trans -Protease Activity and Structural Insights into the Active Form of the Alphavirus Capsid Protease

Abstract

The alphavirus capsid protein (CP) is a serine protease that possesses cis-proteolytic activity essential for its release from the nascent structural polyprotein. The released CP further participates in viral genome encapsidation and nucleocapsid core formation, followed by its attachment to glycoproteins and virus budding. Thus, protease activity of the alphavirus capsid is a potential antialphaviral target to arrest capsid release, maturation, and structural polyprotein processing. However, the discovery of capsid protease inhibitors has been hampered due to the lack of a suitable screening assay and of the crystal structure in its active form. Here, we report the development of a trans-proteolytic activity assay for Aura virus capsid protease (AVCP) based on fluorescence resonance energy transfer (FRET) for screening protease inhibitors. Kinetic parameters using fluorogenic peptide substrates were estimated, and the K m value was found to be 2.63 ؎ 0.62 M while the k cat /K m value was 4.97 ؋ 10 4 M ؊1 min ؊1 . Also, the crystal structure of the trans-active form of AVCP has been determined to 1.81-Å resolution. Structural comparisons of the active form with the crystal structures of available substrate-bound mutant and inactive blocked forms of the capsid protease identify conformational changes in the active site, the oxyanion hole, and the substrate specificity pocket residues, which could be critical for rational drug design. IMPORTANCEThe alphavirus capsid protease is an attractive antiviral therapeutic target. In this study, we have described the formerly unap- , the disordered C-terminal residues after His261, and the presence of a water molecule in the oxyanion hole of AVCP⌬2 (AVCP with a deletion of the last two residues at the C terminus) reveal the effect of the C-terminal Trp267 deletion on enzyme structure. New structural data reported in this study along with the fluorogenic assay will be useful in substrate specificity characterization, high-throughput protease inhibitor screening, and structure-based development of antiviral drugs.

Functional Study of the Retrotransposon-Derived Human PEG10 Protease

“…Activity was not detected when a recombinant protein containing putative autoproteolytic sites was used as the substrate; the lack of trans activity indicated a self-inactivation mechanism for PR PEG10 . The suspected self-inactivation mechanism has already been observed for alphavirus capsid PRs [34]. In these viruses, the nascent structural polyprotein contains an intramolecular (chymotrypsin-like) serine protease that possesses cis activity.…”

“…This autoproteolytic event is a perquisite for capsid assembly and results in PR inactivation. After self-cleavage, the conserved P1 residue of the cleavage site blocks the trans activity by interacting with the catalytic center [34]. Self-inactivation has also been described for the capsid protease of the Aura virus, Chikungunya virus [35], Sindbis virus [36], and Semliki Forest virus [37], but has not been previously reported in the case of retroviral or retroviral-like PRs.…”

The HCPro from thePotyviridaefamily: an enviable multitasking Helper Component that every virus would like to have

“…This observation might indeed explain the exclusive cis-acting mode of HCPro, as the terminal glycine would occupy the space needed for the catalytic site to remain active. Unfortunately, attempts by Guo et al (2011) to remove this amino acid in order to make the proteinase active in trans, as was later performed for the CP serine proteinase of alphaviruses (Aggarwal et al, 2014), were unsuccessful. Second, the overall structure of this domain allowed for accurate comparisons with existing structures of other cysteine-like proteinases, such as papain, indicating that the HCPro atomic arrangement differs significantly from the distinctive papain-like folding.…”

Kinetic characterization of trans-proteolytic activity of Chikungunya virus capsid protease and development of a FRET-based HTS assay

“…Consequently, the alphavirus capsid protease is active only for one proteolytic reaction in the life cycle of virus. Further studies conducted on Aura virus capsid protease (AVCP) and Semliki Forest virus capsid protease (SFCP) demonstrate that the deletion of conserved C-terminal tryptophan leads to the restoration of the proteolytic activity of alphavirus CP 38 39 . The activity of SFCP was determined using nitrophenyl esters of tryptophan and tyrosine as the substrates.…”