Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Dec;73(12):9928-33.
doi: 10.1128/JVI.73.12.9928-9933.1999.

The V protein of simian virus 5 inhibits interferon signalling by targeting STAT1 for proteasome-mediated degradation

L Didcock  1 , D F Young, S Goodbourn, R E Randall
Affiliations

The V protein of simian virus 5 inhibits interferon signalling by targeting STAT1 for proteasome-mediated degradation

L Didcock et al. J Virol. 1999 Dec.

Abstract

To replicate in vivo, viruses must circumvent cellular antiviral defense mechanisms, including those induced by the interferons (IFNs). Here we demonstrate that simian virus 5 (SV5) blocks IFN signalling in human cells by inhibiting the formation of the IFN-stimulated gene factor 3 and gamma-activated factor transcription complexes that are involved in activating IFN-alpha/beta- and IFN-gamma-responsive genes, respectively. SV5 inhibits the formation of these complexes by specifically targeting STAT1, a component common to both transcription complexes, for proteasome-mediated degradation. Expression of the SV5 structural protein V, in the absence of other virus proteins, also inhibited IFN signalling and induced the degradation of STAT1. Following infection with SV5, STAT1 was degraded in the absence of virus protein synthesis and remained undetectable for up to 4 days postinfection. Furthermore, STAT1 was also degraded in IFN-pretreated cells, even though the cells were in an antiviral state. Since pretreatment of cells with IFN delayed but did not prevent virus replication and protein synthesis, these observations suggest that following infection of IFN-pretreated cells, SV5 remains viable within the cells until they eventually go out of the antiviral state.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
SV5 inhibits IFN-α/β (a) and IFN-γ (b) signalling and the formation of ISGF3 (c) and GAF (d) complexes in 2fTGH cells. Cells were transfected with either the IFN-α/β (a)- or IFN-γ (b)-responsive plasmids together with pJATlacZ and at 16 h posttransfection were either mock infected or infected with SV5. At 24 h p.i. the culture medium was supplemented with rHuIFN-αA/D (a) or IFN-γ (b) or left untreated. Four hours later, luciferase and β-galactosidase activities in the cellular lysates were measured. Luciferase activity, expressed in relative light units, was normalized to β-galactosidase activity. For the EMSAs, cells were mock infected or infected with SV5 for 24 h and then were (+) or were not (−) treated with rHuIFN-αA/D (c) or rHuIFN-γ (d) for 1 h. Extracts were prepared and analyzed by EMSAs using either 32P-labelled ISRE (c) or GAS (d) probe.
FIG. 2
FIG. 2
STAT1, but not STAT2, is degraded in SV5-infected human cells. STAT1 and STAT2 were detected by immunoblot analysis in total-cell extracts of mock- or SV5-infected 2fTGH cells (harvested at 20 h p.i.), using antibodies reactive to the C terminus of STAT1 (a) or the N terminus of STAT1 (b) or STAT2 (c). It should be noted that both anti-STAT1 antibodies react with STAT1α and STAT1β (they can be more clearly resolved on lower-percentage SDS-polyacrylamide gels). The lower prominent band, with an estimated molecular mass of 75 kDa, present in both mock-infected and SV5-infected cells (b) has not been identified.
FIG. 3
FIG. 3
The proteasome inhibitor MG132 blocks degradation of STAT1 in SV5-infected 2fTGH cells and of IκBα in TNF-α-stimulated 2fTGH cells. Mock-infected cells (lanes 1) and SV5-infected cells (lanes 2 to 5) were incubated, from the time of infection, in medium that did (lanes 4 and 5) or did not (lanes 1 to 3) contain the proteasome inhibitor MG132 (10 μM). At 8 h p.i., the medium was (lanes 3 and 5) or was not (lanes 1, 2, and 4) further supplemented with TNF-α; 30 min later, the cells were harvested and the levels of STAT1 (a) and IκBα (b) were estimated by immunoblot analysis.
FIG. 4
FIG. 4
The V protein of SV5 induces proteasome-mediated degradation of STAT1. (a) 2fTGH cells were mock infected (lane 1) or infected with recSFV/F (lane 2), recSFV/lacZ (lane 3), or recSFV/V (lane 4) for 8 h, and total-cell extracts were probed for STAT1 as previously described. (Immunofluorescence analysis confirmed that >95% of the cells were expressing the appropriate virus proteins at the time of harvest.) (b) 2fTGH cells were mock infected (lanes 1 and 2) or infected with recSFV/V (lanes 3 and 4). The proteasome inhibitor MG132 (10 μM) was (lanes 2 and 4) or was not (lanes 1 and 3) added to the culture medium at the time of infection. At 8 h p.i., cells were harvested and levels of STAT1 were estimated by immunoblot analysis.
FIG. 5
FIG. 5
The V protein of SV5, but not P, blocks activation of an IFN-α/β-responsive promoter in human cells. 2fTGH cells were transfected with a mixture of plasmids that contained the luciferase gene under the control of either the herpes simplex virus TK promoter (a) or an IFN-α/β-responsive promoter (b; 0.1 μg), together with 0.3 μg of pEFlink2 (control), pEF.SV5-P (expressing the SV5 P protein), or pEF.SV5-V (expressing the SV5 V protein). Also included in the transfection mix was 0.1 μg of plasmid pJATlacZ, in which the lacZ gene is under the control of the rat β-actin promoter. At 40 h posttransfection, the culture medium was (+) or was not (−) supplemented with IFN. Four hours later, luciferase and β-galactosidase activities in cellular lysates were measured. Luciferase activity, expressed in relative light units, was normalized to β-galactosidase activity.
FIG. 6
FIG. 6
STAT1 was rapidly degraded in SV5-infected cells in the absence of virus protein synthesis. (a) Total-cell extracts of 2fTGH cells that were mock infected (lane 1) or infected with SV5 for 1, 2, 4, or 8 h (lanes 2 to 5, respectively) were probed for STAT1 by immunoblot analysis. (b) 2fTGH cells were mock infected (lane 1) or infected with UV-inactivated SV5 (lanes 2 to 6). At 6 (lanes 1 and 2) and 24, 48, 72, and 96 (lanes 3 to 6, respectively) h p.i. total cell extracts were probed for STAT1 by immunoblot analysis. (At no time throughout the latter experiment were cells positive for virus antigens, as judged by immunofluorescence.)
FIG. 7
FIG. 7
Inhibition of transcription and protein synthesis did not prevent the degradation of STAT1 in SV5-infected cells. 2fTGH cells were untreated (Un) or treated with actinomycin D (Act. D; 10 μg/ml) or cycloheximide (Cx; 50 μg/ml) 1 h prior to being mock infected or infected with SV5. Actinomycin D or cycloheximide (as appropriate) was also present throughout infection period. Total-cell extracts were made at 6 h p.i., and the levels of STAT1 were detected by immunoblot analysis.
FIG. 8
FIG. 8
STAT1 was degraded in IFN-pretreated cells. 2fTGH cells were (lanes 2, 4, and 6) or were not (1, 3, and 5) treated with rHuIFN-αA/D for 24 h prior to mock infection (lanes 1 and 2) or infection with SV5 (lanes 3 to 6). Total-cell extracts were made from cells harvested at either 6 (lanes 1 to 4) or 24 (lanes 5 and 6) h p.i., and levels of STAT1 were detected by immunoblot analysis.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alkalay I, Yaron A, Hatzubai A, Orian A, Ciechanover A, Ben-Neriah Y. Stimulation-dependent IκBα phosphorylation marks the NF-κB inhibitor for degradation via the ubiquitin-proteasome pathway. Proc Natl Acad Sci USA. 1995;92:10599–10603. - PMC - PubMed
    1. Boyer S N, Wazer D E, Band V. E7 protein of human papillomavirus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway. Cancer Res. 1996;56:4620–4624. - PubMed
    1. Chin Y E, Kitagawa M, Kuida K, Flavell R A, Fu X Y. Activation of the STAT signaling pathway can cause expression of caspase 1 and apoptosis. Mol Cell Biol. 1997;17:5328–5337. - PMC - PubMed
    1. Choppin P W. Multiplication of a myxovirus (SV5) with minimal cytopathic effects and without interference. Virology. 1964;23:224–233. - PubMed
    1. Delenda C, Hausmann S, Garcin D, Kolakofsky D. Normal cellular replication of Sendai virus without the trans-frame, nonstructural V protein. Virology. 1997;228:55–62. - PubMed

Publication types

MeSH terms

Substances