There are 80 spikes on the surface of Sindbis virus arranged as an icosahedral surface lattice. Each spike consists of three copies of each of the glycoproteins E1 and E2. There are two glycosylation sites on E1 and two on E2. These four sites have been located by removal of the glycosylation recognition motifs using site-specific mutagenesis, followed by cryoelectron microscopy. The positions of these sites have demonstrated that E2 forms the protruding spikes and that E1 must be long and narrow, lying flat on the viral surface, forming an icosahedral scaffold analogous to the arrangement of the E glycoprotein in flaviviruses. This arrangement of E1 leads to both dimeric and trimeric intermolecular contacts, consistent with the observed structural changes that occur on fusion with host cell membranes, suggesting a similar fusion mechanism for alpha- and flaviviruses.

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.

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The alphaviruses are composed of two icosahedral protein shells, one nested within the other. A membrane bilayer derived from the host cell is sandwiched between the protein shells. The protein shells are attached to one another by protein domains which extend one of the proteins of the outer shell through the membrane bilayer to attach to the inner shell. We have examined the interaction of the membrane-spanning domain of one of the membrane glycoproteins with the membrane bilayer and with other virus proteins in an attempt to understand the role this domain plays in virus assembly and function. Through incremental deletions, we have reduced the length of a virus membrane protein transmembrane domain from its normal 26 amino acids to 8 amino acids. We examined the effect of these deletions on the assembly and function of virus particles. We found that progressive truncations in the transmembrane domain profoundly affected production of infectious virus in a cyclic fashion. We also found that membrane composition effects protein-protein and proteinmembrane interactions during virus assembly.Sindbis virus is an alphavirus and a member of the arboviruses, a group of viruses which are propagated in nature via a complicated life cycle involving insect vectors and mammalian hosts. Sindbis virus is simple in its composition but complex in its structure (42). The virion contains three structural proteins, E1, E2, and Capsid (C). These proteins are organized into two geometrically identical Tϭ4 icosahedral shells (32). The outer protein shell is composed of the glycoproteins E1 and E2, organized into trimers of heterodimers (2, 9, 34), and surrounds the inner shell, composed of protein C, which is assembled around the viral RNA. A host-derived membrane bilayer is situated between the concentric shells and is penetrated by the transmembrane (TM) domain anchors of each glycoprotein (19,36,41). The E2 glycoprotein contains a 33-amino-acid endodomain which specifically interacts with a hydrophobic cleft in the capsid protein (17,18,30,31). The interaction between E2 and C gives stability to the structure of the virus and plays a critical role in the formation of the outer virus protein shell around the preformed inner protein shell as the process of envelopment takes place (13). The membrane glycoprotein E1 is incorporated into the icosahedral outer protein shell in a highly constrained, energy-rich conformation (9, 28). This stored energy likely drives the process of cell penetration during infection. Physical and chemical treatments of the virus result in the conversion of the protein to a low-energy, nonnative configuration, which results in the loss of virus infectivity (3,12,28).The three virus structural proteins are synthesized from a subgenomic polycistronic message in the sequence NH-C-PE2

It is widely held that arboviruses such as the alphavirus Sindbis virus gain entry into cells by a process of receptor-mediated endocytosis followed by membrane fusion in the acid environment of the endosome. We have used an approach of direct observation of Sindbis virus entry into cells by electron microscopy and immunolabeling of virus proteins with antibodies conjugated to gold beads. We found that upon attaching to the cell surface, intact RNA-containing viruses became empty shells that could be identified only by antibody labeling. We found that the rate at which full particles were converted to empty particles increased with time and temperature. We found that this entry event takes place at temperatures that inhibit both endosome formation and membrane fusion. We conclude that entry of alphaviruses is by direct penetration of cell plasma membranes through a pore structure formed by virus and, possibly, host proteins.

Cholesterol has been shown to be essential for the fusion of alphaviruses with artificial membranes (liposomes). Cholesterol has also been implicated as playing an essential and critical role in the processes of entry and egress of alphaviruses in living cells. Paradoxically, insects, the alternate host for alphaviruses, are cholesterol auxotrophs and contain very low levels of this sterol. To further evaluate the role of cholesterol in the life cycle of alphaviruses, the cholesterol levels of the alphavirus Sindbis produced from three different mosquito (Aedes albopictus) cell lines; one other insect cell line, Sf21 from Spodoptera frugiperda; and BHK (mammalian) cells were measured. Sindbis virus was grown in insect cells under normal culture conditions and in cells depleted of cholesterol by growth in serum delipidated by using Cab-O-sil, medium treated with methyl-␤-cyclodextrin, or serum-free medium. The levels of cholesterol incorporated into the membranes of the cells and into the virus produced from these cells were determined. Virus produced from these treated and untreated cells was compared to virus grown in BHK cells under standard conditions. The ability of insect cells to produce Sindbis virus after delipidation was found to be highly cell specific and not dependent on the level of cholesterol in the cell membrane. A very low level of cholesterol was required for the generation of wild-type levels of infectious Sindbis virus from delipidated cells. The data show that one role of the virus membrane is structural, providing the stability required for infectivity that may not be provided by the delipidated membranes in some cells. These data show that the amount of cholesterol in the host cell membrane in and of itself has no effect on the process of virus assembly or on the ability of virus to infect cells. Rather, these data suggest that the cholesterol dependence reported for infectivity and assembly of Sindbis virus is a reflection of differences in the insect cell lines used and the methods of delipidation.Sindbis virus, the prototypic Alphavirus, assembles highly symmetrical particles with an associated membrane of host cell origin. The infectious particle is composed of two nested icosahedral shells of Tϭ4 geometry with an intervening membrane bilayer (41). The three structural proteins which comprise the particle, E1, E2, and capsid, are found in a 1:1:1 stoichiometric ratio. The outer shell, composed of glycoproteins E1 and E2, and the nucleocapsid are associated with the outer protein shell through specific interactions of the E2 endodomain with the capsid protein (28)(29)(30)53). Both E1 and E2 are anchored into the membrane bilayer by transmembrane domains (44). During maturation of the virus, the glycoproteins E1 and E2 are processed and oligomerize into trimers of heterodimers and are delivered to the cell surface by the cellular exocytic pathway (6, 39). In mammalian cells, the glycoproteins are trafficked to the plasma membrane to unite with preformed nucleocapsids (5, 12). The maturat...