The M, E, and N Structural Proteins of the Severe Acute Respiratory Syndrome Coronavirus Are Required for Efficient Assembly, Trafficking, and Release of Virus-Like Particles

Abstract

. This suggests that the mechanism of SARS-CoV assembly differs from that of other studied coronaviruses, which only require M and E proteins for VLP formation. When coexpressed, the native envelope trimeric S glycoprotein is incorporated onto VLPs. Interestingly, when a fluorescent protein tag is added to the C-terminal end of N or S protein, but not M protein, the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state-of-the-art imaging technologies. Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS-CoV egress.Coronaviruses are positive-sense RNA enveloped viruses that belong to the Coronaviridae family in the Nidovirales order. These viruses are found in a wide variety of animals and can cause respiratory and enteric disorders of diverse severity (11,18). In the past 5 years, several human and animal coronaviruses have been discovered, including the highly pathogenic virus responsible for the severe acute respiratory syndrome (SARS-CoV) (34,58, 60,64,68,69). Coronavirus particles consist of a helical nucleocapsid structure, formed by the association between nucleocapsid (N) phosphoproteins and the viral genomic RNA, which is surrounded by a lipid bilayer where three or four types of structural proteins are inserted: the spike (S), the membrane (M), and the envelope (E) proteins and, for some coronaviruses only, the hemagglutinin-esterase (HE) protein (for a review, see reference 14). Once sufficient amounts of new genomic RNA and structural proteins have been produced, assembly of particles occurs. Assembly and release of virions are the last stages of the virus life cycle.The triple-spanning membrane glycoprotein M drives the assembly of coronavirus, which bud into the lumen of the endoplasmic reticulum-Golgi intermediary compartment (ERGIC) (32,33,62,63). M is the most abundant envelope protein that sorts viral components to be incorporated into virions. M oligomerization, mainly driven by its transmembrane domain, is believed to allow the formation of a lattice of M proteins at ERGIC membranes (16, 41). S and E membrane proteins are integrated into the lattice through lateral interactions with M, whereas N and viral RNA interact with M C-terminal domain, which is exposed to the cytosol (4,8,15,19,30,36,48,54,55). The coronavirus S protein, responsible for receptor binding and membrane fusion, does not seem to have any major role in coronavirus assembly. Recent studies show that E is a viroporin that forms ion channels (46,66,67). Despite its minor incorporation into virion particles (7,22,40), the small E protein plays an important but not fully understood role in virus morphogenesis and budding (20,35,70). Studies performed on coronaviruses, including the SARS-CoV, demonstrate that depletion of the E gene from coronavirus genome strongly diminish virus growth and particle formation (9,12,35,37,57). The N protein self-associates and encapsidates the RNA genome for incorporation i...

Generation and characterization of a trackable plant-made influenza H5 virus-like particle (VLP) containing enhanced green fluorescent protein (eGFP)

“…A wide range of trackable VLPs has been generated by labeling one or more viral components with a fluorescent protein (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Many VLPs have been labeled after particle production/ purification, usually by chemical coupling to (in)organic Figure 2.…”

Current advancements and potential strategies in the development of MERS-CoV vaccines

“…The route of vaccine administration directly influences both the quality and quantity of vaccine-induced immunity [105–108]. Traditional parenteral administration routes, such as i.m., intradermal, and s.c., have been shown to induce favorable immune responses and/or protection against SARS-CoV and MERS-CoV infections [57, 109–111]. It is demonstrated that i.m.-vaccination with RBD protein of SARS-CoV elicits long-term immune responses with neutralizing activity, protecting immunized mice from SARS-CoV challenge [50].…”

Characterization of an Immunodominant Epitope in the Endodomain of the Coronavirus Membrane Protein

“…The avian infectious bronchitis virus (IBV) M protein contains Golgi-targeting information in its first transmembrane domain [14], whereas the transmembrane domains and the cytoplasmic tail domain of the mouse hepatitis virus (MHV) M protein play important roles in Golgi targeting [15,16]. The M protein interacts with the E, S, and N proteins and plays an essential role in virus assembly [17,18,19]. M is a necessary component of virus-like particles (VLP) during viral assembly [18,20,21,22].…”

“…The M protein interacts with the E, S, and N proteins and plays an essential role in virus assembly [17,18,19]. M is a necessary component of virus-like particles (VLP) during viral assembly [18,20,21,22]. The M proteins interact other M proteins to form homo-oligomers [23].…”