We have previously shown that an Escherichia coli-expressed, denatured spike (S) protein fragment of the severe acute respiratory coronavirus, containing residues 1029 to 1192 which include the heptad repeat 2 (HR2) domain, was able to induce neutralizing polyclonal antibodies (C. The virus-cell membrane fusion event is an essential step in the entry process of all enveloped animal viruses, including important human pathogens such as influenza virus, human immunodeficiency virus (HIV) (8, 23), and the newly emerged severe acute respiratory syndrome coronavirus (SARS-CoV) (9). Following the binding to their receptors on the cell surface, virus-encoded membrane fusion proteins mediate the fusion process. In many but not all cases, the viral fusion proteins are proteolytically processed by host proteases into 2 subunits that remain closely associated with each other: a surface subunit with a receptor-binding site and a transmembrane subunit with a fusion peptide consisting of two or more heptad repeat domains. Upon interaction of the fusion protein with a cellular receptor, the buried fusion peptide is exposed and inserted into the membrane of the target cell. A series of conformational changes trigger virus-cell fusion activity (9) and lead to the unloading of the viral genome into cells. Additionally, many viral fusion proteins also induce cell-cell fusion, i.e., the formation of multinucleated syncytia, facilitating the rapid spread of virus infection.TThe spike (S) protein of coronaviruses is responsible for receptor binding and membrane fusion. It shares similarity with class I virus fusion proteins (2, 3). Typically, it is a type I integral membrane protein, which is N-glycosylated and trimerized in the endoplasmic reticulum. The N-terminal S1 protein contains the receptor-binding site (10,18,22,34). The C-terminal S2 protein is a fusion subunit and anchors on the viral envelope through a transmembrane domain. The S2 protein ectodomain contains two 4,3 hydrophobic heptad repeats (HR1 and HR2) and a putative, internal fusion peptide (3, 23). For the SARS-CoV S protein, the HR2 is located adjacent to the transmembrane domain, whereas the HR1 is 140 to 170 residues upstream of the HR2.Crystallographic, biophysical, and biochemical analysis of the fusion core of SARS-CoV S protein (2,12,19,27,30,35) and other class I fusion proteins (8, 25) supports a model of membrane fusion probably adopted by these enveloped viruses. After the attachment of the receptor-binding subunit to the receptor, the HR1 and HR2 domains in the membrane fusion subunit interact with each other and form a six-helix bundle, a complex consisting of a homotrimeric HR1 coiled coil surrounded by three HR2 helices. The spacer domain (or link, or interhelical domain) between HR1 and HR2 forms a loop and reverses the direction of the polypeptide chain so that the HR2 helices pack against the HR1 coiled coil in an antiparallel manner. This conformational change results in a close apposition of the fusion peptide, already exposed and inserted into th...

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-S⌬10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.A novel coronavirus (CoV) was identified as the etiological agent of severe acute respiratory syndrome (SARS) (8,9,15,20). CoVs are positive-strand RNA viruses, and the virion consists of a nucleocapsid (N) core surrounded by an envelope containing three membrane proteins, spike (S), membrane (M), and envelope (E), that are common to all members of the genus (for reviews, see references 16 and 24). The S protein, which forms morphologically characteristic projections on the virion surface, binds to host receptors and mediates membrane fusion. The M and E proteins are important for viral particle assembly, while N is important for viral RNA packaging.The S protein of CoV is a type 1 integral membrane glycoprotein. It is cotranslationally glycosylated and oligomerized at the endoplasmic reticulum. Its N-linked high-mannose side chains are trimmed and modified and become endoglycosidase H (EndoH) resistant during the transportation to the Golgi apparatus. For some but not all CoVs, the S protein is cleaved into the N-terminal S1 and C-terminal S2 subunits, which contain receptor binding and membrane fusion domains (10, 32), respectively. The mature forms of S are assembled into virions, which release from infected cells. A portion of S is transported to the plasma membrane, resulting in cell-cell fusion or formation of syncytia. The S protein belongs to the class 1 viral fusion proteins and contains two heptad repeat domains (HR1 and HR2) in S2 or the C-...

Here we analyzed the gene expression profile of cells that stably express the severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein to determine its effects on host functions. A lung epithelial cell-line, A549, was chosen for this study because the lung is the primary organ infected by SARS-CoV and fatalities resulted mainly from pulmonary complications. Our results showed that the expression of 3a up-regulates the mRNA levels of all three subunits, A␣, B␤, and ␥, of fibrinogen. Consequently, the intracellular levels as well as the secretion of fibrinogen were increased. We also observed increased fibrinogen levels in SARS-CoV-infected Vero E6 cells.

In this paper we report the cloning and characterization of the erythropoietin (Epo) gene from the pufferfish, Fugu rubripes. This is the first nonmammalian Epo gene to be cloned. The Fugu Epo comprises 5 exons and 4 introns similar to the human EPO, and encodes a 185-amino acid protein that is 32% to 34% identical to Epo from various mammals. The synteny of genes at the Epo locus is conserved between the Fugu and humans. Unlike in mammals in which adult kidney is the primary Epo-producing organ, the heart is the main Epo-producing organ in adult Fugu. In addition to the heart, Fugu Epo is also expressed in the liver and brain similar to the human EPO. Interestingly, the transcripts in the Fugu brain are generated from a distal promoter and include an alternatively spliced first coding exon. No such brain-specific alternative splicing of Epo has been reported in mammals so far. Transient transfection studies in a fish hepatoma cell line (PLHC-1) and a human hepatoma cell line (HepG2) suggest that although the Fugu Epo promoter many not be hypoxia inducible, the gene may be regulated by hypoxia. ( IntroductionErythropoietin (Epo) is a glycoprotein hormone that plays a crucial role in ensuring supply of adequate oxygen to tissues by regulating the production of red blood cells. In mammals, Epo stimulates differentiation and proliferation of erythroid precursor cells in the bone marrow, in response to decreased environmental oxygen concentration or systemic oxygen deficiency caused by anemia. 1 In humans, fetal liver and adult kidney are the primary sites of EPO production. 1 In addition to these tissues, EPO is also produced in the central nervous system, bone marrow, spleen, heart, lung, ovary, testis, and breast cancer cells. [2][3][4][5][6][7] The production of Epo in the kidney, liver, and the central nervous system is greatly induced by hypoxic conditions. 8,9 The gene encoding the human EPO was first cloned in 1985 by 2 groups independently. 10,11 Since then, the Epo gene has been cloned from several mammalian species including nonhuman primates, rodents, ruminants, and felines. [12][13][14][15] The human EPO gene is transcribed from several start sites in the kidney and liver, and is thus independently regulated in these tissues. 16 Expression of the human EPO gene in transgenic mice has indicated that the cis-elements directing expression to the kidney are located between 6 kb and 14 kb upstream of the basal promoter, whereas the liver-specific elements are located in the 3Ј flanking region. 16 A 43-bp cis-element, capable of mediating the hypoxia response in the liver, also resides within the 3Ј flanking region, 120 bp downstream of the polyadenylation (polyA) signal. [16][17][18] Transient transfection studies in human hepatoma cell lines have demonstrated that the 3Ј enhancer induces 15-to 50-fold higher expression of a reporter gene in response to hypoxia.Although an "immunoreactive erythropoietin" that competes with human EPO for reaction with human EPO antibodies has been demonstrated in some teleos...

The genome of the severe acute respiratory syndrome coronavirus encodes for eight accessory viral proteins with no known homologues in other coronaviruses. One of these is the 3b protein, which is encoded by the second open reading frame in subgenomic RNA 3 and contains 154 amino acids. Here, a detailed time-course study was performed to compare the apoptosis and necrosis profiles induced by full-length 3b, a 3b mutant that was deleted by 30 amino acids from the C terminus (3b∆124-154) and the classical apoptosis inducer, Bax. Our results showed that Vero E6 cells transfected with a construct for expressing 3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time-points. At all the time-points analysed, the apoptosis induced by the expression of 3b was less than the level induced by Bax but the level of necrosis was comparable. The 3b∆124-154 mutant behaves in a similar manner indicating that the localization of the 3b protein does not seems to be important for the cell-death pathways since full-length 3b is localized predominantly to the nucleolus, while the mutant is found to be concentrated in the peri-nuclear regions. To our knowledge, this is the first report of the induction of necrosis by a SARS-CoV protein.

We provide a solution of finding optimal measurement strategy for distinguishing between symmetric mixed quantum states. It is assumed that the matrix elements of at least one of the symmetric quantum states are all real and nonnegative in the basis of the eigenstates of the symmetry operator.

Abstract. A 40-kD protein kinase C (PKC)E related activity was found to associate with human epithelial specific cytokeratin (CK) polypeptides 8 and 18. The kinase activity coimmunoprecipitated with CK8 and 18 and phosphorylated immunoprecipitates of the CK. Immunoblot analysis of CK8/18 immunoprecipitates using an anti-PKCE specific antibody showed that the 40-kD species, and not native PKCE (90 kD) associated with the cytokeratins. Reconstitution experiments demonstrated that purified CK8 or CK18 associated with a 40-kD tryptic fragment of purified PKCE, or with a similar species obtained from cells that express the fragment constitutively but do not express CK8/18. C YTOKERATINS (CK)' are a group of intermediate filament (IF) proteins which are expressed primarily in epithelial tissues (Lazarides, 1982 ;Steinert and Roop, 1988 ;Franke et al ., 1981;Osborn and Weber, 1986) . The 30 or so polypeptides which make up the family ofcytokeratin proteins are divided into acidic (type I) and basic/ neutral (type II) keratins. In epithelial cells, CK are found as mosaic noncovalent polymers with an assembly consisting of at least one type I and one type II CK (Steinert and Roop, 1988). For example, "simple" single layer epithelial cells such as intestinal epithelia express CK8 (type II) and CK18 (type I), whereas esophageal epithelial cells express CK4 (type II) and CK13 (type I) predominantly. Cytokeratins are not only important as tissue-specific markers, they also form important markers of cell differentiation .IF proteins, including cytokeratins, undergo several posttranslational modifications such as N112-terminal acetylation (Steinert and Idler, 1975), glycosylation (King and Hounsell, 1989;Roberts and Brunt, 1986), and serine/threonine phosphorylation (Steinert, 1988;Gilmartin et al ., 1984 ;Yeagle et al., 1990;Baribault et al., 1989) . The functional role of IF protein phosphorylation is not well under- A peptide pseudosubstrate specific for PKCE inhibited phosphorylation of CK8/18 in intact cells or in a kinase assay with CK8/18 immunoprecipitates . Tryptic peptide map analysis -of the cytokeratins that were phosphorylated by purified rat brain PKCE or as immunoprecipitates by the associated kinase showed similar phosphopeptides . Furthermore, PKCE immunoreactive species and CK8/18 colocalized using immunofluorescent double staining . We propose that a kinase related to the catalytic fragment of PKCE physically associates with and phosphorylates cytokeratins 8 and 18.

The lck gene encodes a lymphocyte-specific protein-tyrosine kinase that is implicated in T cell maturation and signaling. In mammals, the transcription of the lck gene is regulated by two independent promoters, the proximal promoter, which is active in thymocytes, and the distal promoter, which dominates in mature T cells. In the human and mouse lck gene loci, the two promoter elements are separated by at least 40 kb and 10 kb, respectively. In this study, we have cloned and sequenced 60 kb from the pufferfish (Fugu rubripes) lck locus. The promoter region of the Fugu lck spans only 4.2 kb and contains a proximal and a distal promoter in the 2.3-kb region adjacent to the coding sequence. By generating transgenic mice, we have demonstrated that the compact promoter of the Fugu lck contains regulatory elements that direct expression to lymphoid organs of mice. We were able to localize the regulatory elements to a short region of 830 bp without losing specificity to cultured human T cell line. These results show that the basic mechanisms that mediate lymphocyte-specific expression are conserved between teleosts and mammals. The short promoter of the Fugu lck isolated by us offers a powerful tool for labeling T cells, targeting expression, and manipulating T cell activity in fishes as well as in mammals.T he specificity of gene expression for cell types or developmental stages and the response of genes to physiological stimuli are mediated through a combinatorial interaction of promoter sequences, enhancers, and suppressors. These regulatory elements are composed of short stretches of DNA that are generally found in the promoter region of genes but also can be located in introns or dispersed over many kilobases upstream and downstream of genes (1). Finding these short elements in mammalian genomes is a formidable task because the intergenic regions in these genomes are large and complex. The genome of the pufferfish, Fugu rubripes, which is approximately eight times smaller than the human and mouse genomes, contains compact intergenic and intronic regions that are devoid of repetitive sequences (2, 3). This genome offers an attractive model for the rapid screening of noncoding sequences for conserved putative regulatory elements. Conserved regulatory elements driving cell-and stage-specific expression of developmental control genes such as the Hoxb-4, Otx2, Wnt-1, and Pax6 were identified by this strategy (4-7). Conserved regulatory elements were identified in the Fugu neurohypophyseal gene that express specifically in a subset of neurons in the hypothalamus of transgenic rats (8), and the Fugu tyrosinase gene was shown to contain conserved elements that directed melanocyte and retinal pigment epithelium-specific expression in transgenic mice (9). The hypothesis can be proposed that, where physiological systems show conservation over long stretches of evolutionary time, the genes specifying these processes are likely to be conserved not only in their coding sequences but also in their regulatory sequences. The immun...