A Conserved Region between the Heptad Repeats of Paramyxovirus Fusion Proteins Is Critical for Proper F Protein Folding

Abstract

Paramyxoviruses are a diverse family which utilizes a fusion (F) protein to enter cells via fusion of the viral lipid bilayer with a target cell membrane. Although certain regions of F are known to play critical roles in membrane fusion, the function of much of the protein remains unclear. Sequence alignment of a set of paramyxovirus F proteins and analysis utilizing Block Maker identified a region of conserved amino acid sequence in a large domain between the heptad repeats of F 1 , designated CBF 1 . We employed site-directed mutagenesis to analyze the function of completely conserved residues of CBF 1 in both the simian virus 5 (SV5) and Hendra virus F proteins. The majority of CBF 1 point mutants were deficient in homotrimer formation, proteolytic processing, and transport to the cell surface. For some SV5 F mutants, proteolytic cleavage and surface expression could be restored by expression at 30°C, and varying levels of fusion promotion were observed at this temperature. In addition, the mutant SV5 F V402A displayed a hyperfusogenic phenotype at both 30°C and 37°C, indicating this mutation allows for efficient fusion with only an extremely small amount of cleaved, active protein. The recently published prefusogenic structure of PIV5/SV5 F [Yin, H.S., et al. (2006) Nature 439, 38-44] indicates that residues within and flanking CBF 1 interact with the fusion peptide domain. Together, these data suggest that CBF 1 -fusion peptide interactions are critical for the initial folding of paramyxovirus F proteins from across this important viral family, and can also modulate subsequent membrane fusion promotion.The family Paramyxoviridae comprises many diverse members, including well-known human pathogens such as measles, mumps and respiratory syncytial virus (RSV), as well as animal pathogens like parainfluenza virus 5 (PIV5/SV5), and the newly emerged, zoonotic Hendra and Nipah viruses. Hendra virus first emerged in 1994 and caused an outbreak of severe respiratory illness near Brisbane, Australia. This resulted in the deaths of fourteen horses and two out of three humans infected, succumbing either to respiratory illness or to viral meningoencephalitis (1,2). Nipah virus was responsible for an outbreak of viral encephalitis in Malaysia in 1998, which resulted in the deaths of 105 people and the preventative destruction of over one million swine (3). Hendra and Nipah virus are classified as NIAID priority pathogens and DHHS Select Agents, and no antiviral therapies currently exist for these fatal viruses. Hendra and Nipah are grouped into the Henipavirus genus within the family, due in part to the fact that while they possess 88% homology to each other, they share less than 30% homology with the rest of the family (4,5). † This work was supported by a grant from the National Institute of Allergy and Infectious Diseases (AI-51517) to R.E.D. A.E.G. was supported in part by a predoctoral fellowship from the American Heart Association, Ohio Valley Affiliate (0415223B).* To whom correspondence should be addres...

A Conserved Region in the F 2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation

“…Previous research in multiple laboratories has indicated a role for HRA in folding and fusion (47,49,56). We have recently demonstrated that there is a role for CBF 1 in F protein folding and homotrimerization (15). Two previous studies of paramyxovirus F proteins have included mutations of a small number of residues that fall within CBF 2.…”

Differential Rates of Protein Folding and Cellular Trafficking for the Hendra Virus F and G Proteins: Implications for F-G Complex Formation

“…Thus, slow folding kinetics for Hendra virus G may explain the delayed appearance of an endo H-resistant population. Unlike Hendra virus F, which folds as a trimer (13,14), Hendra virus G is a tetramer composed of two disulfidelinked dimers (7). To examine folding kinetics, cross-linking analysis (13) was performed at various time points post-metabolic labeling.…”

“…(C) Biotinylation analyses of Vero cells expressing both Hendra virus F and Hendra virus G-⌬32 were performed as described previously. (14). (B) Vero cells expressing Hendra virus G-⌬32 were labeled for 30 min and chased for the indicated times, and cross-linking analysis with DTSSP was performed as described previously (14).…”

“…(14). (B) Vero cells expressing Hendra virus G-⌬32 were labeled for 30 min and chased for the indicated times, and cross-linking analysis with DTSSP was performed as described previously (14). ϩ, present; Ϫ, absent.…”

Signal Peptide and Helical Bundle Domains of Virulent Canine Distemper Virus Fusion Protein Restrict Fusogenicity

“…Besides two heptad repeat regions (HRA and HRB) in the F 1 ectodomain, which are well known to play critical roles in paramyxovirus membrane fusion (2,11,30,39), other regions are only poorly characterized for their specific function in the fusion process itself. Interestingly, recently it has been demonstrated that a conserved region, located between both heptad repeats (HRA and HRB) of SV5 and Hendra virus F proteins, was important for regulating F protein expression and activity (13). In addition, single mutations in the globular head and outside both HR domains of SV5 and Newcastle disease virus (NDV) have been shown to allow the F protein to activate the fusion process in the absence of the receptor-binding protein (HN) (20,29).…”