The N-terminus of Bunyamwera orthobunyavirus NSs protein is essential for interferon antagonism - PubMed (original) (raw)

The N-terminus of Bunyamwera orthobunyavirus NSs protein is essential for interferon antagonism

Ingeborg van Knippenberg et al. J Gen Virol. 2010 Aug.

Abstract

Bunyamwera virus NSs protein is involved in the inhibition of cellular transcription and the interferon (IFN) response, and it interacts with the Med8 component of Mediator. A spontaneous mutant of a recombinant NSs-deleted Bunyamwera virus (rBUNdelNSs2) was identified and characterized. This mutant virus, termed mBUNNSs22, expresses a 21 aa N-terminally truncated form of NSs. Like rBUNdelNSs2, mBUNNSs22 is attenuated in IFN-deficient cells, and to a greater extent in IFN-competent cells. Both rBUNdelNSs2 and mBUNNSs22 are potent IFN inducers and their growth can be rescued by depleting cellular IRF3. Strikingly, despite encoding an NSs protein that contains the Med8 interaction domain, mBUNNSs22 fails to block RNA polymerase II activity during infection. Overall, our data suggest that both the interaction of NSs with Med8 and a novel unidentified function of the NSs N-terminus, seem necessary for Bunyamwera virus to counteract host antiviral responses.

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Figures

Fig. 1.

Fig. 1.

Characterization of a mutant Bunyamwera virus that expresses an N-terminally truncated NSs protein. (a) Schematic of S segment RNA sequences surrounding the N and NSs ORF start codons for wtBUNV, rBUNdelNSs2 (delNSs2) and mBUNNSs22 (NSs22). Shown are nt 83–119 and 162–197 of the S (+) RNA. The numbering of the relevant codons is indicated, AUG start codons are underlined and bold, and an asterisk (*) marks a stop codon. Amino acid sequences for N and NSs are shown above and below the RNA sequences, respectively. (b) Western blot analysis of BHK cells infected with wtBUNV (wt) or mBUNNSs22 (NSs22) or mock-infected (M). Strips of the blot were probed with the antibodies indicated on the right; size markers are indicated on the left. (c) Western blot analysis of BHK cells infected with wtBUNV, mBUNNSs22 (22), rBUNdelNSs2 (del) or mock-infected. MG132 (10 μM) was added to the medium at 5 h p.i. and cell extracts were prepared at 12 h p.i. (d, e) Multi-step growth curves of wtBUNV, rBUNdelNSs2 and mBUNNSs22 in BHK cells at 33 °C (d) and 37 °C (e). Shown are mean values of triplicate infections.

Fig. 2.

Fig. 2.

mBUNNSs22 is attenuated in IFN-competent cells and is a potent IFN inducer. (a) Multi-step growth curves of wtBUNV, rBUNdelNSs2 and mBUNNSs22 virus in A549 cells. Shown are mean values of triplicate infections. (b) Levels of IFN induced in A549 cells after 24 h infection with wtBUNV, rBUNdelNSs2 or mBUNNSs22. The relative IFN content of medium from infected cells was measured by comparing the dilution that could protect indicator cells from EMCV infection. (c) Plaque formation in IFN-competent A549 cells (left panels) and IFN-deficient A549-NPro cells (right panels). Cells were infected with wtBUNV, rBUNdelNSs2 or mBUNNSs22 as indicated and were stained for plaque formation after 5 days incubation at 37 °C.

Fig. 3.

Fig. 3.

mBUNNSs22 does not degrade RNAPII or cause shut off of host protein synthesis. (a) Western blot analysis of BHK cells infected with wtBUNV, rBUNdelNSs2, mBUNNS22 or mock-infected, and harvested at the indicated times p.i. Size markers are indicated on the left, and antibodies used on the right. _α_-RNAPII, antibody against RNAPII-CTD, regardless of its phosphorylation state; _α_-Ser2-P, antibody specific for serine-2-phosphorylated CTD of RNAPII. (b) Metabolic labelling of infected cells. A549 cells (top panel) and Vero cells (bottom panel) were infected with wtBUNV, rBUNdelNSs2, mBUNNSs22 or were mock-infected. Cells were labelled with [35S]methionine for 1 h prior to the indicated time p.i., and cell lysates were analysed by SDS-PAGE. Viral proteins and their sizes are indicated on the right.

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