Envelope Protein Palmitoylations Are Crucial for Murine Coronavirus Assembly

Abstract

The coronavirus assembly process encloses a ribonucleoprotein genome into vesicles containing the lipidembedded proteins S (spike), E (envelope), and M (membrane). This process depends on interactions with membranes that may involve palmitoylation, a common posttranslational lipidation of cysteine residues. To determine whether specific palmitoylations influence coronavirus assembly, we introduced plasmid DNAs encoding mouse hepatitis coronavirus (MHV) S, E, M, and N (nucleocapsid) into 293T cells and found that virus-like particles (VLPs) were robustly assembled and secreted into culture medium. Palmitate adducts predicted on cysteines 40, 44, and 47 of the 83-residue E protein were then evaluated by constructing mutant cDNAs with alanine or glycine codon substitutions at one or more of these positions. Triple-substituted proteins (E.Ts) lacked palmitate adducts. Both native E and E.T proteins localized at identical perinuclear locations, and both copurified with M proteins, but E.T was entirely incompetent for VLP production. In the presence of the E.T proteins, the M protein subunits accumulated into detergent-insoluble complexes that failed to secrete from cells, while native E proteins mobilized M into detergent-soluble secreted forms. Many of these observations were corroborated in the context of natural MHV infections, with native E, but not E.T, complementing debilitated recombinant MHVs lacking E. Our findings suggest that palmitoylations are essential for E to act as a vesicle morphogenetic protein and further argue that palmitoylated E proteins operate by allowing the primary coronavirus assembly subunits to assume configurations that can mobilize into secreted lipid vesicles and virions.Coronavirus-infected cells provide models to investigate protein targeting, subcellular protein transport, protein-lipid and protein-protein interactions, and of multiprotein complex assembly. This is because all of these events take place efficiently and in organized temporal fashion within cells to create infectious, lipid-enveloped virus particles. The essential components of infectious coronavirions include three membrane proteins, S (spike), E (envelope), and M (membrane), along with cytoplasmic N (nucleocapsid). These four proteins, along with newly synthesized monopartite plus-strand RNA virus genomes, congregate at endoplasmic reticulum and Golgi membranes, ultimately remodeling the membranes such that they intrude inward toward organelle lumen and then undergo membrane fission to create enveloped virus vesicles. Accomplishing these morphological changes involves several wellknown interactions. N proteins bind viral RNAs to generate helical ribonucleoprotein (RNP) complexes (27), RNPs in turn bind to the cytoplasmic carboxyl-terminal extensions of M proteins (25, 45), M proteins bind to each other (9), and M and S proteins interact at or near their transmembrane-spanning regions (10,16,57). Subcellular targeting signals in the S, E, and M proteins interacting with presumed cellular factors restrict association...

Palmitoylation of Hepatitis C Virus Core Protein Is Important for Virion Production

“…This post-translational modification plays an important role in protein stability (40), intracellular protein trafficking, and targeting to membrane microdomains (39). Palmitoylation is also crucial for assembly and budding of many viruses (41)(42)(43)(44). Unlike other lipid modifications, palmitoylation is a reversible covalent modification, allowing dynamic regulation of multiple complex cellular systems.…”

Supramolecular Architecture of the Coronavirus Particle

“…E proteins are encoded by all known coronavirus genomes and are found at low levels in the virion (Godet et al, 1992;Liu and Inglis, 1991). As pointed out by Kuo and Masters (Kuo et al, 2016), E appears to have three distinct functions that contribute to infection: regulating aggregation-prone M-M interactions (Boscarino et al, 2008), disrupting Golgi organization in a way that produces larger vesicles capable of transporting virions (Machamer and Youn, 2006;Machamer, 2011, 2012), and interacting with host factors in a way that affects pathogenesis (DeDiego et al, 2007Dediego et al, 2008;Nieto-Torres et al, 2015;Regla-Nava et al, 2015;Teoh et al, 2010). E proteins of several coronaviruses have been reported to have ion channel activity (Liao et al, 2004;Madan et al, 2005;Wilson et al, 2004), which appears to enhance viral growth (Wilson et al, 2006;Ye and Hogue, 2007).…”

“…The M protein facilitates viral assembly by interacting with other M (Arndt et al, 2010;de Haan et al, 2000), E (Boscarino et al, 2008;Corse and Machamer, 2003;Lim andLiu, 2001), S (de Haan et al, 1999;Godeke et al, 2000), and N proteins (Escors et al, 2001;Hurst et al, 2005;Narayanan et al, 2000;Sturman et al, 1980). The MHV M protein may also interact with the RNA packaging signal that mediates incorporation of the viral genome into viral particles (Narayanan et al, 2003), but direct M-genome interactions are not efficient enough to rescue viruses in which the C-terminal region of N that interacts with M has been perturbed, suggesting that M-genome interactions are less important than M-N interactions for recovery of infectious virus (Kuo et al, 2016).…”

“…During the assembly process, N protein contributes to the formation of M LONG , narrowing the size range of resulting VLPs (Fig. 2;Neuman et al, 2011) and increasing the efficiency of VLP production (Boscarino et al, 2008;Siu et al, 2008). A study of VLP size and organization showed that MHV VLPs and virions with different components formed particles with a constrained minimum size, and that the size range of particles became narrower and approached the minimum size of VLPs as more structural components were incorporated (Fig.…”

Identification of novel functional regions within the spike glycoprotein of MHV-A59 based on a bioinformatics approach

“…MHV (strain A59) S gene was expressed in the pCAGGS-S plasmid vector (kindly provided by Prof. Thomas M. Gallagher) (Boscarino et al, 2008). All mutations in the S protein were created by using mutagenic primers and overlap-extension PCR (Cormack, 2001), followed by TA cloning of mutagenized amplicons into pGEM-T Easy (Promega) and subsequent sequencing.…”