The 114-day epidemic of the severe acute respiratory syndrome (SARS) swept 29 countries, affected a reported 8,098 people, left 774 patients dead and almost paralyzed the Asian economy. Aggressive quarantine measures, possibly aided by rising summer temperatures, successfully terminated the first eruption of SARS and provided at least a temporal break, which allows us to consolidate what we have learned so far and plan for the future. Here, we review the genomics of the SARS coronavirus (SARS-CoV), its phylogeny, antigenic structure, immune response and potential therapeutic interventions should the SARS epidemic flare up again.

Flaviviruses are assembled intracellularly in an immature form containing heterodimers of two envelope proteins, E and prM. Shortly before the virion exits the cell, prM is cleaved by a cellular enzyme, and this processing step can be blocked by treatment with agents that raise the pH of exocytic compartments. We carried out in vivo and in vitro studies with tick-borne encephalitis (TBE) virus to investigate the possible role of furin in this process as well as the functional consequences of prM cleavage. We found that prM in immature virions can be correctly cleaved in vitro by recombinant bovine furin but that efficient cleavage occurs only after exposure of the virion to mildly acidic pH. The data suggest that exposure to an acidic environment induces an irreversible structural change that renders the cleavage site accessible to the enzyme. Cleavage by furin in vitro resulted in biological activation, as shown by a 100-fold increase in specific infectivity, the acquisition of membrane fusion and hemagglutination activity, and the ability of the envelope proteins to undergo low-pHinduced structural rearrangements characteristic of mature virions. In vivo, prM cleavage was blocked by a furin inhibitor, and infection of the furin-deficient cell line LoVo yielded only immature virions, suggesting that furin is essential for cleavage activation of flaviviruses.

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002, and detailed phylogenetic and epidemiological analyses have suggested that it originated from animals. The spike (S) glycoprotein has been identified as a major component of protective immunity, and 23 different amino acid changes were noted during the expanding epidemic. Using a panel of SARS-CoV recombinants bearing the S glycoproteins from isolates representing the zoonotic and human early, middle, and late phases of the epidemic, we identified 23 monoclonal antibodies (MAbs) with neutralizing activity against one or multiple SARS-CoV spike variants and determined the presence of at least six distinct neutralizing profiles in the SARS-CoV S glycoprotein. Four of these MAbs showed cross-neutralizing activity against all human and zoonotic S variants in vitro, and at least three of these were mapped in distinct epitopes using escape mutants, structure analyses, and competition assays. These three MAbs (S109.8, S227.14, and S230.15) were tested for use in passive vaccination studies using lethal SARS-CoV challenge models for young and senescent mice with four different homologous and heterologous SARS-CoV S variants. Both S227.14 and S230.15 completely protected young and old mice from weight loss and virus replication in the lungs for all viruses tested, while S109.8 completely protected mice from weight loss and clinical signs in the presence of viral titers. We conclude that a single human MAb can confer broad protection against lethal challenge with multiple zoonotic and human SARS-CoV isolates, and we identify a robust cocktail formulation that targets distinct epitopes and minimizes the likely generation of escape mutants.

Cleavage of the mouse hepatitis coronavirus strain A59 spike protein was blocked in a concentrationdependent manner by a peptide furin inhibitor, indicating that furin or a furin-like enzyme is responsible for this process. While cell-cell fusion was clearly affected by preventing spike protein cleavage, virus-cell fusion was not, indicating that these events have different requirements.The surface glycoproteins of many enveloped viruses are initially synthesized as inactive precursors, proteolytic cleavage of which is often required for maturation and full functional activity. In several virus families, this processing step is carried out by cellular proprotein convertases (21), most commonly furin, a component of the constitutive secretory pathway of many different types of cells (9,33). Furin is a membranebound, calcium-dependent subtilisin-like protease whose primary site of action is the trans-Golgi network (TGN), although cycling of furin between TGN and plasma membrane through the exocytic and endocytic pathways has also been demonstrated (6, 28). The enzyme is also secreted from cells in an active soluble form, which is produced by self-cleavage in the TGN (43,45).The mouse hepatitis coronavirus (MHV) spike (S) protein is responsible for attachment to the viral receptor, for virus-cell fusion during viral entry, and for cell-cell fusion during infection. It is a class I fusion protein (5) that is cotranslationally glycosylated to a 150-kDa glycoprotein, which is processed to a 180-kDa form during transport from the endoplasmic reticulum through the Golgi complex. As a late event in maturation, the protein is cleaved into two 90-kDa subunits, S1 and S2 (10,31,34). The S proteins of murine coronaviruses are cleaved to different extents, depending on the strain and the cell line used (10). Cleavage of strain MHV-A59 S protein takes place between residues 717 and 718 at the sequence RRAHR2SVS (26). This sequence resembles the furin consensus sequence motif, RXR/KR (1,21,27). We now demonstrate, for the first time, that furin or a furin-like enzyme is indeed the protease responsible for cleavage of the S protein. Moreover, we investigated the consequences of cleavage, or rather of cleavage inhibition, of the S protein on its fusion activity and on the infectivity and cell entry of the virus.The importance of S protein cleavage for cell-cell fusion has been studied by several groups with inconsistent results. Using a vaccinia virus expression system (11) some investigators found the S proteins from MHV-A59 and MHV-JHM not to require cleavage for the induction of cell-cell fusion but syncytium formation to be delayed in the absence of cleavage (3,38). Others observed that a mutant MHV-JHM S protein, which was not proteolytically cleaved, induced syncytium formation to the same extent as the wild-type S protein (35). In contrast, transient expression of the uncleaved MHV-2 S protein apparently did not result in cell-cell fusion while a cleavable form of this protein did (47). Strikingly, the S protein from a dif...