title: An S101P substitution in the putative cleavage motif of the human metapneumovirus fusion protein is a major determinant for trypsin-independent growth in vero cells and does not alter tissue tropism in hamsters.
abstract: Human metapneumovirus (hMPV), a recently described paramyxovirus, is a major etiological agent for lower respiratory tract disease in young children that can manifest with severe cough, bronchiolitis, and pneumonia. The hMPV fusion glycoprotein (F) shares conserved functional domains with other paramyxovirus F proteins that are important for virus entry and spread. For other paramyxovirus F proteins, cleavage of a precursor protein (F0) into F1 and F2 exposes a fusion peptide at the N terminus of the F1 fragment, a likely prerequisite for fusion activity. Many hMPV strains have been reported to require trypsin for growth in tissue culture. The majority of these strains contain RQSR at the putative cleavage site. However, strains hMPV/NL/1/00 and hMPV/NL/1/99 expanded in our laboratory contain the sequence RQPR and do not require trypsin for growth in Vero cells. The contribution of this single amino acid change was verified directly by generating recombinant virus (rhMPV/NL/1/00) with either proline or serine at position 101 in F. These results suggested that cleavage of F protein in Vero cells could be achieved by trypsin or S101P amino acid substitution in the putative cleavage site motif. Moreover, trypsin-independent cleavage of hMPV F containing 101P was enhanced by the amino acid substitution E93K. In hamsters, rhMPV/93K/101S and rhMPV/93K/101P grew to equivalent titers in the respiratory tract and replication was restricted to respiratory tissues. The ability of these hMPV strains to replicate efficiently in the absence of trypsin should greatly facilitate the generation, preclinical testing, and manufacturing of attenuated hMPV vaccine candidates.
authors.affiliation: MedImmune Vaccines, Inc., 297 N. Bernardo Ave., Mountain View, CA 94043, USA. schicklij@medimmune.com
authors.forename: Jeanne H
authors.initials: JH
authors.lastname: Schickli
authors.forename: Jasmine
authors.initials: J
authors.lastname: Kaur
authors.forename: Nancy
authors.initials: N
authors.lastname: Ulbrandt
authors.forename: Richard R
authors.initials: RR
authors.lastname: Spaete
authors.forename: Roderick S
authors.initials: RS
authors.lastname: Tang
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002522:Chlorocebus aethiops; D006224:Cricetinae; D008647:Mesocricetus; D029121:Metapneumovirus / Q000254:growth & development*; D008969:Molecular Sequence Data; D018523:Tropism; D014357:Trypsin / Q000494:pharmacology*; D014709:Vero Cells; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000502:physiology*; D014779:Virus Replication
chemical_list: D014760:Viral Fusion Proteins; D014357:Trypsin