Summary auto-generated
This study investigates the fusion activity of flaviviruses, particularly tick-borne encephalitis (TBE) and Japanese encephalitis (JE) viruses, using a fusion-from-without assay with mosquito C6/36 cells. The researchers found that both viruses require acidic pH (optimal around 6.2-6.3) to induce membrane fusion and polykaryocyte formation. Two groups of monoclonal antibodies targeting distinct epitopes on the E envelope protein inhibited fusion activity, with one group targeting the highly conserved fusion-active sequence. Analysis of five TBE virus escape mutants revealed that one showed reduced fusion activity while another exhibited altered pH threshold. Critically, immature virions containing the prM precursor protein—generated by growing TBE virus in ammonium chloride or obtained from Langat virus—displayed no fusion activity. These findings suggest that proteolytic processing of prM to mature M protein is necessary for generating fusion-competent virions. The results indicate that flavivirus fusion depends on pH-induced conformational changes in the E protein and requires the maturation step involving prM cleavage.
Key findings
- Flavivirus fusion requires acidic pH (6.2-6.3 optimal) and occurs in mosquito cells at 40°C, indicating pH-dependent conformational changes in the E protein
- Two groups of monoclonal antibodies targeting distinct non-overlapping epitopes on protein E can inhibit fusion, with one targeting the highly conserved fusion-active sequence
- Immature virions containing the prM precursor protein lack fusion activity, suggesting proteolytic cleavage of prM to M is essential for generating fusion-competent virions
- Monoclonal antibody escape mutants revealed that specific amino acid substitutions can alter fusion pH threshold or reduce fusion activity
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Abstract
The fusion activity of flaviviruses [tick-borne encephalitis (TBE) virus and Japanese encephalitis virus] was assessed by inducing fusion from without of C6/36 mosquito cells with purified virus preparations. Membrane fusion and polykaryocyte formation was observed only after incubating the viruses at acidic pH. Two groups of monoclonal antibodies reacting with distinct non-overlapping antigenic domains on the TBE virus protein E inhibited fusion from without. One of these domains contains the most highly conserved and putative fusion-active sequence of the flavivirus protein E. Of five TBE virus monoclonal antibody escape mutants, each defined by a single amino acid substitution in the envelope protein E, one revealed a reduced fusion activity and another one a lower pH threshold. TBE virus grown in the presence of ammonium chloride as well as Langat virus purified from the supernatant of infected chick embryo cells contained the precursor of protein M (prM) rather than M itself. These immature virions did not cause fusion from without, suggesting that the proteolytic processing of prM may be necessary for the generation of fusion-competent virions.
† Present address: Centers for Disease Control, Center for Infectious Diseases, CID, Division of Vector-Borne Viral Diseases, P.O. Box 2087, Fort Collins, Colorado 80522, U.S.A.