The role of M2 proteins of pneumoviruses in transcription regulation, prevention of hypermutation, and activation of the type I interferon pathway.

Abstract

Human metapneumovirus (HMPV) is an important causative agent of respiratory tract disease. Fundamental knowledge of the interaction between HMPV and the innate immune system could lead to the design of novel antiviral therapies. Previously, we demonstrated that HMPV M2-2 deletion mutants had hypermutated genomes and contained defective interfering particles (DIs), which are potent inducers of the IFN response. Here, we investigated the role of the HMPV M2-2 protein as IFN antagonist using chimeric HMPV expressing M2 proteins of other pneumoviruses: respiratory syncytial virus (RSV) and avian metapneumovirus type C (AMPV/C). Chimeric HMPVs expressing the M2 proteins of RSV or AMPV/C were attenuated in HEp-2 cells but did not activate the IFN response, and their genomes were not hypermutated. In contrast, chimeric HMPVs expressing the M2-2 proteins of RSV and AMPV/C, in combination with HMPV M2-1, did activate the IFN response, and their genomes were hypermutated. Investigation of the role of the pneumovirus M2 proteins in transcription regulation demonstrated that the M2-2 protein, only in concerted action with autologous M2-1 protein, acted as a transcription elongation factor. As a second approach, chimeric RSV in which the IFN antagonists NS1 and NS2 were replaced by the HMPV M2-2 gene failed to suppress an IFN response, indicating that the HMPV M2-2 protein is not a potent IFN antagonist. These data indicate that expression of autologous M2-1 and M2-2 proteins is important for the fidelity of the RNA-dependent RNA polymerase, necessary to prevent the accumulation of mutations, and possibly DIs, thereby preventing activation of the IFN responses.IMPORTANCEThe M2-2 protein of human metapneumovirus is suggested to function as a type I interferon antagonist, a function so far not assigned to the M2 proteins of other pneumoviruses. Although M2-2 deletion mutants of HMPV activate the type I interferon pathway, these mutants have hypermutated genomes and contain defective interfering RNAs, known to activate the interferon pathway. Here, we show that the M2-2 protein, in concerted action with autologous M2-1 protein, acts as a transcription elongation factor, which could explain the accumulation of DIs in M2-2 deletion mutants. Additionally, chimeric RSV in which the IFN antagonists NS1 and NS2 were replaced by the HMPV M2-2 gene failed to suppress an IFN response. These data indicate that expression of autologous M2-1 and M2-2 proteins is required for the fidelity of the RNA-dependent RNA polymerase to prevent genome hypermutation and activation of the type I IFN pathway.