Mutagenesis of a conserved fusion peptide-like motif and membrane-proximal heptad-repeat region of hepatitis C virus glycoprotein E1

Abstract

The E1E2 glycoprotein heterodimer of Hepatitis C virus mediates viral entry. E2 attaches the virus to cellular receptors; however, the function of E1 is unknown. We tested the hypothesis that E1 is a truncated class II fusion protein. We mutated amino acids within a predicted fusion peptide (residues 276-286) and a truncated C-terminal stem-like motif, containing a membrane-proximal heptad-repeat sequence (residues 330-347). The fusion peptide mutation F285A abolished viral entry, while mutation of other hydrophobic residues had no effect. Alanine replacement of heptad-repeat residues blocked entry in three of five cases, whereas substitution with the helix breaker, Pro, led to loss of entry function in all cases. The mutations did not affect glycoprotein expression, heterodimerization with E2 or global folding, in contrast to the effects of mutations in the fusion motifs of prototypical class II fusion proteins. Our data suggest that E1 is unlikely to function in an analogous manner to other class II fusion glycoproteins.Virus replication is initiated when viral attachment proteins bind to cellular receptors. In the case of enveloped viruses, water molecules bound to the polar headgroups of viral and cellular membranes exert strong repulsive forces when brought into close proximity. Viral fusion protein(s) overcome this hydration barrier by catalysing membrane fusion. Fusion proteins characteristically comprise two hydrophobic membrane-interactive regions: a C-terminal transmembrane domain (TMD) and a fusion peptide that is located either at the N terminus or internally (Kielian & Rey, 2006). X-ray crystallography has thus far revealed two structural classes of fusion glycoprotein (Kielian, 2006;Kielian & Rey, 2006;Skehel & Wiley, 2000;Stiasny & Heinz, 2006). Class I fusion proteins [e.g. human immunodeficiency virus 1 (HIV-1) gp41, influenza virus HA2] are helical, trimeric rods that project as spikes from the viral envelope. In the fusion-activated state, their N (fusion peptide-proximal) and C (TMD-proximal) termini become juxtaposed at one end of a helical hairpin core domain. Class II fusion glycoproteins (e.g. flavivirus E, alpha virus E1) comprise three domains rich in b-strands that lie roughly parallel to the viral membrane. At neutral pH, the metastable state of E, which has dual receptor-binding and fusion functions, is maintained in a homodimer by monomer-monomer interactions that sequester the fusion loop. In the case of alphaviruses, glycoprotein E2 mediates receptor binding, whereas the associated E1 trimer mediates fusion. E1 metastability is maintained through E1-E2 interactions. At low fusion pH, E and E1 have almost identical trimeric structures where membrane-inserted fusion loops are atop three uptilted protomers. Trimerization creates three surface-exposed hydrophobic grooves along the trimer axis for the antiparallel packing of the TMD-proximal amphipathic a-helical stem to form a hairpin. Thus, hairpin formation is employed by both classes of fusion glycoprotein to appose membrane-associ...

Disulfide Bonds in Hepatitis C Virus Glycoprotein E1 Control the Assembly and Entry Functions of E2 Glycoprotein

“…Furthermore, in contrast to flaviviruses and alphaviruses (14), there is no cleavage of the companion during maturation of the viral particle (6,61), suggesting that this protein could also be involved in virus entry. It has also been shown that E1 provides some help in the fusion process of HCV particles (8,9,10). Here, we show that the companion protein E1 can also affect E2 by potentially modulating its capacity for binding to CD81 coreceptor, although the mechanism is still not clear.…”

“…In contrast, the role of the E1 subunit in HCV entry remains ill defined. However, E1 has also been shown to play a role in the fusion process (8,9,10). HCV has been proposed to contain a class II fusion protein (5).…”

“…In contrast, the role of the E1 companion subunit in HCV remains poorly defined. However, E1 has also been proposed to be involved in the fusion process (8,9,10).…”

Crystal structure of glycoprotein E2 from bovine viral diarrhea virus

“…In the absence of an obvious fusion motif, a key question regarding the pestivirus fusion mechanism is how the viral fusion apparatus anchors itself in the host cell membrane. In HCV, a hydrophobic sequence in E1 (residues 276-286) has been proposed to function as a fusion motif (37). If this is the case and E1 is the fusion protein, E2 is likely to function as a coeffector of fusion providing structural integrity to the fusion complex (Fig.…”

Identification of Interactions in the E1E2 Heterodimer of Hepatitis C Virus Important for Cell Entry

“…Previous studies have never identified this region as important for E1E2 function. Indeed, only the tmd, juxtamembrane region (49), and the potential peptide fusion in E1 (16,17) have been studied in depth. Our studies demonstrate that the AIL motif of E1 is important for the membrane fusion process (Fig.…”