Development ofin VitroPeptide Substrates for Human Rhinovirus-14 2A Protease

1998 ◽  
Vol 356 (1) ◽  
pp. 12-18 ◽  
Author(s):  
Q.May Wang ◽  
Robert B. Johnson ◽  
Wolfgang Sommergruber ◽  
Timothy A. Shepherd
Keyword(s):  
Human Rhinovirus ◽  
2A Protease

Cleavage Specificity of Human Rhinovirus-2 2A Protease for Peptide Substrates

1997 ◽  
Vol 235 (3) ◽  
pp. 562-566 ◽  
Author(s):  
Q.May Wang ◽  
Wolfgang Sommergruber ◽  
Robert B. Johnson
Keyword(s):  
Human Rhinovirus ◽  
Peptide Substrates ◽  
Cleavage Specificity ◽  
2A Protease

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

2016 ◽  
Vol 90 (24) ◽  
pp. 11032-11042 ◽  
Author(s):  
Erin Walker ◽  
Lora Jensen ◽  
Sarah Croft ◽  
Kejun Wei ◽  
Alex J. Fulcher ◽  
...  
Keyword(s):  
Virus Replication ◽  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Human Rhinovirus ◽  
Subcellular Targeting ◽  
Nuclear Targeting ◽  
3C Protease ◽  
2A Protease ◽  
Targeting Ability ◽  
Green Fluorescent

ABSTRACTThe human rhinovirus (HRV) 3C and 2A proteases (3Cproand 2Apro, 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, 3Cprois 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 2Aproactivity is required for 3CD nuclear localization. Using green fluorescent protein (GFP)-tagged forms of 3Cpro, 3D, and mutant derivatives thereof, we show that 3Cprois 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 3Cproactivity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by coexpressing mCherry-2Aprofusion proteins, we demonstrate formally that 2Aproactivity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3Cprois 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 2Aproactivity and not 3Cproactivity, which is observed only later in infection. The results thus define the temporal activities of 2Aproand 3CD/3Cproactivities 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.

scholarly journals Ex Vivo and In Vivo Inhibition of Human Rhinovirus Replication by a New Pseudosubstrate of Viral 2A Protease

2011 ◽  
Vol 86 (2) ◽  
pp. 691-704 ◽  
Author(s):  
N. Falah ◽  
S. Violot ◽  
D. Decimo ◽  
F. Berri ◽  
M.-L. Foucault-Grunenwald ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Human Rhinovirus ◽  
2A Protease

scholarly journals Solution Structure of the 2A Protease from a Common Cold Agent, Human Rhinovirus C2, Strain W12

PLoS ONE ◽  
2014 ◽  
Vol 9 (6) ◽  
pp. e97198 ◽  
Author(s):  
Woonghee Lee ◽  
Kelly E. Watters ◽  
Andrew T. Troupis ◽  
Nichole M. Reinen ◽  
Fabian P. Suchy ◽  
...  
Keyword(s):  
Common Cold ◽  
Solution Structure ◽  
Human Rhinovirus ◽  
2A Protease

scholarly journals Enzymatic Characterization of Refolded Human Rhinovirus Type 14 2A Protease Expressed in Escherichia coli

1998 ◽  
Vol 72 (2) ◽  
pp. 1683-1687 ◽  
Author(s):  
Q. May Wang ◽  
Robert B. Johnson ◽  
Gregory A. Cox ◽  
Elcira C. Villarreal ◽  
Lisa M. Churgay ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Cdna Sequence ◽  
Colorimetric Assay ◽  
Human Rhinovirus ◽  
Bacterial Cells ◽  
Enzymatic Characterization ◽  
Cleavage Specificity ◽  
Microplate Reader ◽  
2A Protease

ABSTRACT Reported here is the production of recombinant human rhinovirus 14 (HRV14) 2A protease from bacterial cells transformed with a heat-inducible plasmid containing the HRV14 2A cDNA sequence. Overexpressed 2A protein partitioned into the inclusion bodies was solubilized in urea and then refolded in the presence of Zn2+. Transition metals were required for the restoration of 2A protease activity as a structural component, but appeared to be inhibitory if added exogenously once the enzyme was refolded. Based on the cleavage specificity studies, a colorimetric assay was developed for the highly purified HRV14 2A protease. A peptide with the sequence RKGDIKSY–p-nitroanilide was found to be cleaved by the 2A protease with a k cat/Km ratio of ∼335 M−1s−1, which allows its activity to be measured continuously with a spectrophotometer or a microplate reader.

scholarly journals Structural view of the 2A protease from human rhinovirus C15

2018 ◽  
Vol 74 (4) ◽  
pp. 255-261
Author(s):  
Hui Ling ◽  
Pan Yang ◽  
Hai Hou ◽  
Yao Sun
Keyword(s):  
Substrate Binding ◽  
Gel Filtration ◽  
Catalytic Triad ◽  
Exchange Chromatography ◽  
Human Rhinovirus ◽  
Signal Pathways ◽  
Molecular Replacement ◽  
The Common ◽  
2A Protease ◽  
Binding Cleft

The majority of outbreaks of the common cold are caused by rhinoviruses. The 2A protease (2Apro) of human rhinoviruses (HRVs) is known to play important roles in the propagation of the virus and the modulation of host signal pathways to facilitate viral replication. The 2Aprofrom human rhinovirus C15 (HRV-C15) has been expressed inEscherichia coliand purified by affinity chromatography, ion-exchange chromatography and gel-filtration chromatography. The crystals diffracted to 2.6 Å resolution. The structure was solved by molecular replacement using the structure of 2Aprofrom coxsackievirus A16 (CVA16) as the search model. The structure contains a conserved His–Asp–Cys catalytic triad and a Zn2+-binding site. Comparison with other 2Aprostructures from enteroviruses reveals that the substrate-binding cleft of 2Aprofrom HRV-C15 exhibits a more open conformation, which presumably favours substrate binding.

Successful in silico discovery of natural inhibitors for human rhinovirus coat protein

Planta Medica ◽  
2007 ◽  
Vol 73 (09) ◽  
Author(s):  
JM Rollinger ◽  
TM Steindl ◽  
K Anrain ◽  
EP Ellmerer ◽  
M Schmidtke ◽  
...  
Keyword(s):  
Coat Protein ◽  
In Silico ◽  
Human Rhinovirus ◽  
Natural Inhibitors
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