 74:4174-4182. - PMC - PubMed
    1. Chatterjee-Kishore, M., A. F. van Den, and G. R. Stark. 2000. Adenovirus E1A down-regulates LMP2 transcription by interfering with the binding of stat1 to IRF1. J. Biol. Chem. 275:20406-20411. - PubMed

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