The leader proteinase (Lpro) of foot‐and‐mouth disease virus (FMDV) initially cleaves itself from the polyprotein. Subsequently, Lpro cleaves the host proteins eukaryotic initiation factor (eIF) 4GI and 4GII. This prevents protein synthesis from capped cellular mRNAs; the viral RNA is still translated, initiating from an internal ribosome entry site. Lpro cleaves eIF4GI between residues G674 and R675. We showed previously, however, that Lpro binds to residues 640–669 of eIF4GI. Binding was substantially improved when the eIF4GI fragment contained the eIF4E binding site and eIF4E was present in the binding assay. Lpro interacts with eIF4GI via residue C133 and residues 183–195 of the C‐terminal extension. This binding domain lies about 25 Å from the active site. Here, we examined the binding of Lpro to eIF4GI fragments generated by in vitro translation to narrow the binding site down to residues 645–657 of human eIF4GI. Comparison of these amino acids with those in human eIF4GII as well as with sequences of eIF4GI from other organisms allowed us to identify two conserved basic residues (K646 and R650). Mutation of these residues was severely detrimental to Lpro binding. Similarly, comparison of the sequence between residues 183 and 195 of Lpro with those of other FMDV serotypes and equine rhinitis A virus showed that acidic residues D184 and E186 were highly conserved. Substitution of these residues in Lpro significantly reduced eIF4GI binding and cleavage without affecting self‐processing. Thus, FMDV Lpro has evolved a domain that specifically recognizes a host cell protein.

Fragile sites are specific genomic loci that are especially prone to chromosome breakage. For the human genome there are 31 rare fragile sites and 88 common fragile sites listed in the National Center for Biotechnology Information database; however, the exact number remains unknown. In this study, unstable DNA sequences, which have been previously tagged with a marker gene, were cloned and provided starting points for the characterization of two aphidicolin inducible common fragile sites. Mapping of these unstable regions with six-color fluorescence in situ hybridization revealed two new fragile sites at 6p21 and 13q22, which encompass genomic regions of 9.3 and 3.1 Mb, respectively. According to the fragile site nomenclature they were consequently entitled as FRA6H and FRA13E. Both identified regions are known to be associated with recurrent aberrations in malignant and nonmalignant disorders. It is conceivable that these fragile sites result in genetic damage that might contribute to cancer phenotypes such as osteosarcoma, breast and prostate cancer. ' 2007 Wiley-Liss, Inc.Key words: fragile site; FRA6H; FRA13E; genomic instability; chromosome rearrangements Fragile sites are specific genomic loci that are especially prone to express genomic instability. They can be visualized as gaps and breaks on metaphase chromosomes after culturing cells under conditions of replication stress. Based on their incidence in the human population, they are divided into rare fragile sites, occurring in less than 5% of all individuals, and common fragile sites being a constitutive feature of the genome of probably all individuals. 1According to the National Center for Biotechnology Information (NCBI), there are 31 rare fragile sites and 88 common fragile sites in the human genome. However, the exact number of fragile sites remains unclear, since there are no stringent criteria for inclusion and there is no regular update of the database. 2The molecular basis for the expression of rare fragile sites is the dynamic mutation of expanding CGG trinucleotide or AT-rich minisatellite sequences that after reaching a certain threshold expansion account for the observed instability. [3][4][5][6] In contrast, the molecular basis for breakage of common fragile sites is still unclear. 12 However, analysis of the identified sequences did not reveal any particular sequence structure; an ATrichness and an enrichment of DNA flexibility structures seem to be the only shared features.13,14 It has been hypothesized that the AT-richness leads to an accumulation of DNA secondary structures, which might cause a delayed replication at fragile sites. [15][16][17][18] This perturbed replication at fragile sites was shown to activate cell cycle checkpoints in an ATR-dependent manner. 19 In line with this, several targets and modifiers of the ATR-pathway, such as BRCA1, SMC1, CHK1 and the Fanconi anemia pathway proteins, were reported to be involved in maintenance of fragile sites stability. [20][21][22][23] The replication perturbation may result in doubl...

The foot-and-mouth disease virus Leader proteinase (L(pro)) frees itself from the growing viral polyprotein by self-processing between its own C-terminus and the N-terminus of the subsequent protein VP4. The ArgLysLeuLys*GlyAlaGlyGln sequence is recognized. The proteinase subsequently cleaves the two isoforms of host cell protein eukaryotic initiation factor (eIF) 4G at the AlaAsnLeuGly*ArgThrThrLeu (eIF4GI) and LeuAsnValGly*SerArgArgSer (eIF4GII) sequences. The enzyme does not, however, recognize the sequence on eIF4GII (AlaAspPheGly*ArgGlnThrPro) which is analogous to that recognized on eIF4GI. To investigate the basis for this specificity, we used site-directed mutagenesis to show that the presence of Phe at the P2 position or Asp at the P3 position severely compromises self-processing. Furthermore, these substitutions also give rise to the production of aberrant cleavage products. As Leu is the preferred amino acid at P2, the specificity of L(pro) is reminiscent of that of cathepsin K. This cellular proteinase can also process collagen through its ability to accept proline at the P2 position. Investigation of the L(pro) substrate specificity showed, however, that in contrast to cathepsin K, L(pro) cannot accept Pro at P2 and does not cleave collagen. Subtle variations in the arrangement of the S2 binding pockets on the enzymes are responsible for these differences in specificity.