Pandemic and epidemic outbreaks of influenza A virus (IAV) infection pose severe challenges to human society. Passive immunotherapy with recombinant neutralizing antibodies can potentially mitigate the threats of IAV infection. With a high throughput neutralizing antibody discovery platform, we produced artificial anti-hemagglutinin (HA) IAV-neutralizing IgGs from phage-displayed synthetic scFv libraries without necessitating prior memory of antibody-antigen interactions or relying on affinity maturation essential for in vivo immune systems to generate highly specific neutralizing antibodies. At least two thirds of the epitope groups of the artificial anti-HA antibodies resemble those of natural protective anti-HA antibodies, providing alternatives to neutralizing antibodies from natural antibody repertoires. With continuing advancement in designing and constructing synthetic scFv libraries, this technological platform is useful in mitigating not only the threats of IAV pandemics but also those from other newly emerging viral infections.

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The grouper iridovirus (GIV) belongs to the family Iridoviridae, whose genome contains an antiapoptotic B-cell lymphoma (Bcl)-2-like gene. This study was carried-out to understand whether GIV blocks apoptosis in its host. UV-irradiated grouper kidney (GK) cells underwent apoptosis. However, a DNA fragmentation assay of UV-exposed GK cells after GIV infection revealed an inhibition of apoptosis. The UV- or heat-inactivated GIV failed to inhibit apoptosis, implying that a gene or protein of the viral particle might contribute to an apoptosis inhibitory function. The DNA ladder assay for GIV-infected GK cells after UV irradiation confirmed that apoptosis inhibition was an early process which occurred as early as 5 min post-infection. A GIV-Bcl sequence comparison showed distant sequence similarities to that of human and four viruses; however, all possessed the putative Bcl-2 homology (BH) domains of BH1, BH2, BH3, and BH4, as well as a transmembrane domain. Northern blot hybridization showed that GIV-Bcl transcription began at 2 h post-infection, and the mRNA level significantly increased in the presence of cycloheximide or aphidicolin, indicating that this GIV-Bcl is an immediate-early gene. This was consistent with the Western blot results, which also revealed that the virion carries the Bcl protein. We observed the localization of GIV-Bcl on the mitochondrial membrane and other defined intracellular areas. By immunostaining, it was proven that GIV-Bcl-expressing cells effectively inhibited apoptosis. Taken together, these results demonstrate that GIV inhibits the promotion of apoptosis by GK cells, which is mediated by the immediate early expressed viral Bcl gene.

Structural origin of substrate-enzyme recognition remains incompletely understood. In the model enzyme system of serine protease, canonical anti-parallel ␤-structure substrate-enzyme complex is the predominant hypothesis for the substrate-enzyme interaction at the atomic level. We used factor Xa (fXa), a key serine protease of the coagulation system, as a model enzyme to test the canonical conformation hypothesis. More than 160 fXa-cleavable substrate phage variants were experimentally selected from three designed substrate phage display libraries. These substrate phage variants were sequenced and their specificities to the model enzyme were quantified with quantitative enzyme-linked immunosorbent assay for substrate phage-enzyme reaction kinetics. At least three substrate-enzyme recognition modes emerged from the experimental data as necessary to account for the sequence-dependent specificity of the model enzyme. Computational molecular models were constructed, with both energetics and pharmacophore criteria, for the substrate-enzyme complexes of several of the representative substrate peptide sequences. In contrast to the canonical conformation hypothesis, the binding modes of the substrates to the model enzyme varied according to the substrate peptide sequence, indicating that an ensemble of binding modes underlay the observed specificity of the model serine protease.Serine proteases recognize peptide sequences as substrates with a spectrum of specificity (1), for which the structural origin remains largely unclear (2, 3). The enzyme family is one of the most important model families in enzymology as about onethird of proteases (coded in about 2% of human genome) can be classified as serine proteases and many of the serine proteases are involved in important physiological regulation and pathogenic dysfunction (1). Ref. 4, and references therein). Still, the enzyme-substrate transition state complex structures have been difficult to be elucidated (5), partly due to the rapid hydrolysis of the substrate and partly due to the rapid dissociation of the products from the active site. Hence the structural origin on the substrate specificity of serine proteases has been incompletely resolved, hampering critical understanding toward enzyme-substrate recognition and its implications in inhibitor design and substrate specificity engineering.Models of enzyme-substrate transition state complexes have been proposed on the basis of the three-dimensional structures of serine protease-inhibitor complexes. Invariably in the models and thus dubbed canonical conformation, the peptide substrate binds to the enzyme in ␤-strand conformation, anti-parallel to the corresponding ␤-strand (residues 214 -217 in chymotrypsin number) in the serine protease (see Refs. 2-6 for reviews). However, accumulating evidence supporting alternative models suggests that the canonical conformation is not universally applicable to serine protease-substrate complexes (5-8). Using factor Xa as a model system, we asked if the canonical conformation hypo...