Mutagenesis of the La Crosse Virus glycoprotein supports a role for Gc (1066–1087) as the fusion peptide

Abstract

The La Crosse Virus (LACV) M segment encodes two glycoproteins (Gn and Gc), and plays a critical role in the neuropathogenesis of LACV infection as the primary determinant of neuroinvasion. A recent study from our group demonstrated that the region comprising the membrane proximal two-thirds of Gc, amino acids 860-1442, is critical in mediating LACV fusion and entry. Furthermore, computational analysis identified structural similarities between a portion of this region, amino acids 970-1350, and the E1 fusion protein of two alphaviruses: Sindbis virus and Semliki Forrest virus (SFV). Within the region 970-1350, a 22-amino-acid hydrophobic segment (1066-1087) is predicted to correlate structurally with the fusion peptides of class II fusion proteins. We performed site-directed mutagenesis of key amino acids in this 22-amino acid segment and determined the functional consequences of these mutations on fusion and entry. Several mutations within this hydrophobic domain affected glycoprotein expression to some extent, but all mutations either shifted the pH threshold of fusion below that of the wild-type protein, reduced fusion efficiency, or abrogated cell-to-cell fusion and pseudotype entry altogether. These results, coupled with the aforementioned computational modeling, suggest that the LACV Gc functions as a class II fusion protein and support a role for the region Gc 1066-1087 as a fusion peptide.

Molecular Architecture of the Bipartite Fusion Loops of Vesicular Stomatitis Virus Glycoprotein G, a Class III Viral Fusion Protein

“…the single tryptophan, Trp-101) were not mutated in this study due to predicted problems in protein transport or assembly. In the case of LaCrosse virus, an alanine substitution of the single tryptophan (Trp-1066) in the predicted fusion loop of the glycoprotein (Gc) did not affect cell surface expression yet completely abrogated fusion (47). Substitutions other than alanine were not tested in this study.…”

Acid-Activated Structural Reorganization of the Rift Valley Fever Virus Gc Fusion Protein

“…Exposure of cells overexpressing RVFV Gn and Gc to low pH results in the formation of multinucleated cells (11). Acid-induced syncytium formation has also been reported for other members of the Bunyaviridae family, including Uukuniemi virus (UUKV, Phlebovirus genus), La Crosse virus (Orthobunyavirus genus), and Hantaan virus (Hantavirus genus) (1,39,42,48). In accordance with the hypothesis of acid-induced fusion activation, bunyaviruses are sensitive to acidotropic agents which raise the pH of intracellular endocytic compartments (55,58).…”

“…The Gc glycoprotein of bunyaviruses has been proposed to be a class II viral fusion protein (15,53). Similar to class II fusion proteins of alphaviruses and flaviviruses, Gc is predicted to be mainly composed of ␤-sheet structures, is synthesized from a polyprotein downstream of a companion protein (Gn), and assembles into a heterodimer in the ER (15,48,62,63).…”

Genetic characterization of orthobunyavirus Melao, strains BE AR633512 and BE AR8033, and experimental infection in golden hamsters (Mesocricetus auratus)

“…The Gc gene encodes glycoprotein Gc which is possibly responsible for viral adsorption to the vertebrate host (hamster) cell and is important in the neuropathogenesis of La Crosse virus (Plassmeyer et al 2007). Although strain BE AR633512 was found to be more closely related to BE AR8033 at the level of the Gc gene, the more intense histopathological alterations observed in hamsters infected with BE AR633512 compared with BE AR8033 might be explained by mutations along the Gc gene.…”