Foot-and-Mouth Disease Virus Leader Proteinase:  Specificity at the P2 and P3 Positions and Comparison with Other Papain-like Enzymes

Abstract

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.

Proteolytic Processing of Turnip Yellow Mosaic Virus Replication Proteins and Functional Impact on Infectivity

“…4B and C), consistent with previous reports regarding cellular and viral papain-like cysteine proteases, such as cathepsin and foot-and-mouth disease virus or Semliki Forest virus protease (22,23,30). The mechanism of cleavage site recognition for TYMV proteinase is unknown, and pursuing the definition of the molecular determinants of its substrate specificity will require further mutagenesis studies.…”

Residue L143 of the Foot-and-Mouth Disease Virus Leader Proteinase Is a Determinant of Cleavage Specificity

“…2B. As reported by Kuehnel et al (13), cleavage products were essentially not detected until 20 min after the start of translation. We then introduced the mutation L143A via site-directed mutagenesis into the plasmid Lb pro VP4/VP2 L200F and examined Lb pro processing in RRLs.…”

“…Comparisons with other papain-like enzymes previously led us to propose that L178 might be important in excluding phenylalanine from the S2 site (13). However, our modeling and minimization experiments did not indicate that substituting L178 would improve the ability of the S2 pocket to accept phenylalanine.…”

“…Kuehnel et al (13) showed previously that in the self-processing reaction, Lb pro does not efficiently recognize the eIF4GII sequence AspPheGly*ArgGlnThr. Analysis of Lb pro cleavage on the sequence AspPheGly*ArgGlnThr showed that the presence of aspartic acid at the P3 position or phenylalanine at the P2 position impaired the Lb pro self-processing reaction (13).…”

“…Analysis of Lb pro cleavage on the sequence AspPheGly*ArgGlnThr showed that the presence of aspartic acid at the P3 position or phenylalanine at the P2 position impaired the Lb pro self-processing reaction (13). The nomenclature of the cleavage sites and the corresponding binding sites on the enzyme is that of Berger and Schechter (2).…”