Point Mutations in the Paramyxovirus F Protein That Enhance Fusion Activity Shift the Mechanism of Complement-Mediated Virus Neutralization

Abstract

bParainfluenza virus 5 (PIV5) activates and is neutralized by the alternative pathway (AP) in normal human serum (NHS) but not by heat-inactivated (HI) serum. We have tested the relationship between the fusion activity within the PIV5 F protein, the activation of complement pathways, and subsequent complement-mediated virus neutralization. Recombinant PIV5 viruses with enhanced fusion activity were generated by introducing point mutations in the F fusogenic peptide (G3A) or at a distal site near the F transmembrane domain (S443P). In contrast to wild-type (WT) PIV5, the mutant G3A and S443P viruses were neutralized by both NHS and HI serum. Unlike WT PIV5, hyperfusogenic G3A and S443P viruses were potent C4 activators, C4 was deposited on NHS-treated mutant virions, and the mutants were neutralized by factor B-depleted serum but not by C4-depleted serum. Antibodies purified from HI human serum were sufficient to neutralize both G3A and S443P viruses in vitro but were ineffective against WT PIV5. Electron microscopy data showed greater deposition of purified human antibodies on G3A and S443P virions than on WT PIV5 particles. These data indicate that single amino acid changes that enhance the fusion activity of the PIV5 F protein shift the mechanism of complement activation in the context of viral particles or on the surface of virus-infected cells, due to enhanced binding of antibodies. We present general models for the relationship between enhanced fusion activity in the paramyxovirus F protein and increased susceptibility to antibody-mediated neutralization.

Identification of Residues in the Human Respiratory Syncytial Virus Fusion Protein That Modulate Fusion Activity and Pathogenesis

“…A study published in 2010 showed that mutation of a single amino acid in Sendai virus F, which created a hyperfusogenic mutant, was also linked to enhanced pulmonary inflammation in vivo (21). A more recent report on parainfluenza virus 5 described a mechanism by which a single amino acid mutation in F causing greater fusion activity also resulted in differential recognition of the virus by the complement pathway (45). We showed that two amino acid changes in F dramatically altered the host response to RSV.…”

The neutralizing capacity of antibodies elicited by parainfluenza virus infection of African Green Monkeys is dependent on complement

“…It is known that there are substantial structural differences between immature and mature forms of the viral glycoproteins (Lamb and Jardetzky, 2007; Mottet et al, 1986). Likewise, point mutations that destabilize the F protein can alter the binding of human antibodies and increase dependence on C’ for neutralization (Johnson et al, 2013). Thus, the inherent conformation of the parainfluenza glycoproteins and epitope accessibility due to virion structures could be a major factor in eliciting antibodies that are initially of low avidity and more dependent on C’ for neutralization.…”

“…It is not clear why, despite strong activation of C’, the pathway does not progress to virion lysis. This could reflect failure to progress through the C’ pathway to MAC formation or lack of a functional membrane attack complex (MAC) due to the presence of C’ inhibitors within the virion (Johnson et al, 2013). …”

“…Furthermore, the rubulavirus F protein can dictate which arm of the C’ pathway is activated. This was evident by our recent finding that a single point mutation in the ectodomain of the PIV5 F protein led to increased fusion activity, but also led to enhanced binding of IgG contained in normal human sera (NHS) and a subsequent shift in C’ activation from the alternative to the classical pathway (Johnson et al, 2013). …”

“…Prior work has shown that the rubulavirus attachment protein (Hemagglutinin-Neuraminidase; HN) and the fusion protein (F) can both contribute to activation of the alternative pathway (McSharry et al, 1981; Hirsch et al, 1986; Johnson et al, 2008; 2013). For PIV5 and MuV, the extent of alternative pathway activation is directly related to the loss of sialic acid on particles due to the presence of neuraminidase activity in the HN protein (McSharry et al, 1981; Hirsch et al, 1986).…”