Modification of the Trypsin-Dependent Cleavage Activation Site of the Human Metapneumovirus Fusion Protein To Be Trypsin Independent Does Not Increase Replication or Spread in Rodents or Nonhuman Primates

Abstract

The contribution of cleavage activation of the fusion F protein of human metapneumovirus (HMPV) to replication and pathogenicity in rodents and nonhuman primates was investigated. Recombinant HMPVs were generated in which the naturally occurring trypsin-dependent cleavage sequence (R-Q-S-R2) was replaced by each of three sequences whose cleavage in vitro does not depend upon added trypsin. Two of these were multibasic sequences derived from avian metapneumovirus type A (R-R-R-R) or type C (R-K-A-R), with the former containing the consensus furin protease cleavage motif (R-X-R/K-R2). The third one (R-Q-P-R) was derived from a recently described trypsin independent HMPV isolate (J. H. Schickli, J. Kaur, N. Ulbrandt, R. R. Spaete, and R. S. Tang, J. Virol. 79:10678-10689, 2005). To preclude the possibility of conferring even greater virulence to this significant human pathogen, the modifications were done in an HMPV variant that was attenuated by the deletion of two of the three envelope glycoproteins, SH and G. Each of the introduced cleavage sequences conferred trypsin independent F cleavage and growth to HMPV in vitro. However, they differed in the efficiency of trypsin independent growth and plaque formation in vitro: R-R-R-R > R-K-A-R > R-Q-P-R > R-Q-S-R. The R-R-R-R mutant was the only one whose growth in vitro was not augmented by added trypsin, indicative of highly efficient trypsin independent cleavage. When inoculated intranasally into hamsters, there was essentially no difference in the magnitude of replication in the upper or lower respiratory tract between the mutants, and virus was not detected in organs outside of the respiratory tract. Evaluation of the most cleavage-efficient mutant, R-R-R-R, in African green monkeys showed that there was no detectable change in the magnitude of replication in the upper and lower respiratory tract or in immunogenicity and protective efficacy against HMPV challenge. These results suggest that cleavage activation is not a major determinant of HMPV virulence.

Frequent Frameshift and Point Mutations in the SH Gene of Human Metapneumovirus Passaged In Vitro

“…Recombinant viruses were recovered as described previously (3,5). Sequence analysis of viral RNA was carried out as described previously (3). In some cases, as indicated in the text, these reverse transcription-PCR (RT-PCR) products were gel purified and cloned using a commercial blunt vector (Perfectly Blunt cloning kit; Novagen) and individual clones were sequenced.…”

“…Recombinant viruses were recovered as described previously (3,5). Sequence analysis of viral RNA was carried out as described previously (3).…”

“…Virus shedding was examined by plaque assay of nasopharyngeal and tracheal lavage samples collected on days 2, 4, 6, and 8 postchallenge. Serum samples were collected on days 0, 28, and 56, and the titers of HMPV-neutralizing antibodies were determined by using an endpoint dilution neutralization assay as described previously (3).…”

Low-pH Triggering of Human Metapneumovirus Fusion: Essential Residues and Importance in Entry

“…One clear distinction between viruses of the Pneumovirinae subfamily and the Paramyxovirinae subfamily with regard to the viral entry mechanism has become apparent in recent years. While the homotypic attachment protein (G, H, or HN) of viruses within the Paramyxovirinae subfamily is required for virus attachment and membrane fusion promotion by the viral F protein, the "attachment" proteins (G) of multiple Pneumovirinae subfamily members were shown to be dispensable for entry in cultured cells and also in vivo in the case of HMPV (7,9,41,48,56). In fact, HMPV with G deleted was infectious in primates (7).…”

“…While the homotypic attachment protein (G, H, or HN) of viruses within the Paramyxovirinae subfamily is required for virus attachment and membrane fusion promotion by the viral F protein, the "attachment" proteins (G) of multiple Pneumovirinae subfamily members were shown to be dispensable for entry in cultured cells and also in vivo in the case of HMPV (7,9,41,48,56). In fact, HMPV with G deleted was infectious in primates (7). Furthermore, the G protein of HMPV did not enhance cell-cell fusion promoted by F in transfected Vero cells (49), suggesting that the HMPV F protein alone is capable of performing both the critical attachment step and efficient membrane fusion.…”