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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.

To study the role of the precursor to the membrane protein (prM) in flavivirus maturation, we inhibited the proteolytic processing of the Murray Valley encephalitis (MVE) virus prM to membrane protein in infected cells by adding the acidotropic agent ammonium chloride late in the virus replication cycle. Viruses purified from supernatants of ammonium chloride-treated cells contained prM protein and were unable to fuse C6/36 mosquito cells from without. When ammonium chloride was removed from the cells, both the processing of prM and the fusion activity of the purified viruses were partially restored. By using monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of MVE virus, we found that at least three epitopes were less accessible to their corresponding antibodies in the prM-containing MVE virus particles. Amino-terminal sequencing of proteolytic fragments of the E protein which were reactive with sequence-specific peptide antisera or MAb enabled us to estimate the site of the E protein interacting with the prM to be within amino acids 200 to 327. Since prM-containing viruses were up to 400-fold more resistant to a low pH environment, we conclude that the E-prM interaction might be necessary to protect the E protein from irreversible conformational changes caused by maturation into the acidic vesicles of the exocytic pathway.