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...
We have expressed and characterized the severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein in cDNA-transfected mammalian cells. The full-length spike protein (S) was newly synthesized as an endoglycosidase H (endo H)-sensitive glycoprotein (gp170) that is further modified into an endo H-resistant glycoprotein (gp180) in the Golgi apparatus. No substantial proteolytic cleavage of S was observed, suggesting that S is not processed into head (S1) and stalk (S2) domains as observed for certain other coronaviruses. While the expressed full-length S glycoprotein was exclusively cell associated, a truncation of S by excluding the C-terminal transmembrane and cytoplasmic tail domains resulted in the expression of an endoplasmic reticulum-localized glycoprotein (gp160) as well as a Golgi-specific form (gp170) which was ultimately secreted into the cell culture medium. Chemical cross-linking, thermal denaturation, and size fractionation analyses suggested that the full-length S glycoprotein of SARS-CoV forms a higher order structure of ϳ500 kDa, which is consistent with it being an S homotrimer. The latter was also observed in purified virions. The intracellular form of the C-terminally truncated S protein (but not the secreted form) also forms trimers, but with much less efficiency than full-length S. Deglycosylation of the full-length homotrimer with peptide N-glycosidase-F under native conditions abolished recognition of the protein by virus-neutralizing antisera raised against purified virions, suggesting the importance of the carbohydrate in the correct folding of the S protein. These data should aid in the design of recombinant vaccine antigens to prevent the spread of this emerging pathogen.
We have recently reported that the sublingual (s.l.) mucosa is an efficient site for inducing systemic and mucosal immune responses. In this study, the potential of s.l. immunization to induce remote Ab responses and CD8+ cytotoxic responses in the female genital tract was examined in mice by using a nonreplicating Ag, OVA, and cholera toxin (CT) as an adjuvant. Sublingual administration of OVA and CT induced Ag-specific IgA and IgG Abs in blood and in cervicovaginal secretions. These responses were associated with large numbers of IgA Ab-secreting cells (ASCs) in the genital mucosa. Genital ASC responses were similar in magnitude and isotype distribution after s.l., intranasal, or vaginal immunization and were superior to those seen after intragastric immunization. Genital, but not blood or spleen, IgA ASC responses were inhibited by treatment with anti-CCL28 Abs, suggesting that the chemokine CCL28 plays a major role in the migration of IgA ASC progenitors to the reproductive tract mucosa. Furthermore, s.l. immunization with OVA induced OVA-specific effector CD8+ cytolytic T cells in the genital mucosa, and these responses required coadministration of the CT adjuvant. Furthermore, s.l. administration of human papillomavirus virus-like particles with or without the CT adjuvant conferred protection against genital challenge with human papillomavirus pseudovirions. Taken together, these findings underscore the potential of s.l. immunization as an efficient vaccination strategy for inducing genital immune responses and should impact on the development of vaccines against sexually transmitted diseases.
Amiodarone interferes with the endocytic pathway, inhibits proteolysis, and causes the formation of vacuoles, but uptake and intracellular distribution of the drug, origin of vacuoles, and functional consequences of amiodarone accumulation remain unclear. Our objective was to study amiodarone uptake, clarify the origin of vacuoles, and investigate the effect of amiodarone on the life cycle of the coronavirus responsible for the Severe Acute Respiratory Syndrome (SARS), which, to enter cells, relies on the proteolytic cleavage of a viral spike protein by the endosomal proteinase cathepsin L. Using alveolar macrophages, we studied uptake of (125)I-amiodarone and (125)I-B2, an analog lacking the lateral group diethylamino-beta-ethoxy, and analyzed the effects of amiodarone on the distribution of endosomal markers and on the uptake of an acidotropic dye. Furthermore, using Vero cells, we tested the impact of amiodarone on the in vitro spreading of the SARS coronavirus. We found that (1) amiodarone associates with different cell membranes and accumulates in acidic organelles; (2) the diethylamino-beta-ethoxy group is an important determinant of uptake; (3) vacuoles forming upon exposure to amiodarone are enlarged late endosomes; (4) amiodarone inhibits the spreading in vitro of SARS coronavirus; and (5) trypsin cleavage of the viral spike protein before infection, which permits virus entry through the plasma membrane, does not impair amiodarone antiviral activity. We conclude that amiodarone alters late compartments of the endocytic pathway and inhibits SARS coronavirus infection by acting after the transit of the virus through endosomes.
A quantitative study was performed to investigate the requirements for secretion of recombinant soluble and particulate forms of the envelope glycoprotein E of tick-borne encephalitis (TBE) virus. Full-length E and a carboxy terminally truncated anchor-free form were expressed in COS cells in the presence and absence of prM, the precursor of the viral membrane protein M. Formation of a heteromeric complex with prM was found to be necessary for efficient secretion of both forms of E, whereas only low levels of anchor-free E were secreted in the absence of prM. The prM-mediated transport function could also be provided by coexpression of prM and E from separate constructs, but a prM-toE ratio of greater than 1:1 did not further enhance secretion. Full-length E formed stable intracellular heterodimers with prM and was secreted as a subviral particle, whereas anchor-free E was not associated with particles and formed a less stable complex with prM, suggesting that prM interacts with both the ectodomain and anchor region of E.
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