The assembly of Sindbis virus

Sindbis virus, the prototype of the alphaviruses, is a precise and complex three dimensional structure. The virion is made up of 240 copies of each of three structural proteins (E1,E2 and C) in a 1:1:1 stoichiometric arrangement, a membrane bilayer and a single copy of plus polarity single stranded RNA. The three virus proteins are organized as a double shelled icosahedron (Paredes et al., 1993; Paredes et al., 1998). The envelope glycoproteins E1 and E2 are organized as heterotrimers. Eighty of the E1-E2 heterotrimers are organized in a T=4 icosahedral lattice through E1-E1 protein associations which interconnect all of the heterotrimers (Anthony and Brown, 1991; Anthony, Paredes, and Brown, 1992). The membrane bilayer is derived from the host cell during virus assembly and is situated between the outer T=4 icosahedral protein shell of E1 and E2 glycoproteins and the inner T=4 icosahedral shell composed of capsid protein (C) (Choi et al., 1991; Coombs and Brown, 1987; Paredes et al., 1993; Paredes, Simon, and Brown, 1992) . The envelope glycoproteins are both type 1 membrane spanning proteins and the E2 glycoprotein has a 33 amino acid endodomain which specifically interacts with the capsid protein locking the outer protein shell to the inner protein shell (Lee and Brown, 1994; Lee et al., 1996; Lopez et al., 1994).  The alphaviruses are not typical of membrane containing viruses the majority of which are well described as membrane bilayers with associated virus proteins. The alphaviruses are protein icosahedra with an associated lipid bilayer.

The assembly of the complex, double shell, membrane containing structure involves numerous and highly specific protein-protein interactions occurring through two separate pathways (Strauss and Strauss, 1994). The virus structural proteins are synthesized from a subgenomic polycistronic RNA with the potential of producing all the structural proteins as a single polypeptide. As the nascent protein is produced, the capsid protein located at the NH terminal end cuts itself from the developing polyprotein utilizing a proteolytic activity contained in its structure. The freed capsid protein assembles together with progeny RNA to produce a nucleocapsid which comprises the inner protein shell. The remainder of the polyprotein containing the sequences of glycoproteins E1 and E2 is integrated into the membranes of the cell endoplasmic reticulum (ER). In the ER the proteins are proteolytically processed to form PE2, the precursor to E2, and E1. The glycoprotein E1 folds from a fully extended form into a more compact form and then binds PE2 to create a heterodimer. The heterodimer forms trimers (3 copies each of E1 and PE2) and E1 continues folding through disulfide-bridged intermediates into a compact, energy rich conformation (Carleton et al., 1997; Mulvey and Brown, 1996). After formation of the heterotrimer, and E1 folding is complete the glycoprotein trimers are exported from the ER to the plasma membrane. En route PE2 is converted to E2 by a furin-like protease in the Trans Golgi Network and E1 is converted from a stable to a metastable form.

The plasma membrane is the point at which the final events in alphavirus assembly take place. The assembled nucleocapsid binds to the endodomain of the E2 glycoprotein in a two step interaction which is highly specific (Liu et al., 1996; Liu and Brown, 1993a; Lopez et al., 1994). The 33 amino acid E2 endodomain is a multi-functional structure. The E2 glycoprotein begins its maturation as PE2 which, in the endoplasmic reticulum, has two membrane spanning domains (Lilijestrom and Garoff, 1991). The first membrane spanning domain is composed of amino acids 365 to 390 and is the anchor domain for this glycoprotein (Rice et al., 1982). The second membrane spanning domain is predicted to be composed of amino acids 405 to 418 (Rost et al., 1995). The second membrane spanning domain contains a sequence which is essential for the specific recognition of virus capsid protein. Binding within this domain is the first of two steps in the binding of nucleocapsids to the E2 endodomain (Lopez et al., 1994). Nucleocapsid binding requires that this domain be removed from the cell membrane and exposed to the cytoplasm. We have demonstrated that this event occurs after the maturation of the spike trimer in the endoplasmic reticulum and its export from the ER (Liu and Brown, 1993b). The precise point in inethe secretory pathway at which the second domain is withdrawn from the cell membrane is not known. The removal of a hydrophobic domain from a membrane bilayer is a very energetically unfavorable event. We have shown that a phosphorylation-dephosphorylation event accompanies this process, however we have not been able to demonstrate that the event is phosphorylation dependent (Liu and Brown, 1993a). The sequence of amino acids connecting the two membrane spanning domains, the cytoplasmic loop, has been implicated as containing the sites for phosphorylation, and as encoding the sequence involved in the second tight binding domain for nucleocapsid binding (Lee et al., 1996). 

The interaction of nucleocapsid with E2 is virus strain specific which implies that a specific capsid protein site exists for E2 binding. Using site-directed mutagenesis we have identified a domain in the capsid protein of Sindbis virus which binds to the E2 endodomain (Lee and Brown, 1994). The mutations in the capsid protein Y180S/ E183G resulted in the production of virus particles with reduced infectivity and an altered interaction of capsid protein with the E2 endodomain. Membrane glycoproteins of the capsid protein mutant had normal functional properties of attachment and membrane fusion which led us to propose that the defect in these mutants was a failure to uncoat the nucleocapsid as the process of virus membrane/host membrane fusion took place. We postulated that this failure resulted from the altered (weakened) E2-capsid association (Lee and Brown, 1994). In the X-ray crystallographic structure produced by Choi et. al (Choi et al., 1991) the capsid protein domain which we identified as critical for E2-capsid association has a fold which places aromatic ring structures on opposite sides of a hydrophobic cleft. A conserved Tyrosine at position 400 in the E2 tail, accessible to binding in the capsid protein hydrophobic cleft suggested that an aromatic interaction was possible between the E2 Y400 and C- Y180/ W247 (Lee et al., 1996). If such an interaction existed it would suggest that the loss of one of the three aromatic structures participating in the association would significantly weaken the strength of the E2-C association. A critical role for the interaction of the aromatic E2 Y400 with capsid protein has been supported by experiments in which Y 400 has been exchanged for other amino acids . Aromatic substitutions supported virus production while non-aromatic substitutions resulted in the failure to assemble mature virions (Gaedigk-Nitschko and Schlesinger, 1991). Molecular modeling of the E2 endodomain into the capsid cleft containing C- Y180 and W 247 indicated that the E2 Y400 could penetrate to a point at which the postulated aromatic interaction could take place (Lee et al., 1996). Molecular modeling also implicated a hydrophobic interaction involving E2- L402 as participating in nucleocapsid binding (Lee et al., 1996).

We have attempted to further assess the role that the aromatic and hydrophobic interactions between residues in E2 and C play in virus maturation by reducing the distance between the point at which the E2 endodomain emerges from the membrane bilayer and E2 Y 400. The hypothesis is that shortening this distance will prevent the specific interaction and prevent virus maturation. To this end we have produced a deletion in the membrane proximal region of the E2 endodomain reducing the distance between E2 Y400 and the membrane by a single amino acid. We have investigated the effects of this deletion on the events leading to the binding of the E2 endodomain to capsid and the ensuing process of envelopment. To measure the critical spanning length of the E2 endodomain which positions the TPY domain into the putative capsid binding cleft, we have constructed a deletion mutant, )K391, in which a nonconserved Lysine (E2 K391) at the membrane/cytoplasm junction of the E2 tail has been deleted (Hernandez, 2000). This mutant was found to produce very low levels of virus from BHK-21 cells due to a defect in an unidentified step in nucleocapsid binding to the E2 endodomain. In contrast, )K391 produced wild type levels of virus from tissue cultured mosquito cells. We propose that the phenotypic differences displayed by this mutant in the two diverse host cells arise from fundamental differences in the lipid composition of the insect cell membranes which affect the physical and structural properties of membranes and thereby virus assembly. The data suggest that these viruses have evolved properties adapted specifically for assembly in the diverse hosts in which they grow.

Referances

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