Structure of the foot-and-mouth disease virus leader protease: a papain-like fold adapted for self-processing and eIF4G recognition.

The leader protease of foot-and-mouth disease virus, as well as cleaving itself from the nascent viral polyprotein, disables host cell protein synthesis by specific proteolysis of a cellular protein: the eukaryotic initiation factor 4G (eIF4G). The crystal structure of the leader protease presented here comprises a globular catalytic domain reminiscent of that of cysteine proteases of the papain superfamily, and a flexible C-terminal extension found intruding into the substrate-binding site of an adjacent molecule. Nevertheless, the relative disposition of this extension and the globular domain to each other supports intramolecular self-processing. The different sequences of the two substrates cleaved during viral replication, the viral polyprotein (at LysLeuLys/GlyAlaGly) and eIF4G (at AsnLeuGly/ArgThrThr), appear to be recognized by distinct features in a narrow, negatively charged groove traversing the active centre. The structure illustrates how the prototype papain fold has been adapted to the requirements of an RNA virus. Thus, the protein scaffold has been reduced to a minimum core domain, with the active site being modified to increase specificity. Furthermore, surface features have been developed which enable C-terminal self-processing from the viral polyprotein.

elF4G and its proteolytic cleavage products: effect on initiation of protein synthesis from capped, uncapped, and IRES-containing mRNAs.

Rhinovirus 2A and foot-and-mouth disease virus Lb proteinases stimulate the translation of uncapped messages and those carrying the rhinovirus and enterovirus Internal Ribosome Entry Segments (IRESes) by a mechanism involving the cleavage of host cell proteins. Here, we investigate this mechanism using an artificial dicistronic RNA containing the human rhinovirus IRES as intercistronic spacer. Because both proteinases cleave eukaryotic initiation factor 4G (eIF4G), we examined whether the cleavage products of eIF4G could stimulate uncapped or IRES-driven translation. Addition of intact eIF4F to translation extracts inhibited IRES-driven translation and reduced the translation stimulation observed in reactions pre-treated with Lb proteinase. Prolonged incubation of translation extracts with Lb proteinase removed all endogenous eIF4G and a substantial amount of the primary C- and N-terminal cleavage products. The translation of all mRNAs was reduced in such extracts. Capped mRNA translation was rescued by the addition of intact eIF4F. In contrast, addition of pre-cleaved eIF4F stimulated translation of uncapped or IRES-bearing messages to the levels seen upon proteinase addition. Furthermore, fractions containing the C-terminal, but not N-terminal, cleavage product of eIF4G stimulated translation moderately. These results demonstrate that the Lb and 2A proteinases stimulate translation of uncapped RNAs and those carrying IRESes by the production of cleavage products of eIF4G that enhance translation and by the removal of intact eIF4G that interferes with this stimulation.

Crystallization and preliminary X-ray diffraction studies of the Lb proteinase from foot-and-mouth disease virus.

Different crystal forms of the C23A mutant from the leader proteinase of foot-and-mouth disease virus were obtained by the hanging drop vapor diffusion technique, using MgCl2 and PEG 6000 as precipitants. Well-developed crystals, with cubic morphology growing to approximately 1.0 mm3 in size, presented a large unit cell parameter of 274.5 A and diffracted to, at most, 5 A resolution. A second type of crystal had a tetragonal appearance and these were obtained in droplets soaked in a silica gel matrix. These crystals, with an approximate size of 0.3 X 0.3 X 0.7 mm3, diffracted to approximately 4.0 A resolution, but presented a strong anisotropic mosaicity around the longest crystal axis. Crystals with a needlelike morphology and reaching sizes of about 0.2 X 0.3 X 1.2 mm3 diffracted beyond 3.5 A resolution and were stable to X-ray radiation for approximately one day when using a conventional source at room temperature. These crystals are orthorhombic with space group I222 (or I2(1)2(1)2(1)) and unit cell dimensions a = 65.9 A, b = 104.3 A, and c = 124.0 A, and appear well suited for high-resolution studies. Density packing considerations are consistent with the presence of two molecules in the asymmetric unit and a solvent content of approximately 54%.

Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. Implications for cap-dependent and cap-independent translational initiation.

Cap-dependent binding of mRNA to the 40 S ribosomal subunit during translational initiation requires the association of eukaryotic initiation factor 4G (eIF4G; formerly eIF-4 gamma and p220) with other initiation factors, notably eIF4E, eIF4A, and eIF3. Infection of cells by picornaviruses results in proteolytic cleavage of eIF4G and generation of a cap-independent translational state. Rhinovirus 2A protease and foot-and-mouth-disease virus L protease were used to analyze the association of eIF4G with eIF4A, eIF4E, and eIF3. Both proteases bisect eIF4G into N- and C-terminal fragments termed cpN and cpC. cpN was shown to contain the eIF4E-binding site, as judged by retention on m7GTP-Sepharose, whereas cpC was bound to eIF3 and eIF4A, based on ultracentrifugal co-sedimentation. Further proteolysis of cpN by L protease produced an 18-kDa polypeptide termed cpN2 which retained eIF4E binding activity and corresponded to amino acid residues 319-479 of rabbit eIF4G. Further proteolysis of cpC yielded several smaller fragments. cpC2 (approximately 887-1402) contained the eIF4A binding site, whereas cpC3 (approximately 480-886) contained the eIF3 binding site. These results suggest that cleavage by picornaviral proteases at residues 479-486 separates eIF4G into two domains, one required for recruiting capped mRNAs and one for attaching mRNA to the ribosome and directing helicase activity. Only the latter would appear to be necessary for internal initiation of picornaviral RNAs.

Foot-and-mouth disease virus Lb proteinase can stimulate rhinovirus and enterovirus IRES-driven translation and cleave several proteins of cellular and viral origin.

Rhinovirus and enterovirus 2A proteinases stimulate translation initiation driven from the cognate internal ribosome entry segment (IRES) (S. J. Hambidge and P. Sarnow, Proc. Natl. Acad. Sci. USA 89:10272-10276, 1992; H.-D. Liebig, E. Ziegler, R. Yan, K. Hartmuth, H. Klump, H. Kowalski, D. Blaas, W. Sommergruber, L. Frasel, B. Lamphear, R. Rhoads, E. Kuechler, and T. Skern, Biochemistry 32:7581-7588, 1993). Given the functional similarities between the foot-and-mouth disease virus (FMDV) L proteinase and these 2A proteinases (autocatalytic excision from the nascent viral polyprotein and cleavage of eIF-4 gamma), we investigated whether the FMDV L proteinase would also be able to stimulate translation initiation. We found that purified recombinant FMDV Lb proteinase could stimulate in vitro translation driven from a rhinovirus or enterovirus IRES by 5- to 10-fold. In contrast, stimulation of translation initiation on a cardiovirus IRES by this proteinase was minimal, and stimulation of translation driven from the cognate FMDV IRES could not be evidenced. Studies using an inhibitor or a mutant Lb proteinase indicated that stimulation of IRES-driven translation is mediated via proteolysis of some cellular component(s). Our studies also demonstrated that the Lb proteinase is capable of stimulating initiation of translation on an uncapped cellular message. Unexpectedly, and in contrast to the 2A proteinases, the Lb proteinase specifically cleaved the products of the two reporter genes used in this study: Xenopus laevis cyclin B2 and influenza virus NS. Therefore, we also set out to investigate the requirements for substrate recognition by the Lb proteinase. Purified recombinant Lb proteinase recognized at least one mengovirus polypeptide and specifically cleaved human cyclin A and poliovirus replicase-related polypeptides. In the latter case, the site(s) of cleavage was located within the N-terminal part of polypeptide 3D. Sequence comparisons revealed no significant primary sequence similarities between the target proteins and the two sites already known to be recognized by the FMDV L proteinase.

Foot-and-mouth disease virus leader proteinase: purification of the Lb form and determination of its cleavage site on eIF-4 gamma.

Many picornaviruses cause a dramatic decrease in the translation of cellular mRNAs in the infected cell, without affecting the translation of their own RNA. Specific proteolysis of protein synthesis initiation factor eIF-4 gamma occurs during infection with rhinoviruses, enteroviruses, and aphthoviruses, apparently leading to an inability of the ribosomes to bind capped mRNAs. Cleavage of eIF-4 gamma in human rhinoviruses and enteroviruses is carried out by the viral 2A proteinase; in aphthoviruses (i.e., foot-and-mouth disease viruses), the leader proteinase is responsible for this reaction. We describe here the purification to homogeneity of the Lb form of the leader proteinase expressed in Escherichia coli. The primary cleavage products of eIF-4 gamma obtained in vitro with purified leader or 2A proteinase are electrophoretically indistinguishable from those found during infection in vivo. However, additional proteolysis products of eIF-4 gamma are observed with the leader proteinase and the human rhinovirus type 2 2A proteinase in vitro. The cleavage site of the leader proteinase in eIF-4 gamma from rabbit reticulocyte was determined by sequencing the purified C-terminal cleavage product by automated Edman degradation. The cleavage site is between Gly-479 and Arg-480 and thus differs from that of rhinovirus and enterovirus 2A proteinases, which cleave between Arg-486 and Gly-487.

Patterns of allele losses suggest the existence of five distinct regions of LOH on chromosome 17 in breast cancer.

Chromosome 17 is a frequent target during breast-cancer formation and progression. It has been shown to be affected by allele losses at multiple sites, as well as by DNA amplification. Our aim was to delineate a map of the genetic alterations on chromosome 17 in a given set of breast tumors. To this end we analyzed 151 pairs of tumor and cognate lymphocyte DNAs by Southern blotting with 5 RFLP or VNTR probes and by PCR at 8 CA repeat polymorphic loci for LOHs. Moreover, we studied DNA amplification of the evi2, erbB2, thraI, gcsf and rara genes. Data presented here point strongly to the existence of 5 distinct regions of allele losses on chromosome 17:2 on 17p, 3 on 17q. Of the 2 regions on 17p, one involves tp53 while the second is located more distally toward the telomere. LOH was found in 45.9% and 58.8% respectively. The 3 regions on 17q are located: (i) on the proximal portion of the long arm band q21, corresponding to the brcaI region; (ii) in a central region defined by the marker D17S74; (iii) on the distal part of 17q (band q25) characterized by losses of the marker D17S24. Each of these regions presented respectively allele losses in 47.5%, 33.3% and 40.8% of the informative tumors. Whereas some tumors presented patterns of LOH consistent with the loss of a complete chromosomal arm or of large portions of the chromosome, a high proportion of the analyzed tumors showed interstitial losses. Amplifications were found in 15% of the tumors and were centered around erbB2.(ABSTRACT TRUNCATED AT 250 WORDS)