The formation of intramolecular disulfide bridges is required for induction of the Sindbis virus mutant ts23 phenotype

Abstract

The Sindbis virus envelope protein spike is a hetero-oligomeric complex composed of a trimer of glycoprotein E1-E2 heterodimers. Spike assembly is a multistep process which occurs in the endoplasmic reticulum (ER) and is required for the export of E1 from the ER. PE2 (precursor to E2), however, can transit through the secretory pathway and be expressed at the cell surface in the absence of E1. Although oligomer formation does not appear to be required for the export of PE2, there is evidence that defects in E1 folding can affect PE2 transit from the ER. Temperature-sensitive mutant ts23 of Sindbis virus contains two amino acid substitutions in E1, while PE2 and capsid protein have the wild-type sequence; however, at the nonpermissive temperature, both E1 and PE2 are retained within the ER and can be isolated in protein aggregates with the molecular chaperone GRP78-BiP. We previously demonstrated that the temperature sensitivity for ts23 was lost as oligomer formation took place at the permissive temperature, suggesting that temperature sensitivity is initiated early in the process of viral spike assembly (M. Carleton and D. T. Brown, J. Virol. 70:952-959, 1996). Experiments described herein investigated the defects in envelope protein maturation that occur in ts23infected cells and which result in retention of both envelope proteins in the ER. The data demonstrate that in ts23-infected cells incubated at the nonpermissive temperature, E1 folding is disrupted early after synthesis, resulting in the rapid incorporation of both E1 and PE2 into disulfide-stabilized aggregates. Furthermore, the aberrant E1 conformation which is responsible for induction of the ts phenotype requires the formation of intramolecular disulfide bridges formed prior to E1 association with PE2 and the completion of E1 folding.

GRP78/BiP/HSPA5/Dna K is a universal therapeutic target for human disease

“…Based on an unbiased search of the National Library of Medicine database it is self evident that GRP78 and evolutionary conserved homologues of GRP78 play essential roles in the biology and life cycles of viruses, bacteria, protosoal and yeast cells as well as higher eukaryotic cells, in particularly tumor cells that express high levels of many "activated" oncogenic signaling proteins (Roux, 1990Earl et al, 1991Anderson et al, 1992;Hogue and Nayak, 1992;Xu et al, 1997;Carleton and Brown, 1997;Xu et al, 1998;Bolt, 2001;Bredèche et al, 2001;Shen et al, 2002;Dimcheff et al, 2004;He, 2006;Spurgers et al, 2010;Moreno and Tiffany-Castiglioni, 2014;Reid et al, 2014). The bacterial GRP78 homologue, Portions of cells were: (i) infected with virus followed 1 h afterwards by treatment with vehicle or with OSU-03012 (1.0 mM) and sildenafil (2 mM).…”

“…In tumor cells with very high protein expression levels or in the reproduction cycle of viruses in higher eukaryotes, the GRP78 protein, together with other chaperone proteins including HSP27, HSP40, HSP60, HSP90, GRP94 and HSP70, is essential for the ATP-dependent correct folding of highly expressed host and viral proteins, including the virus capsid proteins. (Roux, 1990Earl et al, 1991Anderson et al, 1992;Hogue and Nayak, 1992;Xu et al, 1997;Carleton and Brown, 1997;Xu et al, 1998;Bolt, 2001 Fig. 7.…”

“…As noted in the Introduction section, the expression of GRP78 is essential for replication and productive virus release for many "well known" pathogenic viruses such as Ebola; Cytomegalovirus; Chikungunya, Measles; HIV; Influenza; Lassa; and Marburg (Roux, 1990;Earl et al, 1991;Moreno and Tiffany-Castiglioni, 2014;Anderson et al, 1992;Hogue and Nayak, 1992;Xu et al, 1997;Carleton and Brown, 1997;Xu et al, 1998;Bolt, 2001;Bredèche et al, 2001;Shen et al, 2002;Dimcheff et al, 2004;He, 2006;Spurgers et al, 2010;Reid et al, 2014). Some viruses such as Dengue fever virus are reported to actually infect cells through a cell surface expressed GRP78 protein (Quinones et al, 2008).…”

“…GRP78, as a chaperone, also plays an important role in the protein folding processes that occur in the ER including during cancer, liver disease and virus replication. The prokaryotic homologue of GRP78, Dna K, also plays an essential role in bacterial cell biology where it chaperones proteins such as Rec A which is essential for bacterial DNA replication and resistance where engulfed to the respiratory burst of macrophages (Roux, 1990;Earl et al, 1991;Anderson et al, 1992;Hogue and Nayak, 1992;Xu et al, 1997;Carleton and Brown, 1997;Xu et al, 1998;Bolt, 2001;Shen et al, 2002;Dimcheff et al, 2004;He, 2006;Reid et al, 2014;Moreno and Tiffany-Castiglioni, 2014;Spurgers et al, 2010;Bredeche et al, 2001). When high levels of unfolded protein are present in the ER, e.g.…”

Sindbis virus as a model for studies of conformational changes in a metastable virus and the role of conformational changes in in vitro antibody neutralisation

“…The epsilon E1 intermediate is metastable and thought to contain sufficient energy to drive the membrane penetration process and initiate infection [33]. E1 epsilon pairs with pE2 within the ER in the presence of the chaperone protein BiP to form heterodimers which further assemble into a heterotrimeric spike prior to transport to the Golgi [21,31,32,[34][35][36][37]. During the final assembly process of the virion, the heterotrimers are organised by the nucleocapsid into the five-and six-fold arrays which form the outer shell.…”