Proline 137 Is Critical for Adenovirus Protease Encapsidation and Activation but Not Enzyme Activity

Rancourt, Claudine; Keyvani, Hossein; Sircar, Sucheta; Labrecque, Pascale; Weber, Joseph

doi:10.1006/viro.1995.1240

Cited by 52 publications

(43 citation statements)

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“…This peptide is a cleavage product from the C terminus of capsid precursor protein pVI and is likely to be involved in coordinating enzyme activity with capsid assembly and maturation during virus infection (17). The VEGGS peptide was identified as a protease ligand in a phage display library (18).…”

Section: Resultsmentioning

confidence: 99%

Cleavage Efficiency by Adenovirus Protease Is Site-dependent

Diouri

Keyvani

Geoghegan

et al. 1996

Journal of Biological Chemistry

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The adenovirus protease cleaves consensus sequences (M/I/L)XGX-G and (M/I/L)XGG-X. Using purified recombinant protease, we showed that a peptide bearing the GX-G site was hydrolyzed more rapidly than a peptide bearing the GG-X site. The GX-G site was also preferentially cleaved on viral protein pVI which bears both sites of cleavage. Evidence is presented that suggests a biological role for this differential cleavage efficiency.Adenoviruses, like many other viruses, encode an endoproteinase which is required for virus maturation and infectivity (1). The enzyme (AVP) 1 is a cysteine protease that normally cleaves seven viral proteins at two consensus sites: (M/I/ L)XGX-G or (M/I/L)XGG-X (2). Other proteins bearing these sequences are also susceptible to AVP, particularly after denaturation (3-6). In addition to its role in virus maturation, the AVP also appears to have a role in the early phase of infection in decapsidation and release from the endosome (1,7,8). The adenovirus type 2 protease has been co-crystallized with its stimulating peptide pVIc, and its structure was recently reported (9).In the present report we provide evidence that the AVP cleaves the GX-G type consensus site more efficiently than the GG-X site and suggest that this may have a biological role in the course of virus infection. MATERIALS AND METHODS Expression and Purification of AdenovirusProtease-Cloning, expression, and purification of the Ad2 protease using the modified expression vector pRPAd2E3 (pRIT2T from Pharmacia Biotech Inc.), a temperature-inducible protein A gene fusion vector, under the control of the pR promoter, was done essentially as described before (4). In some experiments, a second expression plasmid, pLPV, which contains 14 amino acids fused to the N terminus of the protease, was also used (10).Protease Assay-Unless indicated otherwise, peptide assays were performed as follows. A total reaction volume of 300 l contained 5 M substrate (11), 2-100 pmol of pRPAd2E3 protease, and reaction buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8, 2 M NaCl). The reaction was incubated at 37°C for the indicated times. The synthesis and rationale of the LYRA substrates was described previously (11). The peptide sequence in LYRA2 was ly-AnLRGG-AFSWK-ctmr-R and in LYRA3 was ly-AnLRGA-GFSWK-ctmr-R. Protease activity was also measured by the cleavage of viral polypeptide pVI to iVI (intermediate form of VI) and VI. The source of pVI was ts1 virions grown at 39°C, purified and disrupted with 10% pyridine, and dialyzed with TE buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA), and boiled to denature proteins. Cleavage was detected by staining blots with anti-VI serum (see Western Blotting). Precursor pVI was also prepared by disrupting [35 S]Met-labeled ts1 virus particles produced at 39°C (12). The reaction mixture (40 l) contained 10 l of substrate and 10 l of enzyme in reaction buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA). Incubation was at 37°C for the indicated times.Western Blotting-Proteins were separated by a 15% SDS-PAGE, electroblotted onto a nitroc...

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Section: Resultsmentioning

confidence: 99%

Cleavage Efficiency by Adenovirus Protease Is Site-dependent

Diouri

Keyvani

Geoghegan

et al. 1996

Journal of Biological Chemistry

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“…This mutant has a point mutation in the viral protease, resulting in poor encapsidation of protease and no cleavage of the minor capsid proteins IIIa, VI, and VIII as well as core proteins VII and X [Hassell & Weber, 1978, Rancourt et al, 1995. The consequences of this lack of proteolysis is a hyper-stable particle, severely defective for uncoating.…”

Section: Minor Capsid Proteinsmentioning

confidence: 99%

Current Advances and Future Challenges in Adenoviral Vector Biology and Targeting

Campos¹,

Barry²

2007

CGT

169

121

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Gene delivery vectors based on Adenoviral (Ad) vectors have enormous potential for the treatment of both hereditary and acquired disease. Detailed structural analysis of the Ad virion, combined with functional studies has broadened our knowledge of the structure/function relationships between Ad vectors and host cells/tissues and substantial achievement has been made towards a thorough understanding of the biology of Ad vectors. The widespread use of Ad vectors for clinical gene therapy is compromised by their inherent immunogenicity. The generation of safer and more effective Ad vectors, targeted to the site of disease, has therefore become a great ambition in the field of Ad vector development. This review provides a synopsis of the structure/function relationships between Ad vectors and host systems and summarizes the many innovative approaches towards achieving Ad vector targeting.

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“…The wild-type and tsl Ad2 protease were expressed in E. coil from the pLAM vector Rancourt et al, 1995) and purified as described previously (KeyvaniAmineh et al,I995b). The enzyme was stored in TE buffer (10 mMTris-HC1, pH 8, I mM-EDTA) at --70 °C.…”

Section: Methodsmentioning

confidence: 99%

Adenovirus Type 2 Endoprotease: Isoforms and Redox Effects

Keyvani-Amineh

Diouri

Tihanyi³

et al. 1996

Journal of General Virology

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The cysteine protease encoded by adenovirus type 2 contains eight cysteines, some of which are involved in catalysis and enzyme activation. Here we investigated the effects of oxidation, mercaptoethanol, dithiothreitol, diamide and protein disulphide isomerase on wild-type and mutant enzymes. Three isoforms of the enzyme were detected in infected cells and a fourth in preparations of purified recombinant enzyme. The latter isoform was absent in preparations of enzyme mutated at any of the three conserved cysteines, C-104, C-122 and C-126. Enzyme activity could be stimulated by agents other than the authentic activating peptide (pVIc), such as cysteamine, though less efficiently. Diamide at low concentrations stimulated the activity of the tsl enzyme, but inhibited both tsl and wild-type enzyme at higher concentrations. Protein disulphide isomerase failed to restore enzyme activity to the oxidized isoform. The present studies in combination with previous results using mutants appeared to rule out amino acids C-67, C-122, C-126 and C-127, leaving the two remaining semi-conserved C-17 and C-40 and the conserved C-104 as potential candidates for binding peptide pVIc.

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Proline 137 Is Critical for Adenovirus Protease Encapsidation and Activation but Not Enzyme Activity

Cited by 52 publications

References 0 publications

Cleavage Efficiency by Adenovirus Protease Is Site-dependent

Cleavage Efficiency by Adenovirus Protease Is Site-dependent

Current Advances and Future Challenges in Adenoviral Vector Biology and Targeting

Adenovirus Type 2 Endoprotease: Isoforms and Redox Effects

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