Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

Abstract

Human metapneumovirus (hMPV) is a relatively recently identified paramyxovirus that causes acute upper and lower respiratory tract infection. Entry of hMPV is unusual among the paramyxoviruses, in that fusion is accomplished by the fusion (F) protein without the attachment glycoprotein (G protein). It has been suggested that hMPV F protein utilizes integrin ␣v␤1 as a cellular receptor. Consistent with this, the F proteins of all known hMPV strains possess an integrin-binding motif ( 329 RGD 331 ). The role of this motif in viral entry, infectivity, and pathogenesis is poorly understood. Here, we show that ␣5␤1 and ␣v integrins are essential for cell-cell fusion and hMPV infection. Mutational analysis found that residues R329 and G330 in the 329 RGD 331 motif are essential for cell-cell fusion, whereas mutations at D331 did not significantly impact fusion activity. Furthermore, fusion-defective RGD mutations were either lethal to the virus or resulted in recombinant hMPVs that had defects in viral replication in cell culture. In cotton rats, recombinant hMPV with the R329K mutation in the F protein (rhMPV-R329K) and rhMPV-D331A exhibited significant defects in viral replication in nasal turbinates and lungs. Importantly, inoculation of cotton rats with these mutants triggered a high level of neutralizing antibodies and protected against hMPV challenge. Taken together, our data indicate that (i) ␣5␤1 and ␣v integrins are essential for cell-cell fusion and viral replication, (ii) the first two residues in the RGD motif are essential for fusion activity, and (iii) inhibition of the interaction of the integrin-RGD motif may serve as a new target to rationally attenuate hMPV for the development of live attenuated vaccines. IMPORTANCEHuman metapneumovirus (hMPV) is one of the major causative agents of acute respiratory disease in humans. Currently, there is no vaccine or antiviral drug for hMPV. hMPV enters host cells via a unique mechanism, in that viral fusion (F) protein mediates both attachment and fusion activity. Recently, it was suggested that hMPV F protein utilizes integrins as receptors for entry via a poorly understood mechanism. Here, we show that ␣5␤1 and ␣v integrins are essential for hMPV infectivity and F proteinmediated cell-cell fusion and that the integrin-binding motif in the F protein plays a crucial role in these functions. Our results also identify the integrin-binding motif to be a new, attenuating target for the development of a live vaccine for hMPV. These findings not only will facilitate the development of antiviral drugs targeting viral entry steps but also will lead to the development new live attenuated vaccine candidates for hMPV.

Zinc Binding Activity of Human Metapneumovirus M2-1 Protein Is Indispensable for Viral Replication and Pathogenesis In Vivo

“…However, these tissues were positive for hMPV RNA (data not shown), as determined by realtime RT-PCR, which is not surprising, because real-time RT-PCR was much more sensitive than plaque assay. Similar results were observed for other hMPV mutants, such as receptor binding-defective rhMPVs and methyltransferase-defective rhMPVs, which were defective in receptor binding activity in fusion (F) protein and mRNA cap methyltransferase activity in the large (L) polymerase protein, respectively (31,35). Another possibility is that infectious hMPV has been cleared from nasal turbinate and lungs for these mutants at day 4 postinfection.…”

“…The replication and pathogenesis of rhMPV carrying mutations in the zinc binding motif were determined in cotton rats, the best small-animal model for hMPV (35,36). Briefly, 4-week-old SPF cotton rats were inoculated intranasally with 1.0 ϫ 10 6 PFU of WT rhMPV or rhMPV mutants.…”

“…Thus, the inhibition of zinc binding activity carries great potential as a novel approach to rationally attenuate hMPV for vaccine purposes. The phenotypes (growth kinetics, attenuation, genetic stability, and immunogenicity) of zinc binding-defective rhMPVs in vitro and in vivo were similar to those that have been observed for receptor binding-defective rhMPV and methyltransferase-defective rhMPVs (35,56). Future experiments are required to determine whether cellular immunity is involved in immune protection and to test the safety and immunogenicity of these promising vaccine candidates in nonhuman primates and humans.…”

Methyltransferase-Defective Avian Metapneumovirus Vaccines Provide Complete Protection against Challenge with the Homologous Colorado Strain and the Heterologous Minnesota Strain

“…aMPV-specific neutralizing antibody titers were determined using a plaque reduction neutralizing assay (35). The serum samples from turkeys were heat inactivated at 56°C for 30 min.…”

Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis

“…Similar to hRSV, inactivated vaccines are not suitable for hMPV because it causes enhanced lung damage upon reinfection by the same virus in animal models (47). In contrast, enhanced lung damage has not been observed for live attenuated vaccine candidates in animal models (28,(48)(49)(50). Thus, significant efforts have been devoted to developing live attenuated vaccines for hMPV.…”