Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion

Abstract

Alphaviruses have the ability to induce cell-cell fusion after exposure to acid pH. This observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid pH after incorporation into a cell endosome. We have investigated the requirements for the induction of virus-mediated, low pH-induced cell-cell fusion and cell-virus fusion. We have correlated the pH requirements for this process to structural changes they produce in the virus by electron cryo-microscopy. We found that exposure to acid pH was required to establish conditions for membrane fusion but that membrane fusion did not occur until return to neutral pH. Electron cryo-microscopy revealed dramatic changes in the structure of the virion as it was moved to acid pH and then returned to neutral pH. None of these treatments resulted in the disassembly of the virus protein icosahedral shell that is a requisite for the process of virus membrane-cell membrane fusion. The appearance of a prominent protruding structure upon exposure to acid pH and its disappearance upon return to neutral pH suggested that the production of a "pore"-like structure at the fivefold axis may facilitate cell penetration as has been proposed for polio (J. Virol. 74 (2000) 1342) and human rhino virus (Mol. Cell 10 (2002) 317). This transient structural change also provided an explanation for how membrane fusion occurs after return to neutral pH. Examination of virus-cell complexes at neutral pH supported the contention that infection occurs at the cell surface at neutral pH by the production of a virus structure that breaches the plasma membrane bilayer. These data suggest an alternative route of infection for Sindbis virus that occurs by a process that does not involve membrane fusion and does not require disassembly of the virus protein shell.

Membrane Uncoating of Intact Enveloped Viruses

“…The authors of this paper (43) suggested that classical membrane fusion did not occur but that entry was similar to that seen for poliovirus (19), which has no membrane. The E1 and E2 protein icosahedron cover the alphavirus membrane so it unavailable for contact with the host cell membrane, but changes of the E1 and E2 proteins after binding of the virion may solve that problem.…”

“…In a paper that addressed mainly the effects of pH changes on virus structure, a final section showed transmission electron micrographs of Sindbis viral entry at neutral pH (43). Sindbis virus strain SVHR produces virus with a low particle/ PFU ratio, usually between 1 and 10 (18).…”

“…Sindbis virus strain SVHR produces virus with a low particle/ PFU ratio, usually between 1 and 10 (18). For the studies on Sindbis virus entry at pH 7.2 (43), including the micrographs shown in Fig. 4, the SVHR strain of Sindbis virus was used, and the particle/PFU ratio was 1 (D. T. Brown, personal communication).…”

“…The latter is generally the favorite model (33). After low-pH treatment, alphavirus membranes fuse with cell plasma membranes or with liposomes (which lack receptors) (5,43,53). That alphaviruses can enter the cell surface at neutral pH is supported by the fact that the micrographs show entry at the cell surface at pH 7.2.…”

Characterization of an early-stage fusion intermediate of Sindbis virus using cryoelectron microscopy

“…The structures of these viruses at pH values ∼5.0−5.9 have been examined using cryoelectron microscopy (cryo-EM) and 3D reconstruction techniques (22)(23)(24)(25); however, low-pH-triggered conformational rearrangements of the virus in the context of a target membrane are rarely investigated via these techniques (26). Here we present a cryo-EM study of SINV TE12 in complex with a liposomal membrane at pH 6.4.…”