Bovine respirator syncytial virus nonstructural proteins NS1 and NS2 cooperatively antagonize alpha/beta interferon-induced antiviral response

The functions of bovine respiratory syncytial virus (BRSV) nonstructural pr oteins NS1 and NS2 were studied by generation and analysis of recombinant B RSV carrying single and double gene deletions. Whereas in MDBK cells the la ck of either or both NS genes resulted in a 5,000- to 10,000-fold reduction of virus titers, in Vero cells a moderate (IO-fold) reduction was observed . Interestingly, cell culture supernatants from infected MDBK cells were ab le to restrain the growth of NS deletion mutants in Vero cells, suggesting the involvement of NS proteins in escape from cytokine-mediated host cell r esponses, The responsible factors in MDBK supernatants were identified as t ype I interferons by neutralization of the inhibitory effect with antibodie s blocking the alpha interferon (IFN-alpha) receptor. Treatment of cells wi th recombinant universal IFN-alpha A/D or IFN-beta revealed severe inhibiti on of single and double deletion mutants, whereas growth of full-length BRS V was not greatly affected. Surprisingly, all NS deletion mutants mere equa lly repressed, indicating an obligatory cooperation of NS1 and NS2 in antag onizing IFN-mediated antiviral mechanisms. To verify this finding, we gener ated recombinant rabies virus (rRV) expressing either NS1 or NS2 and determ ined their IFN sensitivity. In cells coinfected with NS1- and NS2-expressin g rRVs, virus replication was resistant to doses of IFN which caused a 1,00 0-fold reduction of replication in cells infected with wild-type RV or with each of the NS-expressing rRVs alone. Thus, BRSV NS proteins have the pote ntial to cooperatively protect an unrelated virus from IFN-alpha/beta media ted antiviral responses. Interestingly, BRSV NS proteins provided a more pr onounced resistance to IFN in the bovine cell line MDBK than in cell lines of other origins, suggesting adaptation to host-specific antiviral response s. The findings described have a major impact on the design of live recombi nant BRSV and HRSV vaccines.