Membrane Fusion Tropism and Heterotypic Functional Activities of theNipah VirusandHendra VirusEnvelope Glycoproteins

Abstract

The paramyxoviruses are enveloped, negative-stranded RNA-containing viruses and include a variety of important human and animal pathogens. These viruses contain two membrane-anchored envelope glycoproteins needed for efficient infection of a receptive host cell: an attachment glycoprotein which may be designated either the hemagglutinin-neuraminidase protein (HN), the hemagglutinin protein (H), or the G protein, depending on the particular paramyxovirus species, and the F glycoprotein, which facilitates the pH-independent membrane fusion event between the virion and host cell during virus infection, resulting in the entry of the nucleocapsid into the cytoplasm (reviewed in references 27 and 29). In a related process, cells expressing the fusion and attachment glycoproteins at their surfaces can mediate the formation of giant cells (syncytia). For most paramyxoviruses, efficient membrane fusion requires the presence of both the fusion and attachment glycoproteins, with the exception of the detectable F-mediated fusion in the absence of HN seen with the simian virus 5 (SV5) system (42). The details of how the attachment and fusion glycoproteins of the paramyxoviruses function in concert in mediating membrane fusion are not fully understood. For the most part, this interaction is type specific, and membrane fusion activity mediated by coexpression (mixing) of the fusion and attachment glycoproteins from different paramyxoviruses (heterotypic) is rarely seen. Although some examples have been noted, the potency of this fusion process is considerably lower than that mediated by the fusion and attachment glycoproteins from the same virus (homotypic) (2, 40).To date, more is known about the important functional domains of the fusion glycoproteins that are involved in driving virion-host cell membrane fusion and their predicted fusogenic conformations than about the attachment glycoproteins. The paramyxovirus fusion proteins are type I membrane glycoproteins existing as trimeric oligomers with considerable hydrophobicity, and the attachment glycoproteins are type II proteins with a tetrameric oligomeric configuration (12,36,37,47). Both proteins contain several potential N-linked glycosylation recognition sequences. Although it is generally presumed that the attachment protein must contact the fusion protein to induce conformational changes in F, evidence of a physical association between these glycoproteins has been observed with limited success and only with Newcastle disease virus (NDV) (49), Human parainfluenza virus (hPIV) (56), and, most recently, Measles virus (MeV) (44).Recently, two newly emerging paramyxoviruses that were identified in cases of severe respiratory and encephalitic diseases in animals and humans have been described; they are now known as Hendra virus (HeV) and Nipah virus (NiV)

The Nipah Virus Fusion Protein Is Cleaved within the Endosomal Compartment

“…Although the NiV F protein has a single arginine at the cleavage site ( Fig. 1), NiV infection is not restricted to the respiratory tract and does not require exogenous trypsin for its growth in cell culture (5,7,13). This suggests that activation of the NiV F protein differs from what is known for other paramyxoviral and orthomyxoviral fusion proteins.…”

“…For this, Vero cells were cultivated for several weeks under serum-free conditions and either infected with NiV or cotransfected with the NiV F and G genes. Only if the F protein is proteolytically processed can fusogenic G/F 1,2 complexes on the surface of infected or transfected cells form and mediate fusion with adjacent receptor-bearing cells (8,13). To analyze cleavage of the NiV F protein under serum-free conditions, transfected cells were metabolically labeled with [ 35 S]methionine and [ 35 S]cysteine for 10 min and incubated for 2 h to allow the protein to be processed.…”

Biochemical, Conformational, and Immunogenic Analysis of Soluble Trimeric Forms of Henipavirus Fusion Glycoproteins

“…The predicted ectodomains of the NiV and HeV F sequences (10) were codon optimized and synthesized by (Geneart Inc., Germany). The NiV and HeV F sequences were synthesized on the basis of sequences cloned early (11,12), which differed from sequences published later. These changes were N67D and N305D in NiV F and D255G and A263T in HeV F. The predicted TM anchor domain (residues 488 to 510) and the C-terminal cytoplasmic tail (CT) domain (residues 511 to 546) (10) of the NiV and HeV F-coding sequences were replaced by either the S-peptide tag (KETAAAKFERQHMDS) or the GCNt motif (MKQIEDKIEEILSKIYHIENEIARIKKLIGE) (33), followed by a factor Xa protease cleavage site (IEGR) and the S-peptide tag, generating NiV or HeV sF and NiV or HeV sF GCNt .…”

“…Recombinant vaccinia viruses expressing full-length NiV F (vKB7) and HeV F (vKB1) have been previously described (11,12). Polyclonal rabbit antisera against HeV F 1 or F 2 that are NiV cross-reactive have been described previously (11,12). Rabbit anti-S-peptide-tag antibody, horseradish peroxidase (HRP) conjugated, was from Bethyl Laboratories, Inc., Montgomery, TX.…”

“…Because sF GCNt could be properly cleaved in vitro by trypsin cleavage, shown earlier, we could examine whether the HeV and NiV sF GCNt could be cleaved and triggered to fold into a postfusion configuration by applying heat (50°C, 15 min) and trypsin. We previously demonstrated that short peptides derived from the HRB sequence of HeV and NiV F (FC2 peptides) were potent inhibitors of virus-mediated cell-cell fusion as well as live virus infection (10,12). Here we used a biotinylated NiV F-derived FC2 peptide and included it during a series of heat and trypsin treatments of sF GCNt carried out in several combinations in an attempt to trap and capture an intermediate form during pre-to postfusion conversion of the sF glycoprotein by complex formation with the HRA domain during sF triggering.…”