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W Gibson – One of the best experts on this subject based on the ideXlab platform.

  • Cytomegalovirus Capsid Protease: Biological Substrates Are Cleaved More Efficiently by Full-Length Enzyme (pUL80a) than by the Catalytic Domain (Assemblin)
    Journal of virology, 2011
    Co-Authors: Steve M. Fernandes, Edward J. Brignole, W Gibson

    Abstract:

    We compared the full-length capsid maturational protease (pPR, pUL80a) of human cytomegalovirus with its proteolytic domain (Assemblin) for the ability to cleave two biological substrates, and we found that pPR is more efficient with both. Affinity-purified, refolded enzymes and substrates were combined under defined reaction conditions, and cleavage was monitored and quantified following staining of the resulting electrophoretically separated fragments. The enzymes were stabilized against self-cleavage by a single point mutation in each cleavage site (ICRMT-pPR and IC-Assemblin). The substrates were pPR itself, inactivated by replacing its catalytic nucleophile (S132A-pPR), and the sequence-related assembly protein precursor (pAP, pUL80.5). Our results showed that (i) ICRMT-pPR is 5- to 10-fold more efficient than Assemblin for all cleavages measured (i.e., the M site of pAP and the M, R, and I sites of S132A-pPR). (ii) Cleavage of substrate S132A-pPR proceeded M>R>I for both enzymes. (iii) Na2SO4 reduced M- and R-site cleavage efficiency by ICRMT-pPR, in contrast to its enhancing effect for both enzymes on I site and small peptide cleavage. (iv) Disrupting oligomerization of either the pPR enzyme or substrate by mutating Leu382 in the amino-conserved domain reduced cleavage efficiency two- to fourfold. (v) Finally, ICRMT-pPR mutants that include the amino-conserved domain, but terminate with Pro481 or Tyr469, retain the enzymatic characteristics that distinguish pPR from Assemblin. These findings show that the scaffolding portion of pPR increases its enzymatic activity on biologically relevant protein substrates and provide an additional link between the structure of this essential viral enzyme and its biological mechanism.

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  • Enzymatic Activities of Human Cytomegalovirus Maturational Protease Assemblin and Its Precursor (pPR, pUL80a): Maximal Activity of pPR Requires Self-Interaction through Its Scaffolding Domain
    Journal of virology, 2007
    Co-Authors: Edward J. Brignole, W Gibson

    Abstract:

    Herpesviruses encode an essential, maturational serine protease whose catalytic domain, Assemblin (28 kDa), is released by self-cleavage from a 74-kDa precursor (pPR, pUL80a). Although there is considerable information about the structure and enzymatic characteristics of Assemblin, a potential pharmacologic target, comparatively little is known about these features of the precursor. To begin studying pPR, we introduced five point mutations that stabilize it against self-cleavage at its internal (I), cryptic (C), release (R), and maturational (M) sites and at a newly discovered “tail” (T) site. The resulting mutants, called ICRM-pPR and ICRMT-pPR, were expressed in bacteria, denatured in urea, purified by immobilized metal affinity chromatography, and renatured by a two-step dialysis procedure and by a new method of sedimentation into glycerol gradients. The enzymatic activities of the pPR mutants were indistinguishable from that of IC-Assemblin prepared in parallel for comparison, as determined by using a fluorogenic peptide cleavage assay, and approximated rates previously reported for purified Assemblin. The percentage of active enzyme in the preparations was also comparable, as determined by using a covalent-binding suicide substrate. An unexpected finding was that, in the absence of the kosmotrope Na2SO4, optimal activity of pPR requires interaction through its scaffolding domain. We conclude that although the enzymatic activities of Assemblin and its precursor are comparable, there may be differences in how their catalytic sites become fully activated.

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  • Cytomegalovirus Assemblin (pUL80a): Cleavage at Internal Site Not Essential for Virus Growth; Proteinase Absent from Virions
    Journal of virology, 2002
    Co-Authors: Chee Kai Chan, Edward J. Brignole, W Gibson

    Abstract:

    The human cytomegalovirus (HCMV) maturational proteinase is synthesized as an enzymatically active 74-kDa precursor that cleaves itself at four sites. Two of these, called the maturational (M) and release (R) sites, are conserved in the homologs of all herpesviruses. The other two, called the internal (I) and cryptic (C) sites, have recognized consensus sequences only among cytomegalovirus (CMV) homologs and are located in the 28-kDa proteolytic portion of the precursor, called Assemblin. I-site cleavage cuts Assemblin in half without detected effect on its enzymatic behavior in vitro. To investigate the requirement for this cleavage during virus infection, we used the CMV-bacterial artificial chromosome system (E. M. Borst, G. Hahn, U. H. Koszinowski, and M. Messerle, J. Virol. 73:8320-8329, 1999) to construct a virus encoding a mutant I site (Ala143 to Val) intended to be blocked for cleavage. Characterizations of the resulting mutant (i) confirmed the presence of the mutation in the viral genome and the inability of the mutant virus to effect I-site cleavage in infected cells; (ii) determined that the mutation has no gross effect on the rate of virus production or on the amounts of extracellular virions, noninfectious enveloped particles (NIEPs), and dense bodies; (iii) established that Assemblin and its cleavage products are present in NIEPs but are absent from CMV virions, an apparent difference from what is found for virions of herpes simplex virus; and (iv) showed that the 23-kDa protein product of C-site cleavage is more abundant in mutant virus-than in wild-type virus-infected cells and NIEPs. We conclude that the production of infectious CMV requires neither I-site cleavage of Assemblin nor the presence of Assemblin in the mature virion.

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D Wade Gibson – One of the best experts on this subject based on the ideXlab platform.

  • Chemical Rescue of I-site Cleavage in Living Cells and in Vitro Discriminates between the Cytomegalovirus Protease, Assemblin, and Its Precursor, pUL80a
    The Journal of biological chemistry, 2005
    Co-Authors: Stephen A. Mccartney, Edward J. Brignole, Keli Kolegraff, Amy N. Loveland, Lashon M. Ussin, D Wade Gibson

    Abstract:

    Abstract Chemical rescue is an established approach that offers a directed strategy for designing mutant enzymes in which activity can be restored by supplying an appropriate exogenous compound. This method has been used successfully to study a broad range of enzymes in vitro, but its application to living systems has received less attention. We have investigated the feasibility of using chemical rescue to make a conditional-lethal mutant of the cytomegalovirus (CMV) maturational protease. The 28-kDa CMV serine protease, Assemblin, has a Ser-His-His catalytic triad and an internal (I) cleavage site near its midpoint. We found that imidazole can restore I-site cleavage to mutants inactivated by replacing the critical active site His with Ala or with Gly, which rescued better. Comparable rescue was observed for counterpart mutants of the human and simian CMV Assemblin homologs and occurred in both living cells and in vitro. Cleavage was established to be at the correct site by amino acid sequencing and proceeded at ∼11%/h in bacteria and ∼30%/h in vitro. The same mutations were unresponsive to chemical rescue in the context of the Assemblin precursor, pUL80a. This catalytic difference distinguishes the two forms of the CMV protease.

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  • Action at the Assemblin dimer interface.
    Nature Structural Biology, 2001
    Co-Authors: D Wade Gibson

    Abstract:

    A recent study of cytomegalovirus Assemblin proteinase suggests that dimer-induced stabilization of the oxyanion hole may activate the enzyme.

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  • Independently cloned halves of cytomegalovirus Assemblin, An and Ac, can restore proteolytic activity to Assemblin mutants by intermolecular complementation.
    Journal of virology, 1997
    Co-Authors: Matthew R T Hall, D Wade Gibson

    Abstract:

    Herpesviruses encode an essential serine proteinase called Assemblin that is responsible for cleaving the precursor assembly protein during the process of capsid formation. In cytomegalovirus (CMV), Assemblin undergoes autoproteolysis at an internal (I) site located near the middle of the molecule. I-site cleavage converts the enzyme to an active two-chain form consisting of the subunits An and Ac. We have recently shown that the recombinant An and Ac subunits can spontaneously associate within eukaryotic cells to yield active two-chain proteinase. This finding indicates that the subunits are able to independently assume their correct functional conformations and led us to test whether they are capable of intermolecular complementation. This was done by coexpressing inactive mutant (point, deletion, and insertion) forms of Assemblin together with the wild-type subunit (either An or Ac) corresponding to the domain of Assemblin that was mutated. Results of these experiments showed that both An and Ac are able to rescue the enzymatic activity of Assemblin mutants. I-site cleavage of the mutated Assemblin occurred during complementation but was not absolutely required, as shown by effective complementation of inactive Assemblins with noncleavable I sites. We have also shown that intermolecular complementation can rescue the activity of an inactive mutant full-length proteinase precursor and can occur between different species of CMV (e.g., human CMV subunit can rescue activity of mutant simian CMV Assemblin). These results indicate that Assemblin is able to form active multimeric structures that may be of functional importance.

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Matthew R T Hall – One of the best experts on this subject based on the ideXlab platform.

  • Assemblin Homolog of Herpes Simplex Virus Type 1 Retains Proteolytic Activity When Expressed as a Recombinant Two-Chain Enzyme
    Virology, 1997
    Co-Authors: Matthew R T Hall, W Gibson

    Abstract:

    The herpes simplex virus type 1 (HSV) maturational proteinase is synthesized as a precursor that undergoes two autoproteolytic cleavages; one at its (M)aturational site, which eliminates its carboxyl ‘‘tail,’’ and a second at its (R)elease site, which separates the amino proteolytic half of the precursor from its nonproteolytic carboxyl half. In cytomegalovirus (CMV) the proteolytic half of the precursor, called Assemblin, undergoes a third cleavage at an (I)nternal site that converts it from a single-chain to a two-chain enzyme that retains activity. The HSV Assemblin homolog has no I site and therefore does not form a counterpart two-chain enzyme. In the work reported here we have cloned and expressed HSV sequences that encode mimics of the An and Ac subunits of two-chain CMV Assemblin. We show that when these HSV sequences are coexpressed in eukaryotic cells, the resulting subunits associate spontaneously to form an active two-chain enzyme. We also show that the two-chain HSV enzyme, like the natural one-chain form, retains its marked preference for HSV over CMV substrates, and that intertypic recombinant two-chain Assemblin (e.g., HSV An/CMV Ac) does not form because the cross-species subunits do not interact. We conclude from these results that (i) there are not intrinsic structural differences in the HSV Assemblin homolog that preclude its functioning as a CMV-like two-chain enzyme, (ii) the substrate selectivity shown by the single-chain HSV enzyme was not noticeably relaxed in the HSV two-chain mimic, and (iii) the interactive domains, through which the An and Ac portions of the single-chain enzymes associate, differ between HSV and CMV. q 1997 Academic Press

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  • Independently cloned halves of cytomegalovirus Assemblin, An and Ac, can restore proteolytic activity to Assemblin mutants by intermolecular complementation.
    Journal of virology, 1997
    Co-Authors: Matthew R T Hall, D Wade Gibson

    Abstract:

    Herpesviruses encode an essential serine proteinase called Assemblin that is responsible for cleaving the precursor assembly protein during the process of capsid formation. In cytomegalovirus (CMV), Assemblin undergoes autoproteolysis at an internal (I) site located near the middle of the molecule. I-site cleavage converts the enzyme to an active two-chain form consisting of the subunits An and Ac. We have recently shown that the recombinant An and Ac subunits can spontaneously associate within eukaryotic cells to yield active two-chain proteinase. This finding indicates that the subunits are able to independently assume their correct functional conformations and led us to test whether they are capable of intermolecular complementation. This was done by coexpressing inactive mutant (point, deletion, and insertion) forms of Assemblin together with the wild-type subunit (either An or Ac) corresponding to the domain of Assemblin that was mutated. Results of these experiments showed that both An and Ac are able to rescue the enzymatic activity of Assemblin mutants. I-site cleavage of the mutated Assemblin occurred during complementation but was not absolutely required, as shown by effective complementation of inactive Assemblins with noncleavable I sites. We have also shown that intermolecular complementation can rescue the activity of an inactive mutant full-length proteinase precursor and can occur between different species of CMV (e.g., human CMV subunit can rescue activity of mutant simian CMV Assemblin). These results indicate that Assemblin is able to form active multimeric structures that may be of functional importance.

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  • Assemblin, an essential herpesvirus proteinase.
    Drug design and discovery, 1997
    Co-Authors: D Wade Gibson, Matthew R T Hall

    Abstract:

    The herpesvirus maturational proteinase, called Assemblin, is essential for the production of infectious virus and represents a new molecular target for the development of antivirals. A brief summary of the synthesis, structure, and function of this fascinating enzyme is presented here.

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