Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I ‫؍‬ 10 mM, 1 mM CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4) was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.

West Nile Virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. The West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins, and its inhibition could represent a potential treatment for viral infections. This article describes the design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide (G 4 SG 4 ) to the N-terminal 184 residues of NS3. A chromogenic assay using synthetic para-nitroanilide (pNA) hexapeptide substrates was used to identify optimal enzyme-processing conditions (pH 9.5, I < 0.1 M, 30% glycerol, 1 mM CHAPS), preferred substrate cleavage sites, and the first competitive inhibitor (Ac-FASGKR-H, IC 50 ϳ1 M). A putative three-dimensional structure of WNV protease, created through homology modeling based on the crystal structures of Dengue-2 and Hepatitis C NS3 viral proteases, provides some valuable insights for structure-based design of potent and selective inhibitors of WNV protease.

By establishing mouse primary keratinocytes (KCs) in culture, we were able, for the first time, to express papillomavirus major capsid (L1) proteins by transient transfection of authentic or codon-modified L1 gene expression plasmids. We demonstrate in vitro and in vivo that gene codon composition is in part responsible for differentiation-dependent expression of L1 protein in KCs. L1 mRNA was present in similar amounts in differentiated and undifferentiated KCs transfected with authentic or codon-modified L1 genes and had a similar half-life, demonstrating that L1 protein production is posttranscriptionally regulated. We demonstrate further that KCs substantially change their tRNA profiles upon differentiation. Aminoacyl-tRNAs from differentiated KCs but not undifferentiated KCs enhanced the translation of authentic L1 mRNA, suggesting that differentiation-associated change to tRNA profiles enhances L1 expression in differentiated KCs. Thus, our data reveal a novel mechanism for regulation of gene expression utilized by a virus to direct viral capsid protein expression to the site of virion assembly in mature KCs. Analysis of two structural proteins of KCs, involucrin and keratin 14, suggests that translation of their mRNAs is also regulated, in association with KC differentiation in vitro, by a similar mechanism.Direction of gene expression to undifferentiated or differentiated cells is classically determined by altered promoter methylation or by production of specific transcription factors or posttranscriptionally by interaction of regulatory mRNA sequences with translational regulators (38). Papillomaviruses (PVs) are a family of double-stranded DNA viruses which replicate exclusively in epithelium, promote cell growth, and affect cellular differentiation, giving rise to benign tumors with, for some virus types, potential for malignancy. mRNA encoding the PV major capsid L1 protein can be transcribed from L1 gene expression plasmids in many types of mammalian cells (20). However, translation of the transcribed mRNA to L1 protein is limited in vivo to differentiated keratinocytes (KCs) (5, 53) and to yeast cells (50,67). Although inhibitory mechanisms have been proposed to explain the blockage of PV L1 gene translation in undifferentiated cells (10, 11), no inhibitory factors have been identified as specific for epithelial cells in vitro or in vivo. Thus, the mechanism for the tight differentiation-specific translation of the PV L1 gene in KCs remains to be determined.PVs, like many mammalian DNA viruses, use relatively few "mammalian consensus" codons to encode their capsid genes, manifesting a high AϩT genome content due to third-nucleotide bias to AϩT (68). In humans, codon-mediated translational controls may play an important role in the differentiation and regulation of tissue-specific gene products (47). Blockage to translation of PV L1 mRNAs has been overcome by codon modification utilizing mammalian preferred codons without changing the protein sequence (36,41,42,69), but it remains unclear whether co...

The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ac-KPGLKRp-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in WNV NS3 protease at S1 (V115A/F, D129A/ E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases.

A new variant of Streptococcus pyogenes serotype M1 (designated ‘M1UK’) has been reported in the United Kingdom, linked with seasonal scarlet fever surges, marked increase in invasive infections, and exhibiting enhanced expression of the superantigen SpeA. The progenitor S. pyogenes ‘M1global’ and M1UK clones can be differentiated by 27 SNPs and 4 indels, yet the mechanism for speA upregulation is unknown. Here we investigate the previously unappreciated expansion of M1UK in Australia, now isolated from the majority of serious infections caused by serotype M1 S. pyogenes. M1UK sub-lineages circulating in Australia also contain a novel toxin repertoire associated with epidemic scarlet fever causing S. pyogenes in Asia. A single SNP in the 5’ transcriptional leader sequence of the transfer-messenger RNA gene ssrA drives enhanced SpeA superantigen expression as a result of ssrA terminator read-through in the M1UK lineage. This represents a previously unappreciated mechanism of toxin expression and urges enhanced international surveillance.

The selection of a suitable scaffold material is important for dentin tissue regeneration, as the characteristics of biomaterials can potentially influence cell proliferation and differentiation. We compared the effects of different scaffolds on dentin regeneration based on dental pulp stem cells (DPSCs) and investigated the regulatory mechanisms of odontogenic differentiation of DPSCs by these scaffolds. Five different scaffolds were tested: demineralized dentin matrix (DDM), ceramic bovine bone (CBB), small intestinal submucosa (SIS), poly-L-lactate-co-glycolate, and collagen-chondroitin sulfate-hyaluronic acid. DPSCs cultured on DDM and CBB exhibited higher levels of alkaline phosphatase (ALP) activity and mRNA expression of bone sialoprotein, osteocalcin, dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) than those cultured on the other three scaffolds. Further, the phosphorylation levels of mitogen-activated protein kinase (MAPK) ERK1/2 and p38 in DPSCs cultured on DDM and CBB were also significantly enhanced compared with the other three scaffolds, and their inhibitors significantly inhibited odontogenic differentiation as assessed by ALP activity and mRNA expression of DSPP and DMP-1. The implantation experiment confirmed these results and showed a large amount of regular-shaped dentin-pulp complex tissues, including dentin, predentin, and odontoblasts only in the DDM and CBB groups. The results indicated that natural mineralized scaffolds (DDM and CBB) have potential as attractive scaffolds for dentin tissue-engineering-promoted odontogenic differentiation of DPSCs through the MAPK signaling pathway.

Salmonella isolates harbour a range of resident prophages which can influence their virulence and ability to compete and survive in their environment. Phage gene profiling of a range of phage types of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) indicates a significant level of correlation of phage gene profile with phage type as well as correlation with genotypes determined by a combination of multi-locus variable-number tandem repeat (VNTR) typing and clustered regularly interspaced short palindromic repeats (CRISPR) typing. Variation in phage gene profiles appears to be partly linked to differences in composition of variants of known prophages. We therefore conducted a study of the distribution of variants of ST64B and Gifsy-1 prophages and coincidently the presence of Gifsy-3 prophage in a range of S. Typhimurium phage types and genotypes. We have discovered two variants of the DT104 variant of ST64B and at least two new variants of Gifsy-1 as well as variants of related phage genes. While there is definite correlation between phage type and the prophage profile based on ST64B and Gifsy-1 variants we find stronger correlation between the VNTR/CRISPR genotype and prophage profile. Further differentiation of some genotypes is obtained by addition of the distribution of Gifsy-3 and a sequence variant of the substituted SB26 gene from the DT104 variant of ST64B. To explain the correlation between genotype and prophage profile we propose that suites of resident prophages promote clonality possibly through superinfection exclusion systems.

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