Hans-Dieter Liebig

The structure of the 2A proteinase from a common cold virus: a proteinase responsible for the shut-off of host-cell protein synthesis

The crystal structure of the 2A proteinase from human rhinovirus serotype 2 (HRV2‐2Apro) has been solved to 1.95 Å resolution. The structure has an unusual, although chymotrypsin‐related, fold comprising a unique four‐stranded β sheet as the N‐terminal domain and a six‐stranded β barrel as the C‐terminal domain. A tightly bound zinc ion, essential for the stability of HRV2‐2Apro, is tetrahedrally coordinated by three cysteine sulfurs and one histidine nitrogen. The active site consists of a catalytic triad formed by His18, Asp35 and Cys106. Asp35 is additionally involved in an extensive hydrogen‐bonding network. Modelling studies reveal a substrate‐induced fit that explains the specificity of the subsites S4, S2, S1 and S1′. The structure of HRV2‐2Apro suggests the mechanism of the cis cleavage and its release from the polyprotein.

Structure and dynamics of coxsackievirus B4 2A proteinase, an enyzme involved in the etiology of heart disease.

ABSTRACT The 2A proteinases (2Apro) from the picornavirus family are multifunctional cysteine proteinases that perform essential roles during viral replication, involving viral polyprotein self-processing and shutting down host cell protein synthesis through cleavage of the eukaryotic initiation factor 4G (eIF4G) proteins. Coxsackievirus B4 (CVB4) 2Apro also cleaves heart muscle dystrophin, leading to cytoskeletal dysfunction and the symptoms of human acquired dilated cardiomyopathy. We have determined the solution structure of CVB4 2Apro (extending in an N-terminal direction to include the C-terminal eight residues of CVB4 VP1, which completes the VP1-2Apro substrate region). In terms of overall fold, it is similar to the crystal structure of the mature human rhinovirus serotype 2 (HRV2) 2Apro, but the relatively low level (40%) of sequence identity leads to a substantially different surface. We show that differences in the cI-to-eI2 loop between HRV2 and CVB4 2Apro translate to differences in the mechanism of eIF4GI recognition. Additionally, the nuclear magnetic resonance relaxation properties of CVB4 2Apro, particularly of residues G1 to S7, F64 to S67, and P107 to G111, reveal that the substrate region is exchanging in and out of a conformation in which it occupies the active site with association and dissociation rates in the range of 100 to 1,000 s−1. This exchange influences the conformation of the active site and points to a mechanism for how self-processing can occur efficiently while product inhibition is avoided.

[40] Picornains 2A and 3C

Publisher Summary This chapter focuses on picornains 2A and 3C. They are synthesized in cells following infection by viruses of the family Picornaviridae. The sequences of the picornains are deduced from genomic sequences. Comparison of these sequences with protein data banks revealed similarity to sequences of trypsin and chymotrypsin-like serine proteinases. The active site nucleophile of the picornains is cysteine. The genetic information encoded in picornaviral genomes is expressed as a single polyprotein of about 2200 amino acids, which undergoes proteolytic processing to give the mature viral proteins. Translation of cloned complementary DNA (cDNA) fragments of picornaviral genomes in rabbit reticulocyte lysates (RRLs) allows the production of radiolabeled proteins corresponding to various parts of the polyprotein. To produce substrates for intermolecular cleavage, regions containing the picornain genes are excluded or only partially present. All picornains 2A and 3C are capable of cleaving oligopeptides with sequences derived from viral cleavage sites. Peptides should contain at least 12 amino acids (6 at either side of the cleavage site) for efficient cleavage.

Rhinovirus 2A proteinase mediated stimulation of rhinovirus RNA translation is additive to the stimulation effected by cellular RNA binding proteins

The internal ribosome entry site (IRES) of enteroviruses, and especially human rhinoviruses (HRV), functions very inefficiently in rabbit reticulocyte lysates, but can be stimulated by addition of HeLa cell extracts. Two HeLa cell activities have been identified: the A-type activity is due to polypyrimidine tract binding protein and the B-type to unr. In addition HRV and enterovirus IRES function requires a third RNA binding protein, poly(rC) binding protein 2, but this is present in reticulocyte lysates in non-limiting amounts. IRES activity can also be stimulated by the cleavage of initiation factor eIF4G mediated by either HRV 2A protease, or foot-and-mouth disease virus (FMDV) L protease. This raises the question of whether this stimulation is independent of that effected by the three RNA binding proteins, or whether cleaved eIF4G functionally mimics one or more of these proteins. It is shown here that the stimulation of HRV IRES activity resulting from cleavage of eIF4G is additive with the stimulation effected by HeLa cell A- and B-type activities. It is proposed that the role of the RNA binding proteins is to maintain or attain the appropriate 3-dimensional structure of the IRES RNA element, whereas the function of eIF4G is to deliver the 40S ribosomal subunit to the correct site on the IRES, a function which, for reasons not yet fully understood, is fulfilled more efficiently by the C-terminal cleavage product of eIF4G than by the intact factor.

A thermosensitive mutant of HRV2 2A proteinase: evidence for direct cleavage of eIF4GI and eIF4GII

Infection of mammalian cells with picornaviruses like entero‐, rhino‐, and aphthoviruses leads to an inhibition of cap‐dependent cellular protein synthesis by the cleavage of both translation initiation factors, eIF4GI and eIF4GII. In entero‐ and rhinovirus infection this cleavage process is mediated by the viral 2A proteinase (2Apro). In order to discriminate between a direct mode of eIF4G cleavage and an indirect cleavage via activation of a cellular proteinase, a thermosensitive 2Apro mutant (ts‐2Apro) of human rhinovirus 2 was employed. Temperature shift experiments of cytoplasmic HeLa cell extracts incubated with ts‐2Apro strongly support a direct mode of cleavage of eIF4GI and eIF4GII by the viral 2Apro.