Respiratory syncytial virus (RSV) nonstructural (NS) proteins as host range determinants: a chimeric bovine RSV with NS genes from human RSV is attenuated in interferon-competent bovine cells

Abstract

Bovine respiratory syncytial virus (BRSV) escapes from cellular responses to alpha/beta interferon (IFN-alpha/beta) by a concerted action of the two viral nonstructural proteins, NS1 and NS2. Here we show that the NS proteins of human RSV (HRSV) are also able to counteract IFN responses and that they have the capacity to protect replication of an unrelated rhabdovirus. Even combinations of BRSV and HRSV NS proteins showed a protective activity, suggesting common mechanisms and cellular targets of HRSV and BRSV NS proteins. Although able to cooperate, NS proteins from BRSV and HRSV showed differential protection capacity in cells from different hosts. A chimeric BRSV with HRSV NS genes (BRSV h1/2) was severely attenuated in bovine IFN competent MDBK and Klu cells, whereas it replicated like BRSV in IFN-incompetent Vero cells or in IFN-competent human HEp-2 cells. After challenge with exogenous IFN-alpha, BRSV h1/2 was better protected than wild-type BRSV in human HEp-2 cells. In contrast, in cells of bovine origin, BRSV h1/2 was much less resistant to exogenous IFN than wild-type BRSV. These data demonstrate that RSV NS1 and NS2 proteins are major determinants of host range. The differential IFN escape capacity of RSV NS proteins in cells from different hosts provides a basis for rational development of attenuated live RSV vaccines.

FIG. 1.

(A) Organization of rRV containing HRSV (Long) NS1 or NS2 open reading frames between the RV G and L genes. (B) Western blot analysis of HRSV NS1 and HRSV NS2 expression in infected BSR cells. NS1 and NS2 proteins were detected using a serum raised against a C-terminal peptide of NS1 that cross-reacts with NS2 (kindly provided by J. A. Melero, Madrid, Spain). The two bands representing the HRSV nonstructural proteins are indicated. Lane 1, mock infection; lane 2, SAD hNS1; lane 3, SAD hNS2.

FIG. 1.

(A) Organization of rRV containing HRSV (Long) NS1 or NS2 open reading frames between the RV G and L genes. (B) Western blot analysis of HRSV NS1 and HRSV NS2 expression in infected BSR cells. NS1 and NS2 proteins were detected using a serum raised against a C-terminal peptide of NS1 that cross-reacts with NS2 (kindly provided by J. A. Melero, Madrid, Spain). The two bands representing the HRSV nonstructural proteins are indicated. Lane 1, mock infection; lane 2, SAD hNS1; lane 3, SAD hNS2.

FIG. 2.

IFN resistance of RV in cells coinfected with RVs expressing HRSV NS1 or NS2. (A) Infections of MDBK cells with wild-type SAD VB (VB wild type), SAD hNS1 (H1), or SAD hNS2 (H2) or coinfections with SAD bNS1 (B1) and SAD bNS2 (B2) or SAD hNS1 and SAD hNS2. Indicated concentrations of recombinant IFN-α A/D were added immediately after seeding. Virus titers were determined 2 days postinfection. Results represent the mean values of three independent experiment, with error bars indicating standard deviation. (B) Infection of MDBK cells using various combinations of RVs expressing BRSV or HRSV nonstructural proteins.

FIG. 3.

Construction of recombinant BRSVs. The locations of the protein-encoding frames (shaded bars) are shown relative to the viral genome (vRNA, black bar). In the enlargement, the organization of wild-type BRSV and the recombinant BRSV carrying the HRSV nonstructural proteins, rBRSV h1/2, is depicted. Leader RNA is marked by diagonal hatching, and the relative positions of the corresponding nucleotides and restriction sites used for cloning are indicated.

FIG. 4.

Growth of BRSV h1/2 is attenuated in bovine cells. Vero (A), HEp-2 (B), MDBK (C), and Klu (D) cells were infected in suspension with either rBRSV wild type (▪) or rBRSV h1/2 (□) at an MOI of 0.1. Virus was harvested at the indicated time points, and virus titers were determined by limiting dilution. Bars show standard deviation for at least two independent experiments.

FIG. 5.

IFN resistance of BRSV h1/2 is cell type dependent. Vero (A), MDBK (B), or HEp-2 (C) cells were infected at an MOI of 0.1 with the indicated viruses. Recombinant IFN-α A/D was added to concentrations up to 10,000 U/ml directly after seeding. Virus titers were determined 3 days postinfection. Bars show standard deviation for at least two independent experiments.