Three-dimensional domain swapping as a mechanism to lock the active conformation in a super-active octamer of SARS-CoV main protease

Proteolytic processing of viral polyproteins is indispensible for the lifecycle of coronaviruses. The main protease (M^pro) of SARS-CoV is an attractive target for anti-SARS drug development as it is essential for the polyprotein processing. M^pro is initially produced as part of viral polyproteins and it is matured by autocleavage. Here, we report that, with the addition of an N-terminal extension peptide, M^pro can form a domain-swapped dimer. After complete removal of the extension peptide from the dimer, the mature M^pro self-assembles into a novel super-active octamer (AO-M^pro). The crystal structure of AO-M^pro adopts a novel fold with four domain-swapped dimers packing into four active units with nearly identical conformation to that of the previously reported M^pro active dimer, and 3D domain swapping serves as a mechanism to lock the active conformation due to entanglement of polypeptide chains. Compared with the previously well characterized form of M^pro, in equilibrium between inactive monomer and active dimer, the stable AO-M^pro exhibits much higher proteolytic activity at low concentration. As all eight active sites are bound with inhibitors, the polyvalent nature of the interaction between AO-M^pro and its polyprotein substrates with multiple cleavage sites, would make AO-M^pro functionally much more superior than the M^pro active dimer for polyprotein processing. Thus, during the initial period of SARS-CoV infection, this novel active form AO-M^pro should play a major role in cleaving polyproteins as the protein level is extremely low. The discovery of AO-M^pro provides new insights about the functional mechanism of M^pro and its maturation process.     

Liberation of SARS-CoV main protease from the viral polyprotein: N-terminal autocleavage does not depend on the mature dimerization mode

The main protease (M^pro) plays a vital role in proteolytic processing of the polyproteins in the replicative cycle of SARS coronavirus (SARS-CoV). Dimerization of this enzyme has been shown to be indispensable for trans-cleavage activity. However, the auto-processing mechanism of M^pro, i.e. its own release from the polyproteins through autocleavage, remains unclear. This study elucidates the relationship between the N-terminal autocleavage activity and the dimerization of “immature” M^pro. Three residues (Arg4, Glu290, and Arg298), which contribute to the active dimer conformation of mature M^pro, are selected for mutational analyses. Surprisingly, all three mutants still perform N-terminal autocleavage, while the dimerization of mature protease and trans-cleavage activity following auto-processing are completely inhibited by the E290R and R298E mutations and partially so by the R4E mutation. Furthermore, the mature E290R mutant can resume N-terminal autocleavage activity when mixed with the “immature” C145A/E290R double mutant whereas its trans-cleavage activity remains absent. Therefore, the N-terminal auto-processing of M^pro appears to require only two “immature” monomers approaching one another to form an “intermediate” dimer structure and does not strictly depend on the active dimer conformation existing in mature protease. In conclusion, an auto-release model of M^pro from the polyproteins is proposed, which will help understand the auto-processing mechanism and the difference between the autocleavage and trans-cleavage proteolytic activities of SARS-CoV M^pro.     

SARS冠状病毒主蛋白酶抑制剂的筛选及抑制动力学研究

对SARS冠状病毒主蛋白酶(SARS-CoV M^pro)进行异源重组表达与提纯,并以其为靶点,利用基于荧光共振能量转移(FRET)技术的体外药物筛选模型,对蛋白酶抑制剂聚焦库96种化合物进行了体外抑制活性的评价,并从动力学的角度探讨筛选出的阳性化合物对SARS-CoV M^pro的抑制能力与机制。结果表明:通过筛选获得抑制率>80%、淬灭率<20%的化合物5种,为P-1-08、P-1-19、P-2-24、P-2-28、P-2-54,其半数有效抑制浓度(IC₅₀)分别为:0.69±0.05μmol/L、1.19±0.41μmol/L、0.14±0.01μmol/L、1.36±0.07μmol/L、0.36±0.03μmol/L。其中化合物P-1-08、P-1-19、P-2-24、P-2-54对SARS冠状病毒主蛋白酶的抑制作用为不可逆抑制,化合物P-2-28的抑制作用为可逆抑制。根据Lineweaver-Burk图和Dixon图的研究,发现化合物P-2-28对SARS冠状病毒主蛋白酶呈竞争性抑制,抑制常数Ki为0.81μmol/L。通过对底物浓度,IC₅₀值及Ki值关系的研究,进一步验证了P-2-28的抑制作用为竞争性抑制。该抑制剂的发现为SARS冠状病毒主蛋白酶抑制剂的研究打下基础,为抗SARS病毒药物开发提供了先导化合物。     

Screening of electrophilic compounds yields an aziridinyl peptide as new active-site directed SARS-CoV main protease inhibitor

The coronavirus main protease, M^pro, is considered a major target for drugs suitable to combat coronavirus infections including the severe acute respiratory syndrome (SARS). In this study, comprehensive HPLC- and FRET-substrate-based screenings of various electrophilic compounds were performed to identify potential M^pro inhibitors. The data revealed that the coronaviral main protease is inhibited by aziridine- and oxirane-2-carboxylates. Among the trans-configured aziridine-2,3-dicarboxylates the Gly-Gly-containing peptide 2c was found to be the most potent inhibitor.     

Expression, purification and characterization of recombinant severe acute respiratory syndrome coronavirus non-structural protein 1

The coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS), SARS-CoV, encodes two large polyproteins (pp1a and pp1ab) that are processed by two viral proteases to yield mature non-structural proteins (nsps). Many of these nsps have essential roles in viral replication, but several have no assigned function and possess amino acid sequences that are unique to the CoV family. One such protein is SARS-CoV nsp1, which is processed from the N-terminus of both pp1a and pp1ab. The mature SARS-CoV protein is present in cells several hours post-infection and co-localizes to the viral replication complex, but its function in the viral life cycle remains unknown. Furthermore, nsp1 sequences are highly divergent across the CoV family, and it has been suggested that this is due to nsp1 possessing a function specific to viral interactions with its host cell or acting as a host specific virulence factor. In order to initiate structural and biophysical studies of SARS-CoV nsp1, a recombinant expression system and a purification protocol have been developed, yielding milligram quantities of highly purified SARS-CoV nsp1. The purified protein was characterized using circular dichroism, size exclusion chromatography, and multi-angle light scattering.     

Without its N-finger, the main protease of severe acute respiratory syndrome coronavirus can form a novel dimer through its C-terminal domain

The main protease (M(pro)) of severe acute respiratory syndrome coronavirus (SARS-CoV) plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target for anti-SARS drug development. It was found that SARS-CoV M(pro) exists in solution as an equilibrium of both monomeric and dimeric forms, and the dimeric form is the enzymatically active form. However, the mechanism of SARS-CoV M(pro) dimerization, especially the roles of its N-terminal seven residues (N-finger) and its unique C-terminal domain in the dimerization, remain unclear. Here we report that the SARS-CoV M(pro) C-terminal domain alone (residues 187 to 306; M(pro)-C) is produced in Escherichia coli in both monomeric and dimeric forms, and no exchange could be observed between them at room temperature. The M(pro)-C dimer has a novel dimerization interface. Meanwhile, the N-finger deletion mutant of SARS-CoV M(pro) also exists as both a stable monomer and a stable dimer, and the dimer is formed through the same C-terminal-domain interaction as that in the M(pro)-C dimer. However, no C-terminal domain-mediated dimerization form can be detected for wild-type SARS-CoV M(pro). Our study results help to clarify previously published controversial claims about the role of the N-finger in SARS-CoV M(pro) dimerization. Apparently, without the N-finger, SARS-CoV M(pro) can no longer retain the active dimer structure; instead, it can form a new type of dimer which is inactive. Therefore, the N-finger of SARS-CoV M(pro) is not only critical for its dimerization but also essential for the enzyme to form the enzymatically active dimer.     

Production of authentic SARS-CoV M(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction

The viral proteases have proven to be the most selective and useful for removing the fusion tags in fusion protein expression systems. As a key enzyme in the viral life-cycle, the main protease (M(pro)) is most attractive for drug design targeting the SARS coronavirus (SARS-CoV), the etiological agent responsible for the outbreak of severe acute respiratory syndrome (SARS) in 2003. In this study, SARS-CoV M(pro) was used to specifically remove the GST tag in a new fusion protein expression system. We report a new method to produce wild-type (WT) SARS-CoV M(pro) with authentic N and C termini, and compare the activity of WT protease with those of three different types of SARS-CoV M(pro) with additional residues at the N or C terminus. Our results show that additional residues at the N terminus, but not at the C terminus, of M(pro) are detrimental to enzyme activity. To explain this, the crystal structures of WT SARS-CoV M(pro) and its complex with a Michael acceptor inhibitor were determined to 1.6 Angstroms and 1.95 Angstroms resolution respectively. These crystal structures reveal that the first residue of this protease is important for sustaining the substrate-binding pocket and inhibitor binding. This study suggests that SARS-CoV M(pro) could serve as a new tag-cleavage endopeptidase for protein overproduction, and the WT SARS-CoV M(pro) is more appropriate for mechanistic characterization and inhibitor design.     

Identification and characterization of the putative fusion peptide of the severe acute respiratory syndrome-associated coronavirus spike protein

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a newly identified member of the family Coronaviridae and poses a serious public health threat. Recent studies indicated that the SARS-CoV viral spike glycoprotein is a class I viral fusion protein. A fusion peptide present at the N-terminal region of class I viral fusion proteins is believed to initiate viral and cell membrane interactions and subsequent fusion. Although the SARS-CoV fusion protein heptad repeats have been well characterized, the fusion peptide has yet to be identified. Based on the conserved features of known viral fusion peptides and using Wimley and White interfacial hydrophobicity plots, we have identified two putative fusion peptides (SARS(WW-I) and SARS(WW-II)) at the N terminus of the SARS-CoV S2 subunit. Both peptides are hydrophobic and rich in alanine, glycine, and/or phenylalanine residues and contain a canonical fusion tripeptide along with a central proline residue. Only the SARS(WW-I) peptide strongly partitioned into the membranes of large unilamellar vesicles (LUV), adopting a beta-sheet structure. Likewise, only SARS(WW-I) induced the fusion of LUV and caused membrane leakage of vesicle contents at peptide/lipid ratios of 1:50 and 1:100, respectively. The activity of this synthetic peptide appeared to be dependent on its amino acid (aa) sequence, as scrambling the peptide rendered it unable to partition into LUV, assume a defined secondary structure, or induce both fusion and leakage of LUV. Based on the activity of SARS(WW-I), we propose that the hydrophobic stretch of 19 aa corresponding to residues 770 to 788 is a fusion peptide of the SARS-CoV S2 subunit.     

Persistent replication of severe acute respiratory syndrome coronavirus in human tubular kidney cells selects for adaptive mutations in the membrane protein

Severe acute respiratory syndrome (SARS) is a systemic disease characterized by both lung pathology and widespread extrapulmonary virus dissemination causing multiple organ injuries. In this regard, renal dysfunction is an ominous sign in patients with SARS. Indeed, clusters of SARS coronavirus (SARS-CoV) particles have been detected in the cytoplasm of renal tubular epithelial cells in postmortem studies, explaining the presence of infectious virus in the urine of SARS patients. In order to investigate the potential SARS-CoV kidney tropism, we have evaluated the susceptibility of human renal cells of tubular and glomerular origin to in vitro SARS-CoV infection. Immortalized cultures of differentiated proximal tubular epithelial cells (PTEC), glomerular mesangial cells (MC), and glomerular epithelial cells (podocytes) were found to express the SARS-CoV receptor angiotensin-converting enzyme 2 on their surface. Productive infection, however, occurred only in PTEC but not in glomerular cells. A transient infection with poor virus production was observed in MC, whereas podocytes were not permissive to SARS-CoV infection. In contrast to the cytopathic infection of the Vero E6 cell line, SARS-CoV did not cause overt cytopathic effects in PTEC or MC. Of interest, PTEC, but not MC, maintained stable levels of SARS-CoV production in serial subcultures, suggesting a persistent state of infection. In this regard, a SARS-CoV variant with increased replication capacity in PTEC was selected after four serial subculture passages. This SARS-CoV variant acquired a single nonconservative amino acid change from glutamic acid (E) to alanine (A) at position 11 in the viral membrane (M) protein. The E11A point mutation was sufficient for enhanced SARS-CoV replication and persistence in PTEC when introduced in a SARS-CoV recombinant infectious clone. These findings indicate that human PTEC may represent a site of SARS-CoV productive and persistent replication favoring the emergence of viral variants with increased replication capacity, at least in these kidney cells.     

Binding interaction of quercetin-3-beta-galactoside and its synthetic derivatives with SARS-CoV 3CL(pro): structure-activity relationship studies reveal salient pharmacophore features

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for discovery of drugs against SARS, because of its critical role in the viral life cycle. In this study, a natural compound called quercetin-3-β-galactoside was identified as an inhibitor of the protease by molecular docking, SPR/FRET-based bioassays, and mutagenesis studies. Both molecular modeling and Q189A mutation revealed that Gln189 plays a key role in the binding. Furthermore, experimental evidence showed that the secondary structure and enzymatic activity of SARS-CoV 3CL^pro were not affected by the Q189A mutation. With the help of molecular modeling, eight new derivatives of the natural product were designed and synthesized. Bioassay results reveal salient features of the structure–activity relationship of the new compounds: (1) removal of the 7-hydroxy group of the quercetin moiety decreases the bioactivity of the derivatives; (2) acetoxylation of the sugar moiety abolishes inhibitor action; (3) introduction of a large sugar substituent on 7-hydroxy of quercetin can be tolerated; (4) replacement of the galactose moiety with other sugars does not affect inhibitor potency. This study not only reveals a new class of compounds as potential drug leads against the SARS virus, but also provides a solid understanding of the mechanism of inhibition against the target enzyme.     

pH-dependent conformational flexibility of the SARS-CoV main proteinase (M(pro)) dimer: molecular dynamics simulations and multiple X-ray structure analyses

The SARS coronavirus main proteinase (M(pro)) is a key enzyme in the processing of the viral polyproteins and thus an attractive target for the discovery of drugs directed against SARS. The enzyme has been shown by X-ray crystallography to undergo significant pH-dependent conformational changes. Here, we assess the conformational flexibility of the M(pro) by analysis of multiple crystal structures (including two new crystal forms) and by molecular dynamics (MD) calculations. The MD simulations take into account the different protonation states of two histidine residues in the substrate-binding site and explain the pH-activity profile of the enzyme. The low enzymatic activity of the M(pro) monomer and the need for dimerization are also discussed.     

Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a pH- and cysteine protease-independent FcγR pathway

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 Fcγ 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.     

Novel Inhibitors of Severe Acute Respiratory Syndrome Coronavirus Entry That Act by Three Distinct Mechanisms

Severe acute respiratory syndrome (SARS) is an infectious and highly contagious disease that is caused by SARS coronavirus (SARS-CoV) and for which there are currently no approved treatments. We report the discovery and characterization of small-molecule inhibitors of SARS-CoV replication that block viral entry by three different mechanisms. The compounds were discovered by screening a chemical library of compounds for blocking of entry of HIV-1 pseudotyped with SARS-CoV surface glycoprotein S (SARS-S) but not that of HIV-1 pseudotyped with vesicular stomatitis virus surface glycoprotein G (VSV-G). Studies on their mechanisms of action revealed that the compounds act by three distinct mechanisms: (i) SSAA09E2 {N-[[4-(4-methylpiperazin-1-yl)phenyl]methyl]-1,2-oxazole-5-carboxamide} acts through a novel mechanism of action, by blocking early interactions of SARS-S with the receptor for SARS-CoV, angiotensin converting enzyme 2 (ACE2); (ii) SSAA09E1 {[(Z)-1-thiophen-2-ylethylideneamino]thiourea} acts later, by blocking cathepsin L, a host protease required for processing of SARS-S during viral entry; and (iii) SSAA09E3 [N-(9,10-dioxo-9,10-dihydroanthracen-2-yl)benzamide] also acts later and does not affect interactions of SARS-S with ACE2 or the enzymatic functions of cathepsin L but prevents fusion of the viral membrane with the host cellular membrane. Our work demonstrates that there are at least three independent strategies for blocking SARS-CoV entry, validates these mechanisms of inhibition, and introduces promising leads for the development of SARS therapeutics.     

Mice transgenic for human angiotensin-converting enzyme 2 provide a model for SARS coronavirus infection

To establish a small animal model of severe acute respiratory syndrome (SARS), we developed a mouse model of human severe acute respiratory syndrome coronavirus (SARS-CoV) infection by introducing the human gene for angiotensin-converting enzyme 2 (hACE2) (the cellular receptor of SARS-CoV), driven by the mouse ACE2 promoter, into the mouse genome. The hACE2 gene was expressed in lung, heart, kidney, and intestine. We also evaluated the responses of wild-type and transgenic mice to SARS-CoV inoculation. At days 3 and 7 postinoculation, SARS-CoV replicated more efficiently in the lungs of transgenic mice than in those of wild-type mice. In addition, transgenic mice had more severe pulmonary lesions, including interstitial hyperemia and hemorrhage, monocytic and lymphocytic infiltration, protein exudation, and alveolar epithelial cell proliferation and desquamation. Other pathologic changes, including vasculitis, degeneration, and necrosis, were found in the extrapulmonary organs of transgenic mice, and viral antigen was found in brain. Therefore, transgenic mice were more susceptible to SARS-CoV than were wild-type mice, and susceptibility was associated with severe pathologic changes that resembled human SARS infection. These mice will be valuable for testing potential vaccine and antiviral drug therapies and for furthering our understanding of SARS pathogenesis.     

Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans

It has been reported that lactoferrin (LF) participates in the host immune response against Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) invasion by enhancing NK cell activity and stimulating neutrophil aggregation and adhesion. We further investigated the role of LF in the entry of SARS pseudovirus into HEK293E/ACE2-Myc cells. Our results reveal that LF inhibits SARS pseudovirus infection in a dose-dependent manner. Further analysis suggested that LF was able to block the binding of spike protein to host cells at 4°C, indicating that LF exerted its inhibitory function at the viral attachment stage. However, LF did not disrupt the interaction of spike protein with angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV. Previous studies have shown that LF colocalizes with the widely distributed cell-surface heparan sulfate proteoglycans (HSPGs). Our experiments have also confirmed this conclusion. Treatment of the cells with heparinase or exogenous heparin prevented binding of spike protein to host cells and inhibited SARS pseudovirus infection, demonstrating that HSPGs provide the binding sites for SARS-CoV invasion at the early attachment phase. Taken together, our results suggest that, in addition to ACE2, HSPGs are essential cell-surface molecules involved in SARS-CoV cell entry. LF may play a protective role in host defense against SARS-CoV infection through binding to HSPGs and blocking the preliminary interaction between SARS-CoV and host cells. Our findings may provide further understanding of SARS-CoV pathogenesis and aid in treatment of this deadly disease.     

Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) can be proteolytically activated by cathepsins B and L upon viral uptake into target cell endosomes. In contrast, it is largely unknown whether host cell proteases located in the secretory pathway of infected cells and/or on the surface of target cells can cleave SARS S. We along with others could previously show that the type II transmembrane protease TMPRSS2 activates the influenza virus hemagglutinin and the human metapneumovirus F protein by cleavage. Here, we assessed whether SARS S is proteolytically processed by TMPRSS2. Western blot analysis revealed that SARS S was cleaved into several fragments upon coexpression of TMPRSS2 (cis-cleavage) and upon contact between SARS S-expressing cells and TMPRSS2-positive cells (trans-cleavage). cis-cleavage resulted in release of SARS S fragments into the cellular supernatant and in inhibition of antibody-mediated neutralization, most likely because SARS S fragments function as antibody decoys. trans-cleavage activated SARS S on effector cells for fusion with target cells and allowed efficient SARS S-driven viral entry into targets treated with a lysosomotropic agent or a cathepsin inhibitor. Finally, ACE2, the cellular receptor for SARS-CoV, and TMPRSS2 were found to be coexpressed by type II pneumocytes, which represent important viral target cells, suggesting that SARS S is cleaved by TMPRSS2 in the lung of SARS-CoV-infected individuals. In summary, we show that TMPRSS2 might promote viral spread and pathogenesis by diminishing viral recognition by neutralizing antibodies and by activating SARS S for cell-cell and virus-cell fusion.     

Participation of both host and virus factors in induction of severe acute respiratory syndrome (SARS) in F344 rats infected with SARS coronavirus

To understand the pathogenesis and develop an animal model of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV), the Frankfurt 1 SARS-CoV isolate was passaged serially in young F344 rats. Young rats were susceptible to SARS-CoV but cleared the virus rapidly within 3 to 5 days of intranasal inoculation. After 10 serial passages, replication and virulence of SARS-CoV were increased in the respiratory tract of young rats without clinical signs. By contrast, adult rats infected with the passaged virus showed respiratory symptoms and severe pathological lesions in the lung. Levels of inflammatory cytokines in sera and lung tissues were significantly higher in adult F344 rats than in young rats. During in vivo passage of SARS-CoV, a single amino acid substitution was introduced within the binding domain of the viral spike protein recognizing angiotensin-converting enzyme 2 (ACE2), which is known as a SARS-CoV receptor. The rat-passaged virus more efficiently infected CHO cells expressing rat ACE2 than did the original isolate. These results strongly indicate that host and virus factors such as advanced age and virus adaptation are critical for the development of SARS in rats.     

Apical entry and release of severe acute respiratory syndrome-associated coronavirus in polarized Calu-3 lung epithelial cells

Severe acute respiratory syndrome (SARS), caused by a novel coronavirus (CoV) known as SARS-CoV, is a contagious and life-threatening respiratory illness with pneumocytes as its main target. A full understanding of how SARS-CoV would interact with lung epithelial cells will be vital for advancing our knowledge of SARS pathogenesis. However, an in vitro model of SARS-CoV infection using relevant lung epithelial cells is not yet available, making it difficult to dissect the pathogenesis of SARS-CoV in the lungs. Here, we report that SARS-CoV can productively infect human bronchial epithelial Calu-3 cells, causing cytopathic effects, a process reflective of its natural course of infection in the lungs. Indirect immunofluorescence studies revealed a preferential expression of angiotensin-converting enzyme 2 (ACE-2), the functional receptor of SARS-CoV, on the apical surface. Importantly, both ACE-2 and viral antigen appeared to preferentially colocalize at the apical domain of infected cells. In highly polarized Calu-3 cells grown on the membrane inserts, we found that cells exposed to virus through the apical rather than the basolateral surface showed high levels of viral replication. Progeny virus was released into the apical chamber at titers up to 5 logs higher than those recovered from the basolateral chambers of polarized cultures. Taken together, these results indicate that SARS-CoV almost exclusively entered and was released from the apical domain of polarized Calu-3 cells, which might provide important insight into the mechanism of transmission and pathogenesis of SARS-CoV.     

SARS CoV main proteinase: The monomer-dimer equilibrium dissociation constant

The SARS coronavirus main proteinase (SARS CoV main proteinase) is required for the replication of the severe acute respiratory syndrome coronavirus (SARS CoV), the virus that causes SARS. One function of the enzyme is to process viral polyproteins. The active form of the SARS CoV main proteinase is a homodimer. In the literature, estimates of the monomer-dimer equilibrium dissociation constant, KD, have varied more than 65,0000-fold, from <1 nM to more than 200 microM. Because of these discrepancies and because compounds that interfere with activation of the enzyme by dimerization may be potential antiviral agents, we investigated the monomer-dimer equilibrium by three different techniques: small-angle X-ray scattering, chemical cross-linking, and enzyme kinetics. Analysis of small-angle X-ray scattering data from a series of measurements at different SARS CoV main proteinase concentrations yielded KD values of 5.8 +/- 0.8 microM (obtained from the entire scattering curve), 6.5 +/- 2.2 microM (obtained from the radii of gyration), and 6.8 +/- 1.5 microM (obtained from the forward scattering). The KD from chemical cross-linking was 12.7 +/- 1.1 microM, and from enzyme kinetics, it was 5.2 +/- 0.4 microM. While each of these three techniques can present different, potential limitations, they all yielded similar KD values.     

The aetiology of SARS: Koch's postulates fulfilled

Proof that a newly identified coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV) is the primary cause of severe acute respiratory syndrome (SARS) came from a series of studies on experimentally infected cynomolgus macaques (Macaca fascicularis). SARS-CoV-infected macaques developed a disease comparable to SARS in humans; the virus was re-isolated from these animals and they developed SARS-CoV-specific antibodies. This completed the fulfilment of Koch's postulates, as modified by Rivers for viral diseases, for SARS-CoV as the aetiological agent of SARS. Besides the macaque model, a ferret and a cat model for SARS-CoV were also developed. These animal models allow comparative pathogenesis studies for SARS-CoV infections and testing of different intervention strategies. The first of these studies has shown that pegylated interferon-alpha, a drug approved for human use, limits SARS-CoV replication and lung damage in experimentally infected macaques. Finally, we argue that, given the worldwide nature of the socio-economic changes that have predisposed for the emergence of SARS and avian influenza in Southeast Asia, such changes herald the beginning of a global trend for which we are ill prepared.