Overexpression of the M2-2 protein of respiratory syncytial virus inhibits viral replication

Abstract

The M2-2 protein of respiratory syncytial virus (RSV) is involved in regulation of viral RNA transcription and replication. Encoded by the next-to-last gene of RSV, the M2-2 open reading frame (ORF) overlaps with the upstream M2-1 ORF, suggesting that the production of the M2-2 protein might be tightly regulated during virus replication. To evaluate the effect of M2-2 overexpression on RSV replication, the M2-2 gene was separated from M2-1 by leaving it at the position prior to the M2-1 or moving it to the promoter proximal position as an independent transcriptional unit in the RSV A2 genome. Although recombinant viruses bearing the shuffled M2-2 gene were recovered and expressed higher levels of M2-2, most of these viruses grew poorly in HEp-2 cells. Sequence analysis revealed that various mutations (substitution, insertion, and deletion) occurred in the M2-2 gene, resulting in reduced M2-2 activity as measured by the RSV minigenome system. Further examination of the M2-2 sequence and its function showed that either one of the first two AUG codons located at the 5' end of M2-2 could be used to produce a functional M2-2 protein and that deletion of the first six amino acids from its N terminus or four amino acids from its C terminus greatly reduced its function. The effect of M2-2 protein on RSV replication was also studied by examining RSV replication in cells transiently expressing M2-2. The M2-2 protein expressed at a high level completely inhibited RSV replication. These results strongly suggested that the level of the M2-2 protein produced in the infected cells is critical to RSV replication.

FIG. 1.

Construction of RSV antigenomic cDNA with M2-2 inserted at the first or ninth position. To move the M2-2 ORF to the 3′ promoter proximal position, the M2-2 sequence together with the created GE sequence for M2-2 and the GS sequence for NS1 was cloned into the KpnI site that was inserted after the original NS1 GS sequence. The antigenomic cDNA containing the 3-nt (TCT) IGR between the inserted M2-2 and NS1 was designated pM2-2G1S. To increase the length of the IGR between M2-2 and NS1, an EcoRI site was introduced at the M2-2 GE signal, and the cDNA containing the SH-G (nt 4499 to 4673) IGR flanked by the EcoRI sites was inserted such that the SH GE sequence with an additional a nucleotide would be used by M2-2. This antigenomic cDNA was designated pM2-2G1L. To insert M2-2 between the F and M2 genes to create antigenomic cDNA pM2-2G9, a BglII site was introduced into the F/M2 intergenic region at nt 7552 and inserted with the M2-2 ORF flanked by the GS and GE sequence.

FIG. 2.

Growth kinetics of the recombinant viruses in Vero and HEp-2 cells. Vero and HEp-2 cells were infected with each virus in duplicate at an MOI of 0.2. At 24-h intervals, the infected culture supernatants were harvested, and virus titers were determined by plaque assay in Vero cells. The mean titers and standard deviations (bars) are shown.

FIG. 3.

Viral RNA and protein synthesis in M2-2 recombinant virus-infected cells. (A) Northern blot analysis of viral RNA expression. Total RNA was extracted from virus-infected cells at 24 h postinfection. The membranes were hybridized with riboprobes specific to the NS1, M2-1, or N gene. (B) Western blot analysis of the viral protein expression. Protein polypeptides from the infected cells were separated in 4 to 20% polyacrylamide gel containing 0.1% SDS, and the blots were detected with antibodies specific to the N, NS1, or M2-1 proteins.

FIG. 4.

Expression of M2-2 in M2-2 recombinant-infected cells. Total RNA was extracted from virus-infected HEp-2 cells at 24 h postinfection for Northern blotting with a riboprobe specific to the M2-2 gene (A), and the infected cells were also radiolabeled with [³⁵S]methionine and [³⁵S]cysteine and subjected to immunoprecipitation with M2-2-specific antibody (B).

FIG. 5.

Overexpression of M2-2 inhibited RSV infection. BSR T7/T5 cells were transfected with ICP4-tagged M2-2 plasmid or the NP plasmid of influenza virus. After incubation at 37°C for 3 h, the cells were superinfected with A2 at an MOI of 10.0. At 16 h postinfection, the cell monolayers were fixed and immunostained with a mixture of anti-ICP4 monoclonal and anti-NS1 rabbit polyclonal antibodies or anti-NP monoclonal and anti-NS1 rabbit polyclonal antibodies. The secondary anti-rabbit antibody conjugated with Texas Red was used to detect NS1 for RSV infection, and the anti-mouse antibody conjugated with fluorescein isothiocyanate was used to detect ICP4-M2-2 or influenza NP expressed from the transfected plasmids.