Site‐specific mutagenesis of cDNA clones expressing a poliovirus proteinase

Abstract

The cleavage of poliovirus precursor polypeptides occurs at specific amino acid pairs that are recognized by viral proteinases. Most of the polio-specific cleavages occur at glutamine-glycine (Q-G) pairs that are recognized by the viral-encoded proteinase 3C (formerly called P3-7c). In order to carry out a defined molecular genetic study of the enzymatic activity of protein 3C, we have made cDNA clones of the poliovirus genome. The cDNA region corresponding to protein 3C was inserted into an inducible bacterial expression vector. This recombinant plasmid (called pIN-III-C3-7c) utilizes the bacterial lipoprotein promoter to direct the synthesis of a precursor polypeptide that contains the amino acid sequence of protein 3C as well as the amino- and carboxy-terminal Q-G cleavage signals. These signals have been previously shown to allow autocatalytic production of protein 3C in bacteria transformed with plasmid pIN-III-C3-7c. We have taken advantage of the autocatalytic cleavage of 3C in a bacterial expression system to study the effects of site-specific mutagenesis on its proteolytic activity. One mutation that we have introduced into the cDNA region encoding 3C is a single amino acid insertion near the carboxy-terminal Q-G cleavage site. The mutant recombinant plasmid (designated pIN-III-C3-mu 10) directs the synthesis of a bacterial-polio precursor polypeptide that is like the wild-type construct (pIN-III-C3-7c). However, unlike the wild-type precursor, the mutant precursor cannot undergo autocatalytic cleavage to generate the mature proteinase 3C. Rather, the precursor is able to carry out cleavage at the amino-terminal Q-G site but not at the carboxy-terminal site. Thus, we have generated an altered poliovirus proteinase that is still able to carry out at least part of its cleavage activities but is unable to be a suitable substrate for self-cleavage at its carboxy-terminal Q-G pair.

Cellular mRNA Decay Protein AUF1 Negatively Regulates Enterovirus and Human Rhinovirus Infections

“…Wildtype FLAG-3CD protein was expressed, thereby producing both FLAG-3CD proteinase and its FLAG-3C cleavage product by the autocatalytic activity of 3C. Additionally, FLAG-3CD, which is incapable of autocleavage into 3C and 3D due to a mutation at the 3C/3D junction but retains catalytic activity (24,34), was unable to effect relocalization of AUF1 in transfected cells (data not shown). To determine the requirement for proteinase activity in the redistribution of AUF1 by 2A, a previously described mutated 2A protein lacking proteinase activity (2A-L98P) was expressed, and localization of AUF1 was examined (24,35,36).…”

Modulation of the RNA Binding and Protein Processing Activities of Poliovirus Polypeptide 3CD by the Viral RNA Polymerase Domain

“…This plasmid encodes a histidine-tagged 3CD protein containing a serine insertion proximal to the 3C/3D junction that essentially eliminates auto-processing at this site without affecting proteinase and RNA binding activities (11,23).…”

Viral Proteinase Requirements for the Nucleocytoplasmic Relocalization of Cellular Splicing Factor SRp20 during Picornavirus Infections

“…The 3CD proteinase is found at higher concentrations in the cell during infection than 3C proteinase, and has self-cleavage properties to generate 3C proteinase and 3D polymerase. To prevent self-cleavage and assess only 3CD activity, we utilized a mutated version of 3CD, containing an amino acid insertion adjacent to the cleavage site to render the protein deficient in self-cleavage while retaining proteolytic activity (3CD10) (41). FLAG-3CD10 (or empty vector) and GFPSRp20 plasmids were cotransfected into cells, and 3CD10 expression was controlled using an inducible promoter (described in Materials and Methods).…”