title: Deletion of M2 gene open reading frames 1 and 2 of human metapneumovirus: effects on RNA synthesis, attenuation, and immunogenicity.
abstract: The M2 gene of human metapneumovirus (HMPV) contains two overlapping open reading frames (ORFs), M2-1 and M2-2. The expression of separate M2-1 and M2-2 proteins from these ORFs was confirmed, and recombinant HMPVs were recovered in which expression of M2-1 and M2-2 was ablated individually or together [rdeltaM2-1, rdeltaM2-2, and rdeltaM2(1+2)]. Each M2 mutant virus directed efficient multicycle growth in Vero cells. The ability to recover HMPV lacking M2-1 contrasts with human respiratory syncytial virus, for which M2-1 is an essential transcription factor. Expression of the downstream HMPV M2-2 ORF was not reduced when translation of the upstream M2-1 ORF was silenced, indicating that it is initiated separately. The rdeltaM2-2 mutants exhibited a two- to fivefold increase in the accumulation of mRNA, normalized to the genome template, suggesting that M2-2 has a role in regulating RNA synthesis. Replication and immunogenicity were tested in hamsters. Animals infected intranasally with rdeltaM2-1 or rdeltaM2(1+2) did not have recoverable virus in the lungs or nasal turbinates on days 3 or 5 postinfection and did not develop HMPV-neutralizing serum antibodies or resistance to HMPV challenge. Thus, M2-1 appears to be essential for significant virus replication in vivo. In animals infected with rdeltaM2-2, virus was recovered from only 1 of 12 animals and only in the nasal turbinates on a single day. However, all of the animals developed a high titer of HMPV-neutralizing serum antibodies and were highly protected against challenge with wild-type HMPV. The HMPV rdeltaM2-2 virus is a promising and highly attenuated HMPV vaccine candidate.
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8007, USA. ubuchholz@niaid.nih.gov
authors.forename: Ursula J
authors.initials: UJ
authors.lastname: Buchholz
authors.forename: Stéphane
authors.initials: S
authors.lastname: Biacchesi
authors.forename: Quynh N
authors.initials: QN
authors.lastname: Pham
authors.forename: Kim C
authors.initials: KC
authors.lastname: Tran
authors.forename: Lijuan
authors.initials: L
authors.lastname: Yang
authors.forename: Cindy L
authors.initials: CL
authors.lastname: Luongo
authors.forename: Mario H
authors.initials: MH
authors.lastname: Skiadopoulos
authors.forename: Brian R
authors.initials: BR
authors.lastname: Murphy
authors.forename: Peter L
authors.initials: PL
authors.lastname: Collins
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004279:DNA, Viral / Q000235:genetics; D017353:Gene Deletion; D005814:Genes, Viral; D006801:Humans; D008647:Mesocricetus; D029121:Metapneumovirus / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity / Q000502:physiology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D018184:Paramyxoviridae Infections / Q000276:immunology / Q000821:virology; D012367:RNA, Viral / Q000096:biosynthesis; D011995:Recombination, Genetic; D014709:Vero Cells; D014763:Viral Matrix Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000096:biosynthesis; D014765:Viral Vaccines / Q000235:genetics; D014774:Virulence / Q000235:genetics; D014779:Virus Replication / Q000235:genetics
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; D014763:Viral Matrix Proteins; D014764:Viral Proteins; D014765:Viral Vaccines