title: Identification of human parainfluenza virus type 2 (HPIV-2) V protein amino acid residues that reduce binding of V to MDA5 and attenuate HPIV-2 replication in nonhuman primates.
abstract: Human parainfluenza virus type 2 (HPIV-2), an important pediatric respiratory pathogen, encodes a V protein that inhibits type I interferon (IFN) induction and signaling. Using reverse genetics, we attempted the recovery of a panel of V mutant viruses that individually contained one of six cysteine-to-serine (residues 193, 197, 209, 211, 214, and 218) substitutions, one of two paired charge-to-alanine (R175A/R176A and R205A/K206A) substitutions, or a histidine-to-phenylalanine (H174F) substitution. This mutagenesis was performed using a cDNA-derived HPIV-2 virus that expressed the V and P coding sequences from separate mRNAs. Of the cysteine substitutions, only C193S, C214S, and C218S yielded viable virus, and only the C214S mutant replicated well enough for further analysis. The H174F, R175A/R176A, and R205A/K206A mutants were viable and replicated well. The H174F and R205A/K206A mutants did not differ from the wild-type (WT) V in their ability to physically interact with MDA5, a cytoplasmic sensor of nonself RNA that induces type I IFN. Like WT HPIV-2, these mutants inhibited IFN-β induction and replicated efficiently in African green monkeys (AGMs). In contrast, the C214S and R175A/R176A mutants did not bind MDA5 efficiently, did not inhibit interferon regulatory factor 3 (IRF3) dimerization or IFN-β induction, and were attenuated in AGMs. These findings indicate that V binding to MDA5 is important for HPIV-2 virulence in nonhuman primates and that some V protein residues involved in MDA5 binding are not essential for efficient HPIV-2 growth in vitro. Using a transient expression system, 20 additional mutant V proteins were screened for MDA5 binding, and the region spanning residues 175 to 180 was found to be essential for this activity.
authors.affiliation: LID, NIAID, NIH, 50 South Drive, Room 6511, MSC 8007, Bethesda, MD 20892, USA.
authors.forename: Anne
authors.initials: A
authors.lastname: Schaap-Nutt
authors.forename: Caraline
authors.initials: C
authors.lastname: Higgins
authors.forename: Emerito
authors.initials: E
authors.lastname: Amaro-Carambot
authors.forename: Sheila M
authors.initials: SM
authors.lastname: Nolan
authors.forename: Christopher
authors.initials: C
authors.lastname: D'Angelo
authors.forename: Brian R
authors.initials: BR
authors.lastname: Murphy
authors.forename: Peter L
authors.initials: PL
authors.lastname: Collins
authors.forename: Alexander C
authors.initials: AC
authors.lastname: Schmidt
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D008253:Macaca mulatta; D050296:Microbial Viability; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010223:Parainfluenza Virus 2, Human / Q000235:genetics / Q000472:pathogenicity*; D011485:Protein Binding; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D000596:Amino Acids; D050505:Mutant Proteins; C068854:V protein, human parainfluenza virus type 1; D014764:Viral Proteins; D037521:Virulence Factors; D053487:DEAD-box RNA Helicases