Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: IV. viral proteinase slides along DNA to locate and process its substrates

Harvard University, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2012; 288(3). DOI: 10.1074/jbc.M112.407460
Source: PubMed


Precursor proteins used in the assembly of adenovirus virions must be processed by the virally encoded adenovirus proteinase
(AVP) before the virus particle becomes infectious. An activated adenovirus proteinase, the AVP-pVIc complex, was shown to
slide along viral DNA with an extremely fast one-dimensional diffusion constant, 21.0 ± 1.9 × 106 bp2/s. In principle, one-dimensional diffusion can provide a means for DNA-bound proteinases to locate and process DNA-bound
substrates. Here, we show that this is correct. In vitro, AVP-pVIc complexes processed a purified virion precursor protein in a DNA-dependent reaction; in a quasi in vivo environment, heat-disrupted ts-1 virions, AVP-pVIc complexes processed five different precursor proteins in DNA-dependent
reactions. Sliding of AVP-pVIc complexes along DNA illustrates a new biochemical mechanism by which a proteinase can locate
its substrates, represents a new paradigm for virion maturation, and reveals a new way of exploiting the surface of DNA.

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Available from: Carmen San Martín, Jul 30, 2015
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    • "Complete activation of AVP requires both DNA binding and pVI [8] [9]. In immature virus particles, pVI slides along the viral genome through interactions of the C-terminus of pVI with the DNA [10] [11]. As pVI encounters AVP, AVP cleaves the 11 C-terminal residues from pVI (pVIc), which then binds and covalently links to AVP via a disulfide bridge yielding maximum AVP activity [8,10–16]. "
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    ABSTRACT: Mature human adenovirus particles contain four minor capsid proteins, in addition to the three major capsid proteins (penton base, hexon and fiber) and several proteins associated with the genomic core of the virion. Of the minor capsid proteins, VI plays several crucial roles in the infection cycle of the virus, including hexon nuclear targeting during assembly, activation of the adenovirus proteinase (AVP) during maturation and endosome escape following cell entry. VI is translated as a precursor (pVI) that is cleaved at both N- and C-termini by AVP. Whereas the role of the C-terminal fragment of pVI, pVIc, is well established as an important co-factor of AVP, the role of the N-terminal fragment, pVIn, is currently elusive. In fact, the fate of pVIn following proteolytic cleavage is completely unknown. Here, we use a combination of proteomics-based peptide identification, native mass spectrometry and hydrogen-deuterium exchange mass spectrometry to show that pVIn is associated with mature human adenovirus, where it binds at the base of peripentonal hexons in a pH-dependent manner. Our findings suggest a possible role for pVIn in targeting pVI to hexons for proper assembly of the virion and timely release of the membrane lytic mature VI molecule.
    Journal of Molecular Biology 05/2014; 426(9). DOI:10.1016/j.jmb.2014.02.022 · 4.33 Impact Factor
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    ABSTRACT: SUMMARY Late in an adenovirus infection, the viral proteinase (AVP) becomes activated to process virion precursor proteins used in virus assembly. AVP is activated by two cofactors- the viral DNA and pVIc, an 11-amino acid peptide originating from the C-terminus of the precursor protein pVI. There is a conundrum in the activation of AVP in that AVP and pVI are sequence independent DNA binding proteins with nM equilibrium dissociation constants such that in the virus particle they are predicted to be essentially irreversibly bound to the viral DNA. Here we resolve that conundrum by showing that activation of AVP takes place on the one-dimensional contour of DNA. In vitro, pVI, a substrate, slides on DNA via one-dimensional diffusion, D(1) = 1.45 x 10(6) (bp)(2)/s, until it binds to AVP also on the same DNA molecule. AVP, partially activated by being bound to DNA, excises pVIc which binds to the AVP molecule that cut it out. pVIc then forms a disulfide bond with AVP forming the fully active AVP-pVIc complex bound to DNA. In vivo, in heat-disrupted immature virus, AVP was also activated by pVI in DNA-dependent reactions. This activation mechanism illustrates a new paradigm for virion maturation and a new way, by sliding on DNA, for bimolecular complexes to form among proteins not involved in DNA metabolism.
    Journal of Biological Chemistry 10/2012; 288(3). DOI:10.1074/jbc.M112.407312 · 4.57 Impact Factor
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    ABSTRACT: The adenovirus proteinase (AVP), the first member of a new class of cysteine proteinases, is essential for the production of infectious virus, and here we report its structure at 0.98 Å-resolution. AVP, initially synthesized as an inactive enzyme, requires two cofactors for maximal activity- pVIc, an 11 amino acid peptide, and the viral DNA. Comparison of the structure of AVP with that of an active form the AVP-pVIc complex reveals why AVP is inactive. Both forms have an α + β fold; the major structural differences between them lie in the β-sheet domain. In AVP-pVIc, the general base His54 Nδ is 3.9 Å from the Cys122 Sγ thereby rendering it nucleophilic. In AVP, however, His54 Nδ is 7.0 Å away Cys122 Sγ, too far away to be able to abstract the proton from Cys122. In AVP-pVIc, Tyr84 forms a cation-π interaction with His54 that should raise the pKa of His54 and freeze the imidazole ring in the place optimal for forming an ion pair with Cys122. In AVP, however, Tyr84 is more than 11 Å away from its position in AVP-pVIc. Based on the structural differences between AVP and AVP-pVIc, we present a model which postulates activation of AVP by pVIc occurs via a 62-amino acid long activation pathway in which the binding of pVIc initiates contiguous conformational changes, like falling dominos: There is a common pathway that branches into a pathway that leads to the repositioning of His54 and another pathway that leads to the repositioning of Tyr84.
    Journal of Biological Chemistry 10/2012; 288(3). DOI:10.1074/jbc.M112.407429 · 4.57 Impact Factor
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