Structural basis for substrate specificity of alphavirus nsP2 proteases

Abstract

The alphavirus nsP2 protease is essential for correct processing of the alphavirus nonstructural polyprotein (nsP1234) and replication of the viral genome. We have combined molecular dynamics simulations with our structural studies to reveal features of the nsP2 protease catalytic site and S1'-S4 subsites that regulate the specificity of the protease. The catalytic mechanism of the nsP2 protease appears similar to the papain-like cysteine proteases, with the conserved catalytic dyad forming a thiolate-imidazolium ion pair in the nsP2-activated state. Substrate binding likely stabilizes this ion pair. Analysis of bimolecular complexes of Venezuelan equine encephalitis virus (VEEV) nsP2 protease with each of the nsP1234 cleavage sites identified protease residues His 510 , Ser 511 , His 546 , and Lys 706 as critical for cleavage site recognition. Homology modelling and molecular dynamics simulations of diverse alphaviruses and their cognate cleavage site sequences revealed general features of substrate recognition that operate across alphavirus strains as well as strain specific covariance between binding site and cleavage site residues. For instance, compensatory changes occurred in the P3 and S3 subsite residues to maintain energetically favourable complementary binding surfaces. These results help explain how alphavirus nsP2 proteases recognize different cleavage sites within the non-structural polyprotein and discriminate between closely related cleavage targets.

Differences in Processing Determinants of Nonstructural Polyprotein and in the Sequence of Nonstructural Protein 3 Affect Neurovirulence of Semliki Forest Virus

“…Merits, and A. Lulla, unpublished data). Functional linkage between position P4 of the 1/2 site (residue 534 of ns polyprotein) and amino acid residue 515 of nsP2 (residue 1052 of the ns polyprotein), which is part of the protease S4 subsite, was also predicted by Russo et al (47). Therefore, it has been proposed that amino acid residues 534 and 1052, which are His and Val in L10 and SFV6 but Arg and Glu in A7(74) and A774wt (Table 1), may represent additional determinants for SFV virulence.…”

Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease’s active site cysteine residue

“…Most likely this reflects differences in the basic enzymatic activities of the proteases from different alphaviruses. CHIKV nsP2 harbours a Glu residue in position 515 that is predicted to be part of the S4 subsite of the enzyme22. In contrast, the SFV protease has Val515 that was shown to increase the ability of nsP2 to process P34 polyprotein in infected cells43.…”

“…Subsequent molecular modelling revealed the features of the catalytic site and the S1′–S4 subsites22. A peptidomimetic inhibitor of nsP2 protease binds at the interface of the protease and MTL domains resulting in conformational change that most probably assists in leaving group departure of either the amine or Cys thiolate during the catalytic cycle23; the binding mode of natural substrates is most likely similar.…”

Hypervariable Domain of Nonstructural Protein nsP3 of Venezuelan Equine Encephalitis Virus Determines Cell-Specific Mode of Virus Replication

“…Alphavirus nsP1, nsP2, and nsP4 were assigned multiple roles in viral RNA synthesis and modification of the intracellular environment (22,23,48,(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62). However, one of the four nonstructural proteins, nsP3, remains insufficiently explored, and to date, its functions remain obscure.…”