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2 The abbreviations used are: S, spike; SARS-CoV, severe acute respiratory syndrome coronavirus; MLV, murine leukemia virus; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-gubbing non-integrin, ACE2, argiotensin I conserting enzyme 2. FIGURE 4. Introduction of alanine residues in the S2 region modulates elastase-induced virus entry mediated by SARS-CoV S. A and B, pseudotyped virion infections were performed as described for Fig. 2. Infection was induced by treatment with elastase (A) or trypsin (B). Results are presented as relative light units (RLU). Error bars represent the S.E. of three independent experiments. C, cleavage of SARS-CoV by elastase. HEK293T cells expressing the wild type (WT) or mutant SARS-CoV spike protein were treated with 50 g/ml elastase. Cell surface proteins were biotinylated and precipitated, and cleavage products of the SARS-CoV spike protein were analyzed in Western blot with an antibody recognizing the C9 tag at the C terminus of the protein.
Elastase-mediated Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein at Discrete Sites within the S2 Domain * □ S

Phenanthroquinolizidine alkaloids a b s t r a c t The stereoselective synthesis of epi-thieno analogues of the phenanthroquinolizidine bioactive alkaloids (À)-Cryptopleurine and (À)-(15R)-Hydroxycryptopleurine was achieved in five steps starting from easily available enantiopure (S)-2-aminoadipic acid used as chiral pool and nitrogen atom source. During these investigations, both p-cationic cyclization of chiral N-thienylmethyl-6-oxopipecolinic acids into pure (S)keto-lactams and theirs regioselective and diastereoselective reduction, considered as key steps of this sequence, were studied. Of particular interest, the FriedeleCrafts cyclization using (CF 3 CO) 2 O/BF 3 $Et 2 O show that near the expected keto-lactams, enamides and enamidones containing trifluoromethyl residue were isolated. A mechanism leading to the latter products with high synthetic potential was discussed.
Use of chiral-pool approach into epi-thieno analogues of the scarce bioactive phenanthroquinolizidine alkaloids

The severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) is one of the predominant antigenic protein and the most abundant shed antigen throughout the SARS-CoV infection. This feature makes it a suitable molecular target for diagnostic applications. In this study the full length codon optimized NP gene and its subfragment gene segment was cloned in a bacterial expression vector. The full length NP could be expressed in E. coli at very high level within inclusion bodies. The inclusion bodies were successfully solubilized, purified under denaturing conditions employing IMAC column and refolded. The non-glycosylated NP was used to immunize mice for hybridoma development. The polyclonal antiserum from animals immunized with this recombinant NP protein was found to specifically recognize the NP and its subfragments, thus demonstrating the immunogenic nature of the recombinant protein. The NP antigen or a subfragment could be useful for developing a sensitive serum diagnostic assay to monitor SARS-CoV outbreaks by detecting the early human anti-SARS antibodies. In addition, the availability of the NP fragments could facilitate epitope mapping of anti-NP monoclonals for identifying suitable sandwich pairs.

Copious production of SARS-CoV nucleocapsid protein employing codon optimized synthetic gene