. 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...

The E protein of the Severe Acute Respiratory Syndrome Coronavirus binds the tight junction protein PALS1 through a novel PDZ domain-binding motif/PDZ domain interaction. Expression of E delays formation of tight junctions and alters cyst morphogenesis of MDCKII epithelial cells.

BackgroundEntry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process.Methodology/Principal FindingsWe performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S-pseudotyped particles and potentiated S-dependent membrane fusion.Conclusions/SignificanceEzrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.