Evidence against the Human Metapneumovirus G, SH, and M2-2 Proteins as Bona Fide Interferon Antagonists

J Virol. 2022 Sep 14;96(17):e0072322. doi: 10.1128/jvi.00723-22. Epub 2022 Aug 17.


The production of type I interferon (IFN) is the hallmark of the innate immune response. Most, if not all, mammalian viruses have a way to circumvent this response. Fundamental knowledge on viral evasion of innate immune responses may facilitate the design of novel antiviral therapies. To investigate how human metapneumovirus (HMPV) interacts with the innate immune response, recombinant viruses lacking G, short hydrophobic (SH), or M2-2 protein expression were assessed for IFN induction in A549 cells. HMPV lacking G or SH protein expression induced similarly low levels of IFN, compared to the wild-type virus, whereas HMPV lacking M2-2 expression induced significantly more IFN than the wild-type virus. However, sequence analysis of the genomes of M2-2 mutant viruses revealed large numbers of mutations throughout the genome. Over 70% of these nucleotide substitutions were A-to-G and T-to-C mutations, consistent with the properties of the adenosine deaminase acting on RNA (ADAR) protein family. Knockdown of ADAR1 by CRISPR interference confirmed the role of ADAR1 in the editing of M2-2 deletion mutant virus genomes. More importantly, Northern blot analyses revealed the presence of defective interfering RNAs (DIs) in M2-2 mutant viruses and not in the wild-type virus or G and SH deletion mutant viruses. DIs are known to be potent inducers of the IFN response. The presence of DIs in M2-2 mutant virus stocks and hypermutated virus genomes interfere with studies on HMPV and the innate immune response and should be addressed in future studies. IMPORTANCE Understanding the interaction between viruses and the innate immune response is one of the barriers to the design of antiviral therapies. Here, we investigated the role of the G, SH, and M2-2 proteins of HMPV as type I IFN antagonists. In contrast to other studies, no IFN-antagonistic functions could be observed for the G and SH proteins. HMPV with a deletion of the M2-2 protein did induce type I IFN production upon infection of airway epithelial cells. However, during generation of virus stocks, these viruses rapidly accumulated DIs, which are strong activators of the type I IFN response. Additionally, the genomes of these viruses were hypermutated, which was prevented by generating stocks in ADAR knockdown cells, confirming a role for ADAR in hypermutation of HMPV genomes or DIs. These data indicate that a role of the HMPV M2-2 protein as a bona fide IFN antagonist remains elusive.

Keywords: ADAR; HMPV G protein; HMPV M2-2 protein; HMPV SH protein; human metapneumovirus; innate immunity; interferon antagonist.

MeSH terms

  • A549 Cells
  • Adenosine Deaminase
  • Antiviral Agents / metabolism
  • Humans
  • Immunity, Innate*
  • Interferon Type I* / antagonists & inhibitors
  • Interferon Type I* / immunology
  • Metapneumovirus* / genetics
  • Metapneumovirus* / metabolism
  • RNA-Binding Proteins
  • Viral Proteins* / genetics
  • Viral Proteins* / metabolism


  • Antiviral Agents
  • Interferon Type I
  • RNA-Binding Proteins
  • Viral Proteins
  • ADAR protein, human
  • Adenosine Deaminase