Proteolytic activation of tick-borne encephalitis virus by furin

Abstract

Flaviviruses are assembled intracellularly in an immature form containing heterodimers of two envelope proteins, E and prM. Shortly before the virion exits the cell, prM is cleaved by a cellular enzyme, and this processing step can be blocked by treatment with agents that raise the pH of exocytic compartments. We carried out in vivo and in vitro studies with tick-borne encephalitis (TBE) virus to investigate the possible role of furin in this process as well as the functional consequences of prM cleavage. We found that prM in immature virions can be correctly cleaved in vitro by recombinant bovine furin but that efficient cleavage occurs only after exposure of the virion to mildly acidic pH. The data suggest that exposure to an acidic environment induces an irreversible structural change that renders the cleavage site accessible to the enzyme. Cleavage by furin in vitro resulted in biological activation, as shown by a 100-fold increase in specific infectivity, the acquisition of membrane fusion and hemagglutination activity, and the ability of the envelope proteins to undergo low-pHinduced structural rearrangements characteristic of mature virions. In vivo, prM cleavage was blocked by a furin inhibitor, and infection of the furin-deficient cell line LoVo yielded only immature virions, suggesting that furin is essential for cleavage activation of flaviviruses.

Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control

“…CHIKV infection of cultured human cells was also shown to be inhibited by impairing the maturation of the CHIKV E2 surface glycoprotein using furin inhibitors (Ozden et al, 2008). A similar observation was previously reported using the flavivirus Tick-borne encephalitis virus (Stadler et al, 1997).…”

Structures and mechanisms in flavivirus fusion

“…The maturation cleavage results in the removal of slightly more than half of the N-terminal end of prM and occurs at a site corresponding to the consensus sequence for furin and related proteases (RXRIKR), which is also found in many other viral glycoproteins that require proteolytic cleavage for activation (reviewed by Klenk and Garten, 1994). The mechanism of cleavage has been investigated in some detail in vitro using immature TBE virus (secreted from cells treated with ammonium chloride to raise the pH in the TGN) and recombinant bovine furin (Stadler et al, 1997). These experiments confirmed the requirement for a slightly acidic pH (6.7 or lower) for cleavage and further showed that the exposure of immature virions to low pH apparently induced an irreversible conformational change to make the cleavage site accessible to the enzyme.…”

“…Therefore, even without exposure to acidic pH, the contacts at 3-fold axes might be strong enough (and those at the 2-fold axes weak enough) to allow the isolation of trimers under certain experimental conditions. In the TBE virus system, however, there is clearly a quantitative oligomeric switch from Triton X-100-stable dimers at neutral pH to trimers at acidic pH, and in all the experiments carried out so far, this change was irreversible (Allison et al, 1995a;Stiasny et al, 1996;Schalich et al, 1996;Stadler et al, 1997).…”

“…Immature virions containing uncleaved prM proteins can be isolated from infected cells by cell lysis (Wengler, 1989), but they can also be obtained in a secreted form from the supernatants of cells treated after infection with acidotropic agents, apparently because increasing the acidic pH in the TGN prevents the cleavage of prM (Shapiro et al, 1973;Randolph et al, 1990b;Guirakhoo et al, 1992;Heinz et al, 1994). prM-containing particles of several different flaviviruses have been characterized (Wengler, 1989;Randolph et al, 1990b;Guirakhoo et al, 1991;Guirakhoo et al, 1992;Heinz et al, 1994;Stadler et al, 1997), and their properties differ from those of mature virions in several important aspects. They exhibit significantly lower specific infectivity, HA activity, and fusion activity; and, in contrast to mature virions, acidic pH does not induce a change in the epitope reactivity pattern or in the oligomeric structure, suggesting that these changes are prevented by the presence of prM in immature virions.…”

An inactivated West Nile Virus vaccine derived from a chemically synthesized cDNA system

“…4b). The predominant band in these preparations is the envelope antigen (E) migrating in the 60 kDa range, the fainter bands representing the pre-membrane (prM) and the dimeric membrane (M) proteins (see also [26]).…”