The Missing Link in Coronavirus Assembly

Abstract

One missing link in the coronavirus assembly is the physical interaction between two crucial structural proteins, the membrane (M) and envelope (E) proteins. In this study, we demonstrate that the coronavirus infectious bronchitis virus E can physically interact, via a putative peripheral domain, with M. Deletion of this domain resulted in a drastic reduction in the incorporation of M into virus-like particles. Immunofluorescent staining of cells coexpressing M and E supports that E interacts with M and relocates M to the same subcellular compartments that E resides in. E was retained in the pre-Golgi membranes, prior to being translocated to the Golgi apparatus and the secretory vesicles; M was observed to exhibit similar localization and translocation profiles as E when coexpressed with E. Deletion studies identified the C-terminal 6-residue RDKLYS as the endoplasmic reticulum retention signal of E, and site-directed mutagenesis of the ؊4 lysine residue to glutamine resulted in the accumulation of E in the Golgi apparatus. The third domain of E that plays a crucial role in virus budding is a putative transmembrane domain present at the N-terminal region, because deletion of the domain resulted in a free distribution of the mutant protein and in dysfunctional viral assembly.Morphogenesis and assembly of mammalian enveloped RNA viruses are complex processes. During these processes, the viral core, consisting of viral RNA and core proteins, becomes wrapped in a membranous structure (viral envelope) derived from host cell membranes to form virion particles. The assembly process, which is referred to as budding, usually occurs at either the plasma or intracellular membranes. The formation of viral core, envelope, and the assembly of virus particles would involve interaction between viral proteins and host membrane components, among viral structural proteins and between viral proteins and viral RNA. Characterization of these events at the molecular level has been greatly facilitated by the understanding that viral structural proteins may contain all of the necessary information to dictate the assembly process. In fact, it was observed that coexpression of viral structural proteins would result in the formation of virus-like particles (VLPs) 1 in many different viral systems (1-9). In this study, we exploit the coronavirus VLP system to study the interaction between two structural proteins during coronavirus assembly and the implication of this interaction in the assembly and release of coronavirus particles. Coronavirus is the largest RNA virus known so far. It has a positive-sense, single-strand RNA genome of 27-30 kilobases in length. Despite of the huge genome size, coronavirus typically contains four structural proteins, i.e. a type I spike (S) glycoprotein required for infectivity, a phosphorylated nucleocapsid (N) protein that interacts with the viral genome to form a helical core, a major type III integral membrane (M) protein, and a minor type III envelope (E) protein (10 -15). A fifth protein, the hemagglut...

Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity

“…It has been shown for several coronaviruses that coexpression of S, M, and E results in the formation of virus-like particles that bud from infected cells (19,(26)(27)(28). Also, whereas M expressed alone was retained in the Golgi network, coexpression of M and E resulted in expression at more distal cell membranes (19,20,27,29). Thus, the expression of these combinations in addition to the single inserts might increase the opportunity for producing the proteins in an immunogenic form.…”

Protein Kinase C β Is Required for Human Monocyte Chemotaxis to MCP-1

“…More selective inhibitors have been used to investigate the contribution of mitogen-activated protein kinases, and although several investigators have speculated that PKC is required, this has not been definitively shown (23,24,33). In primary human monocytes, PKC has been shown to be part of the signaling pathways controlling the respiratory burst (29,34,35). PKC has been proposed to sustain calcium influx and be involved in the second phase diacylglycerol accumulation during chemotaxis (36,37).…”

“…Treatment of Cells with ODN-The antisense ODN used in our studies were derived from our prior published work, which reported their efficacy (28,29). The sequence for cPKC-antisense ODN was 5Ј-CCC CAG ATG AAG TCG GTG CA-3Ј, and its control-sense ODN sequence was 5Ј-GGG GTC TAC TTC AGC CAC GT-3Ј.…”

Protein Kinase Cα Regulates Human Monocyte O·̄2Production and Low Density Lipoprotein Lipid Oxidation

“…We routinely measure monocyte-mediated LDL oxidation at 24 h after monocyte activation, a time when considerable oxidation products have accumulated and the oxidation begins to plateau. In our earlier studies, PKC activity was shown to contribute to LDL lipid oxidation both early and late during the 24-h incubation period (7).…”