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-R downward arrow) 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-R downward arrow). 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.

FIG. 1.

Construction, recovery, and plaque morphologies of the HMPV F cleavage site mutants in the ΔSH/G background. (A) The complete rHMPV genome is shown at the top, drawn approximately to scale. The NheI and BsiWI sites that had been added (5, 6) are shown, as well as a naturally occurring unique Acc65I site. Underneath is the rHMPVΔSH/G genome in which the sequence of the F cleavage site was manipulated. (B) Sequences of the F cleavage sites in recovered ΔSH/G viruses and plaque size in Vero cells. The electropherograms show the nucleotide and amino acid sequences surrounding the F protein cleavage site (nt 3355 to 3381 of the HMPV genome and amino acids 97 to 105 of the F protein). The red letters indicate mutated nucleotides and amino acids, and the arrow shows the presumed cleavage site. On the right are photomicrographs showing the plaque sizes of the recovered viruses in Vero cells in the presence or absence of added trypsin in methylcellulose culture medium taken on day 6 postinfection, after fixation and immunostaining with polyclonal antibodies raised against gradient-purified HMPV.

FIG. 2.

Comparison of the multistep growth kinetics of the F cleavage site mutations in the presence or absence of trypsin. Monolayer cultures of Vero cells were infected at an input MOI of 0.01 PFU per cell with the indicated HMPVΔSH/G virus. Three conditions were evaluated: the multicycle growth and plaque assay were both performed in the presence of trypsin (A); multicycle growth was in the absence of trypsin, and the plaque assay was performed with trypsin (B); and multicycle growth and the plaque assay were both performed in the absence of trypsin (C). Supernatant aliquots (0.5 ml out of a total medium volume of 2 ml per well) were taken on the indicated days postinfection and replaced by an equivalent volume of fresh medium with or without trypsin as appropriate. The samples were flash frozen and analyzed later in parallel by plaque assay. Each time point was represented by two wells, and each virus titration was done in duplicate. Means are shown. The standard errors were calculated but the bars are not shown, because the errors were very small and the bars were obscured by the symbols. w/o, without.

FIG. 3.

Western blot analysis of F protein expressed in Vero cells infected with the rHMPVΔSH/G F cleavage site mutants in the presence or absence of trypsin. Cell lysates (A) or clarified medium supernatants (B) were analyzed to detect intracellular and virion-associated F protein, respectively. The samples were denatured, reduced, and analyzed on 4 to 12% polyacrylamide gels. Following electrotransfer, membranes were incubated with a hamster antiserum raised against an HPIV1 vector expressing the HMPV F protein (27) to detect the F protein (A, upper panel, and B) or with a rabbit antiserum raised against sucrose-purified HMPV virions (A, lower panel) to detect the M2-1 protein as a loading control. Bound antibodies were detected with a peroxidase-conjugated goat anti-rabbit or anti-hamster immunoglobulin G and were visualized by chemiluminescence. The HMPV proteins are indicated on the right, and the positions of molecular markers (in kilodaltons) are shown on the left.

FIG. 4.

Kinetics of replication of rHMPV and the ΔSH/G-F_WT and ΔSH/G-F_AMPV-A mutants in the upper and lower respiratory tracts of African green monkeys. Two animals (rHMPV group) or four animals (ΔSH/G-F_WT and ΔSH/G-F_AMPV-A groups) were each inoculated on day 0 by the combined intranasal and intratracheal routes with a 1-ml inoculum per site containing 10⁶ PFU of the indicated virus. Results from 10 additional animals that were inoculated with rHMPV under identical conditions in previous experiments were included. The nasopharyngeal swab (A) and tracheal lavage (B) specimens were taken on the indicated days, and the titers of shed virus were quantified by plaque assay. The detection limit was 0.7 log₁₀ PFU/ml.