Structure of the Autocatalytic Cysteine Protease Domain of Potyvirus Helper-component Proteinase

Abstract

The helper-component proteinase (HC-Pro) of potyvirus is involved in polyprotein processing, aphid transmission, and suppression of antiviral RNA silencing. There is no high resolution structure reported for any part of HC-Pro, hindering mechanistic understanding of its multiple functions. We have determined the crystal structure of the cysteine protease domain of HC-Pro from turnip mosaic virus at 2.0 Å resolution. As a protease, HC-Pro only cleaves a Gly-Gly dipeptide at its own C terminus. The structure represents a postcleavage state in which the cleaved C terminus remains tightly bound at the active site cleft to prevent trans activity. The structure adopts a compact ␣/␤-fold, which differs from papain-like cysteine proteases and shows weak similarity to nsP2 protease from Venezuelan equine encephalitis alphavirus. Nevertheless, the catalytic cysteine and histidine residues constitute an active site that is highly similar to these in papain-like and nsP2 proteases. HC-Pro recognizes a consensus sequence YXVGG around the cleavage site between the two glycine residues. The structure delineates the sequence specificity at sites P1-P4. Structural modeling and covariation analysis across the Potyviridae family suggest a tryptophan residue accounting for the glycine specificity at site P1 . Moreover, a surface of the protease domain is conserved in potyvirus but not in other genera of the Potyviridae family, likely due to extra functional constrain. The structure provides insight into the catalysis mechanism, cis-acting mode, cleavage site specificity, and other functions of the HC-Pro protease domain.

A Novel Role of the Potyviral Helper Component Proteinase Contributes To Enhance the Yield of Viral Particles

“…Furthermore, the cysteine and histidine catalytic residues were mapped by site-directed mutagenesis to the C-terminal half of the protein, supporting the hypothesis that HCPro closely resembles members of the papain-type family of cysteine proteases (17). The first high-resolution crystal structure of an HCPro protease domain was recently solved, giving new and important insights into its unique protein folding, self-cleavage mechanism, and substrate specificity (18).…”

“…Hence, the well-described interaction between the PTK motif of HCPro and CP, which is involved in aphid transmission, does not appear to be necessary for the novel function (see below). In addition, the proteinase activity is not required, since while the HCPro protease domain works only in cis (18), the ability of HCPro to stabilize CP can be supplied in trans (Fig. 1 to 4), and it is not affected by a point mutation in the crucial cysteine of the proteinase catalytic triad (C344A) (Fig.…”

viral silencing suppressors: Tools forged to fine-tune host-pathogen coexistence

“…some suppressors may target endogenous regulators of the silencing to modulate host defense. Potyviral helper-component protease (HCPro) is a multifunctional protein involved in many aspects of virus infection (Anandalakshmi et al, 1998;Carrington et al, 1989;Guo et al, 2011;Kasschau et al, 1997;Lakatos et al, 2006;Mallory et al, 2001). Tobacco etch virus (TEV) HC-Pro suppresses silencing through vsiRNA-sequestration and interferes with vsiRNA methylation (Lozsa et al, 2008).…”

Tobacco etch virus Protein P1 Traffics to the Nucleolus and Associates with the Host 60S Ribosomal Subunits during Infection

“…HC-Pro, a cysteine proteinase that self-cleaves from the viral polyprotein, suppresses the host defensive RNA silencing pathways (4). It is also involved in aphid transmission (5). P3N-PIPO and CI play roles in virus cell-to-cell movement (6).…”