. 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...

Public health measures successfully contained outbreaks of the severe acute respiratory syndrome coronavirus (SARS-CoV) infection. However, the precursor of the SARS-CoV remains in its natural bat reservoir, and reemergence of a human-adapted SARS-like coronavirus remains a plausible public health concern. Vaccination is a major strategy for containing resurgence of SARS in humans, and a number of vaccine candidates have been tested in experimental animal models. We previously reported that antibody elicited by a SARS-CoV vaccine candidate based on recombinant full-length Spike-protein trimers potentiated infection of human B cell lines despite eliciting in vivo a neutralizing and protective immune response in rodents. These observations prompted us to investigate the mechanisms underlying antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro. We demonstrate here that anti-Spike immune serum, while inhibiting viral entry in a permissive cell line, potentiated infection of immune cells by SARS-CoV Spike-pseudotyped lentiviral particles, as well as replication-competent SARS coronavirus. Antibody-mediated infection was dependent on Fc␥ receptor II but did not use the endosomal/lysosomal pathway utilized by angiotensin I converting enzyme 2 (ACE2), the accepted receptor for SARS-CoV. This suggests that ADE of SARS-CoV utilizes a novel cell entry mechanism into immune cells. Different SARS vaccine candidates elicit sera that differ in their capacity to induce ADE in immune cells despite their comparable potency to neutralize infection in ACE2-bearing cells. Our results suggest a novel mechanism by which SARS-CoV can enter target cells and illustrate the potential pitfalls associated with immunization against it. These findings should prompt further investigations into SARS pathogenesis.

Vaccine-induced antibodies can prevent or, in the case of feline infectious peritonitis virus, aggravate infections by coronaviruses. We investigated whether a recombinant native full-length S-protein trimer (triSpike) of severe acute respiratory syndrome coronavirus (SARS-CoV) was able to elicit a neutralizing and protective immune response in animals and analyzed the capacity of anti-S antibodies to mediate antibody-dependent enhancement (ADE) of virus entry in vitro and enhancement of replication in vivo. SARS-CoV-specific serum and mucosal immunoglobulins were readily detected in immunized animals. Serum IgG blocked binding of the S-protein to the ACE2 receptor and neutralized SARS-CoV infection in vitro. Entry into human B cell lines occurred in a FcgammaRII-dependent and ACE2-independent fashion indicating that ADE of virus entry is a novel cell entry mechanism of SARS-CoV. Vaccinated animals showed no signs of enhanced lung pathology or hepatitis and viral load was undetectable or greatly reduced in lungs following challenge with SARS-CoV. Altogether our results indicate that a recombinant trimeric S protein was able to elicit an efficacious protective immune response in vivo and warrant concern in the safety evaluation of a human vaccine against SARS-CoV.