California serogroup viruses: Gb mediated fusion and entry

Members of the California serogroup of orthobunyaviruses, including La Crosse (LAC) and Tahyna (TAH) viruses, are significant human pathogens in areas where their mosquito vectors are endemic. Previous studies using wild-type LAC and TAH181/57, a highly neurovirulent strain with low neuroinvasiveness have demonstrated that the neuroinvasive phenotype maps to the M segment. To further define the role of the M segment in fusion and entry, a panel of recombinant M segment constructs were prepared using LAC, TAH181/57, and V22F, a monoclonal-resistant variant of LAC with deficient fusion function. These M segment constructs were tested in two surrogate assays for virus entry: a cell-to-cell fusion assay based on T7-luciferase expression, and a pseudotype transduction assay based on the incorporation of the bunyavirus glycoproteins on an Murine Leukemia Virus (MLV) backbone. Both assays demonstrated that Gc is the principal determinant of virus fusion and cell entry. Further, the region delineated by amino acids 860-1442, which corresponds to the membrane proximal two-thirds of Gc, is key to these processes. These results, coupled with structural modeling suggesting homologies between the carboxy region of Gc and the fusion protein (E1) from two alphaviruses (Semliki Forest virus and Sindbis virus), suggest that the LAC Gc functions as a type II fusion protein. Using the predicted structural model of Gc, an amino acid region (1066-1087) that shares a homologous structural location with the fusion domain of SFV E1 was identified as the putative fusion domain for LAC Gc. To examine the role of this region in fusion and entry, a panel of LAC point mutant M segments were prepared and examined for total and surface expression by IFA and flow cytometry. These constructs were then examined in our cell-to-cell fusion and pseudotype transduction assays. The results demonstrated mutations within this putative fusion domain either disrupted or abrogated fusion. In total, these results combined with computational modeling, suggest the hydrophobic region (1066-1087) of LAC Gc functions as the fusion peptide. These studies provided a better understanding of LAC virus entry and highlighted similarities between disparate viral fusion proteins