Expression and Purification of Recombinant Rhinovirus 14 3CD Proteinase and Its Comparison to the 3C Proteinase

Gj, Davis; Wang, QM; Ga, Cox; Rb, Johnson; Wakulchik, Mark; Ca, Dotson; Ec, Villarreal

doi:10.1006/abbi.1997.0291

Cited by 24 publications

(16 citation statements)

References 18 publications

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“…Cleavage of PABP by 3CD was less efficient than that by 3C pro (Fig. 1B, compare the PABP cleavage product in the lane labeled GFP-3C with that in the lane labeled GFP-3Cuc), consistent with previous reports (23).…”

Section: Nuclear Localization Of Noncleavable 3cd Of Hrv-infected Celsupporting

confidence: 91%

Rhinovirus 16 2A Protease Affects Nuclear Localization of 3CD during Infection

Walker

Jensen

Croft

et al. 2016

J Virol

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The human rhinovirus (HRV) 3C and 2A proteases (3C pro and 2A pro , respectively) are critical in HRV infection, as they are required for viral polyprotein processing as well as proteolysing key host factors to facilitate virus replication. Early in infection, 3C pro is present as its precursor 3CD, which, although the mechanism of subcellular targeting is unknown, is found in the nucleus as well as the cytoplasm. In this study, we use transfected and infected cell systems to show that 2A pro activity is required for 3CD nuclear localization. Using green fluorescent protein (GFP)-tagged forms of 3C pro , 3D, and mutant derivatives thereof, we show that 3C pro is located in the cytoplasm and the nucleus, whereas 3CD and 3D are localized predominantly in the cytoplasm, implying that 3D lacks nuclear targeting ability and that 3C pro activity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by coexpressing mCherry-2A pro fusion proteins, we demonstrate formally that 2A pro activity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3C pro is insufficient to allow 3CD access to the nucleus. Finally, we confirm the relevance of these results to HRV infection by demonstrating that nuclear localization of 3CD correlates with 2A pro activity and not 3C pro activity, which is observed only later in infection. The results thus define the temporal activities of 2A pro and 3CD/3C pro activities in HRV serotype16 infection. IMPORTANCEThe human rhinovirus genome encodes two proteases, 2A and 3C, as well as a precursor protease, 3CD. These proteases are essential for efficient virus replication. The 3CD protein is found in the nucleus early during infection, though the mechanism of subcellular localization is unknown. Here we show that 2A protease is required for this localization, the 3C protease activity of 3CD is not sufficient to allow 3CD entry into the nucleus, and 3D lacks nuclear targeting ability. This study demonstrates that both 2A and 3C proteases are required for the correct localization of proteins during infection and defines the temporal regulation of 2A and 3CD/3C protease activities during HRV16 infection.

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Section: Nuclear Localization Of Noncleavable 3cd Of Hrv-infected Celsupporting

confidence: 91%

Rhinovirus 16 2A Protease Affects Nuclear Localization of 3CD during Infection

Walker

Jensen

Croft

et al. 2016

J Virol

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“…Several proteases can be selected to remove the tag, including SUMO protease, enteropeptidase, thrombin, factor Xa, PreScission and tobacco etch virus (TEV) protease. Among these, SUMO protease only cleaves SUMO tags [92], enteropeptidase and thrombin are incompatible with buffers containing reducing agents [114], factor Xa should not be used in the presence of chelating agents because it binds calcium ions [115], and PreScission leaves behind a Gly-Pro dipeptide on the N-terminus of the recombinant protein after digestion [116]. TEV protease is not inhibited by reducing agents, exhibits very high specificity, is inexpensive, and in most cases cleaves recombinant proteins in a manner that leaves the native protein intact [98,114].…”

Section: Expression Vectors For High-throughput Protein Expressionmentioning

confidence: 99%

High-throughput recombinant protein expression in Escherichia coli : current status and future perspectives

Jia¹,

Jeon²

2016

Open Biol.

236

160

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The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for producing recombinant proteins. In this post-genomic era, challenges remain to rapidly express and purify large numbers of proteins for academic and commercial purposes in a high-throughput manner. In this review, we describe several state-of-the-art approaches that are suitable for the cloning, expression and purification, conducted in parallel, of numerous molecules, and we discuss recent progress related to soluble protein expression, mRNA folding, fusion tags, post-translational modification and production of membrane proteins. Moreover, we address the ongoing efforts to overcome various challenges faced in protein expression in E. coli, which could lead to an improvement of the current system from trial and error to a predictable and rational design.

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“…However, unlike the proteinase alone, the proteinase-polymerase fusion did not cleave within p37 (Table 1). Proteolysis with differing substrate specificity by the 3C and 3CD proteins of picornaviruses has also been demonstrated (5,6,14,34). The 3CD protein of poliovirus is multifunctional.…”

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confidence: 99%

trans Activity of the Norovirus Camberwell Proteinase and Cleavage of the N-Terminal Protein Encoded by ORF1

Seah

Marshall

Wright

2003

J Virol

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The virus-encoded proteinase of Camberwell virus, a genogroup 2 norovirus, was synthesized in Escherichia coli. The purified proteinase had correct N and C termini and showed trans activity in cell-free assays. trans activity was also demonstrated in COS cells transfected with constructs encoding either the proteinase or a proteinase-polymerase fusion. The N-terminal protein of ORF1 was cleaved in COS cells, possibly at the site E 194 /S.

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Expression and Purification of Recombinant Rhinovirus 14 3CD Proteinase and Its Comparison to the 3C Proteinase

Cited by 24 publications

References 18 publications

Rhinovirus 16 2A Protease Affects Nuclear Localization of 3CD during Infection

Rhinovirus 16 2A Protease Affects Nuclear Localization of 3CD during Infection

High-throughput recombinant protein expression in Escherichia coli : current status and future perspectives

trans Activity of the Norovirus Camberwell Proteinase and Cleavage of the N-Terminal Protein Encoded by ORF1

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