Expression of Semliki Forest Virus E1 Protein inEscherichia coli

Abstract

Exposure of Semliki Forest virus 1 to mildly acidic conditions results in conformational changes of the viral spike proteins, which in turn leads to a pore formation across its membrane. The ability to form a pore has been ascribed to the ectodomain of the Semliki Forest virus (SFV) E1 spike protein. To elucidate whether the E1 protein per se is sufficient for low pH-dependent pore formation, we expressed E1 in Escherichia coli in an inducible manner using the pET11c expression system. The data obtained clearly showed that the E1 protein was expressed in the bacterial cell membrane and that exposure of E. coli expressing the SFV E1 protein to low pH (<6.2) resulted in a permeability change of the membrane. Thus, we conclude that the E1 protein of SFV per se is sufficient to promote pore formation under mildly acidic conditions.The entry of a virus into a host cell is an essential step in the chain of events leading to infection. A multitude of viruses use the endocytotic pathway to access host cells. As a model for the entry of enveloped animal viruses into cells, the ␣ virus Semliki Forest virus has been extensively studied (1). Once attached, the virus is internalized via coated vesicles and transferred to the endosome. Due to the acidic conditions within this organelle, the lipid envelope of SFV fuses with the endosomal membrane of the target cell (2). This low pH-induced fusion is mediated by the so called virus spikes (3-5). Each spike is a heterotrimer, being composed of the type I integral membrane glycoproteins E1 (50.786 kDa) and E2 (51.855 kDa), plus the peripheral glycoprotein E3 (11.369 kDa), which is associated with E2 (6). Several functions have been ascribed to the spike proteins; e.g. the E2 and E3 precursor protein p62 forms a heterodimer with E1 in the endoplasmic reticulum and is responsible for the transport of the complex to the plasma membrane (6). The E1 protein is involved in the acid-induced fusion of the viral and endosomal membranes (7-9).Under mildly acidic conditions (pH 5.8) the spike proteins undergo an irreversible conformational change that results in the dissociation of the E1/E2/E3 complex, the formation of an E1 homotrimer and the exposure of a fusion peptide on the E1 protein (7). This conformational change also leads to the formation of a pore, which causes an alteration in the permeability of the virion membrane or of a cell membrane expressing the spike proteins (10 -14). It has been suggested that this acid-induced pore formation plays a crucial role in the penetration and uncoating process of SFV (15).Several experiments have shown that pore formation is dependent on the ectodomain of the E1 spike protein (14,16,17).It has been speculated that the E1 protein per se would be sufficient for triggering acid-induced pore formation. So far, all attempts to express isolated E1 protein on the surface of eukaryotic cells have failed. The E1 protein was synthesized but retained in the endoplasmic reticulum, since efficient transport of the glycoproteins to the plasma membrane req...

Entry of alphaviruses at the plasma membrane converts the viral surface proteins into an ion-permeable pore that can be detected by electrophysiological analyses of whole-cell membrane currents

“…SF virus particles from which lipid and the E2 protein were removed by protease treatment in the presence of detergent did retain a residual infectivity (Omar & Koblet, 1988). Furthermore, it has been shown that SF virus E1 protein expressed in E. coli is incorporated into the bacterial plasma membrane and that these membranes become permeable to [ 3 H]choline at low pH (Nyfeler et al, 2001). Comparable results have been obtained in similar experiments using eukaryotic cells (Dick et al, 1996).…”

“…The molecular basis of this process has remained elusive. A series of analyses of changes in membrane permeability in cells that had accumulated alphavirus membrane proteins have been made by C. Kempf and coworkers (Kempf et al, 1987;Schlegel et al, 1991;Dick et al, 1996;Käsermann & Kempf, 1996;Nyfeler et al, 2001). These authors studied the permeability of the plasma membrane of cells late in infection.…”

“…3 H]choline as the water-soluble molecule (Nyfeler et al, 2001). Fusion of alphaviruses with membranes has a special requirement for cholesterol Ahn et al, 2002) and sphingolipid (Nieva et al, 1994).…”

The membrane proteins of flaviviruses form ion-permeable pores in the target membrane after fusion: identification of the pores and analysis of their possible role in virus infection

“…In vitro analyses of the disassembly of alphavirus cores indicated that a flow of protons through this pore from the endosome into the cytoplasm may be involved in the regulation of disassembly of alphavirus cores in vivo (Wengler & Wengler, 2002). These results and important earlier experiments (Lanzrein et al, 1993;Dick et al, 1996;Nyfeler et al, 2001) led us to conclude that the E1 protein of alphaviruses is not only responsible for fusion but probably has an additional function, namely the ability to form an ion-permeable pore in the target membrane after fusion (Wengler et al, 2003).…”

A short treatment of cells with the lanthanide ions La3+, Ce3+, Pr3+ or Nd3+ changes the cellular chemistry into a state in which RNA replication of flaviviruses is specifically blocked without interference with host-cell multiplication

“…The fusion proteins of alphaviruses (family Togaviridae ; reviewed by Schlesinger & Schlesinger, 2001) and flaviviruses (family Flaviviridae ; reviewed by Lindenbach & Rice, 2001) are class II fusion proteins. These proteins have two additional functions: they generate an icosahedral lattice on the viral surface (Lescar et al , 2001; Pletnev et al , 2001) and they form ion-permeable pores in the target membrane during virus entry (Carrasco, 1995; Nyfeler et al , 2001; Wengler et al , 2003). Alphaviruses can be adsorbed to cells, and exposure to low pH activates virus entry at the plasma membrane (White et al , 1980; Paredes et al , 2004).…”