Site-directed mutagenesis of proteinase 3C results in a poliovirus deficient in synthesis of viral RNA polymerase

Dewalt, Patricia Gillis; Semler, Bert L.

doi:10.1128/jvi.61.7.2162-2170.1987

Cited by 58 publications

(21 citation statements)

References 34 publications

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“…Since poliovirus encodes its own proteinases, we tested to see if mutations in a viral proteinase would affect the formation of complex III. Poliovirus mutant Sel3C-02 (kindly provided by Dr Bert Semler) has a site-directed valine to alanine substitution at amino acid 54 of the poliovirus 3CPro gene (Dewalt and Semler, 1987). This virus produces less of the viral-encoded RNA dependent RNA polymerase (3DP°l) than wild-type poliovirus and very little 3CPro.…”

Section: Resultsmentioning

confidence: 99%

Poliovirus proteinase 3C converts an active form of transcription factor IIIC to an inactive form: a mechanism for inhibition of host cell polymerase III transcription by poliovirus.

et al. 1991

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In HeLa cells, RNA polymerase III (pol III)‐mediated transcription is severely inhibited by poliovirus infection. This is due primarily to a reduction in the transcriptional activity of TFIIIC, a transcription factor which binds in a sequence specific manner to the internal promoter of pol III genes. Using gel retardation assays, we have shown previously that inhibition of pol III transcription by poliovirus is correlated with disappearance of a transcriptionally active form of TFIIIC (complex I) concomitant with the appearance of a faster mobility, transcriptionally inactive form of TFIIIC (complex III). We show here that a poliovirus with a point mutation in the proteinase 3C (3Cpro) region failed to produce complex III and is limited in its ability to inhibit pol III transcription compared with the wild‐type virus. Incubation of purified 3Cpro, expressed in Escherichia coli, with transcriptionally active TFIIIC (complex I) in vitro resulted in generation of the transcriptionally inactive complex III form of TFIIIC. In an in vitro transcription assay, treatment of the complex I form of TFIIIC with 3Cpro almost completely inhibited pol III transcription. Finally expression of the 3Cpro gene in transfected HeLa cells resulted in significant inhibition of pol III‐mediated transcription. The results presented here suggest that proteolysis of the transcriptionally active form of TFIIIC by poliovirus 3Cpro is a mechanism by which poliovirus inhibits host cell RNA pol III transcription.

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Section: Resultsmentioning

confidence: 99%

Poliovirus proteinase 3C converts an active form of transcription factor IIIC to an inactive form: a mechanism for inhibition of host cell polymerase III transcription by poliovirus.

et al. 1991

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“…This arrangement of /~-strands is compatible with recently reported data on site-directed and random mutagenesis of this protease. Specifically, substitution of Val or Ala for GIy 51 (hereafter in this section the poliovirus numbering is used), presumably disrupting a/~-turn, was lethal, whereas substitution of Asp (a residue frequently occurring in flturns [23]) in the same position resulted in a viable virus [24]. Substitutions in strands E and F which could cause local deformations of the/3-sheet exerted relatively mild effects on viral reproduction [24][25][26], and a substitution of Ser for Cys 153 in strand J appeared to be without effect on the activity of 3C P'° expressed in E. coli [9].…”

Section: A Chymotrypsin-like Structural Fold In 3c Pr°mentioning

confidence: 99%

“…Specifically, substitution of Val or Ala for GIy 51 (hereafter in this section the poliovirus numbering is used), presumably disrupting a/~-turn, was lethal, whereas substitution of Asp (a residue frequently occurring in flturns [23]) in the same position resulted in a viable virus [24]. Substitutions in strands E and F which could cause local deformations of the/3-sheet exerted relatively mild effects on viral reproduction [24][25][26], and a substitution of Ser for Cys 153 in strand J appeared to be without effect on the activity of 3C P'° expressed in E. coli [9]. Moreover, the processing defects inflicted by substitutions in strands E and F were similar (namely, impairment of the cleavage at the C-terminus of 3C Pr° itself [24,25]), in accord with our proposal that these strands might interact with each other in native 3C Pt°.…”

Section: A Chymotrypsin-like Structural Fold In 3c Pr°mentioning

confidence: 99%

“…Substitutions in strands E and F which could cause local deformations of the/3-sheet exerted relatively mild effects on viral reproduction [24][25][26], and a substitution of Ser for Cys 153 in strand J appeared to be without effect on the activity of 3C P'° expressed in E. coli [9]. Moreover, the processing defects inflicted by substitutions in strands E and F were similar (namely, impairment of the cleavage at the C-terminus of 3C Pr° itself [24,25]), in accord with our proposal that these strands might interact with each other in native 3C Pt°.…”

Section: A Chymotrypsin-like Structural Fold In 3c Pr°mentioning

confidence: 99%

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Cysteine proteases of positive strand RNA viruses and chymotrypsin‐like serine proteases

et al. 1989

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Evidence is presented, based on sequence comparison and secondary structure prediction, of structural and evolutionary relationship between chymotrypsin-like serine proteases, cysteine proteases of positive strand RNA viruses (3C proteases of picornaviruses and related enzymes of come-, nepo-and potyviruses) and putative serine protease of a sobemovirus. These observations lead to re-identification of principal catalytic residues of viral proteases. Instead of the pair of Cys and His, both located in the C-terminal part of 3C proteases, a triad of conserved His, Asp(Glu) and Cys(Ser) has been identified, the first two residues resident in the N-terminal, and Cys in the C-terminal fl-barrel domain. These residues are suggested to form a charge-transfer system similar to that formed by the catalytic triad of chymotrypsin-like proteases. Based on the structural analogy with chymotrypsin-like proteases, the His residue previously implicated in catalysis, together with two partially conserved Gly residues, is predicted to constitute part of the substrate-binding pocket of 3C proteases. A partially conserved ThrLys/Arg dipeptide located in the loop preceding the catalytic Cys is suggested to confer the primary cleavage specificity of 3C toward Glx/Gly(Ser) sites. These observations provide the first example of relatedness between proteases belonging, by definition, to different classes.

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“…A final maturational cleavage occurs in the assembled virion by an unknown mechanism. Studies of chimeric and mutant picornaviruses have demonstrated that interruption of 3C proteolytic processing prevents the formation of new virions (Dewalt & Semler, 1987;Kean et al, 1988Kean et al, , 1990Dewalt et al, 1989Dewalt et al, , 1990Mirzayan et al, 1991).…”

mentioning

confidence: 99%

The picornaviral 3C proteinases: Cysteine nucleophiles in serine proteinase folds

Malcolm

1995

Protein Science

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The 3C proteinases are a novel group of cysteine proteinases with a serine proteinase-like fold that are responsible for the bulk of polyprotein processing in the Picornaviridae. Because members of this viral family are to blame for several ongoing global pandemic problems (rhinovirus, hepatitis A virus) as well as sporadic outbreaks of more serious pathologies (poliovirus), there has been continuing interest over the last two decades in the development of antiviral therapies. The recent determination of the structure of two of the 3C proteinases by X-ray crystallography opens the door for the application of the latest advances in computer-assisted identification and design of anti-proteinase therapeutickhemoprophylactic agents. Keywords: antiviral; computer-assisted drug design; inhibitor; proteinase PicornavirusesThe Picornaviridae are a family of small, closely related RNA viruses responsible for a variety of human and animal pathologies. The most famous member of the family is the well-studied poliovirus (HPV), the cause of poliomyelitis. In addition, the family includes rhinovirus (HRV), the etiologic agent for over 50% of common colds; hepatitis A virus (HAV), which produces a usually benign form of hepatitis that is endemic to many lessdeveloped parts of the world; encephalomyocarditis virus (EMCV), responsible for a relatively rare inflammation of the myocardium; and foot and mouth disease virus (FMDV), a highly contagious livestock pathogen, to name but one example from each of the five genera. A tremendous number of studies have been published over the last 20,years to establish the details of picornavirus replication and proteolytic maturation. These have been thoroughly reviewed (Krausslich & Wimmer, 1988;Lawson & Semler, 1990;Palmenberg, 1990;Dougherty & Semler, 1993) and will not be discussed here. It is sufficient to state that, although there are many subtle but profound differences between the genomes of the five genera of picornaviruses, they all nevertheless require the action of a 3C proteinase at some point in their maturation in order to generate new virions.The life cycle of the Picornaviridae can be summarized quite briefly for the purposes of this discussion (for a detailed review Reprint requests to: Bruce A. Malcolm, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; e-mail: bruce-malcolm@darwin.biochem.ualberta.ca.see Krausslich & Wimrner, 1988, and references therein). The virus attaches to and enters the cell via some form of endocytosis. The virus then uncoats, releasing its positive sense singlestranded RNA into the cytosol, where the latter functions as a messenger RNA to direct the synthesis of a single polyprotein of approximately 250 KO (Fig. 1). This polyprotein undergoes a co-translational cleavage into a capsid (Pl) and nonstructural protein (P2-P3) precursor. In the case of the entero-and rhinoviruses, this is mediated by the 2A proteinase (Toyoda et al., 1986; Sommergruber et al., 1989). In other Picornaviridae, how this cleava...

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Site-directed mutagenesis of proteinase 3C results in a poliovirus deficient in synthesis of viral RNA polymerase

Cited by 58 publications

References 34 publications

Poliovirus proteinase 3C converts an active form of transcription factor IIIC to an inactive form: a mechanism for inhibition of host cell polymerase III transcription by poliovirus.

Poliovirus proteinase 3C converts an active form of transcription factor IIIC to an inactive form: a mechanism for inhibition of host cell polymerase III transcription by poliovirus.

Cysteine proteases of positive strand RNA viruses and chymotrypsin‐like serine proteases

The picornaviral 3C proteinases: Cysteine nucleophiles in serine proteinase folds

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