Abstract
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 × 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.
Highlights
PVIc, an 11-amino acid peptide from the C terminus of adenovirus precursor protein pVI, activates the adenovirus proteinase (AVP)
DNA Is Required for the Activation of AVP by pVI in Vitro— Which components are required for the activation of AVP by pVI to produce the enzymatically active AVP-pVIc complex? If only a simple bimolecular interaction between AVP and pVI were needed, mixing purified AVP (8) with purified pVI (32) should result in the cleavage of pVI to yield pVIc followed by the formation of active AVP-pVIc complexes
We have shown how AVP can be activated by pVI in the core of the immature virion, i.e. how an AVP-pVIc complex can form in a dense DNA environment
Summary
AVP-pVIc, noncovalent or covalently linked heterodimer of AVP and pVIc; N-VI, pVI from which the C-terminal peptide, amino acids 240 –250, was cleaved; pVIc, 11-amino acid cofactor (GVQSLKRRRCF) originating from the C terminus of virion precursor protein pVI; pVIn, the 33 amino acid peptide originating from the N terminus of virion precursor protein pVI; VI-C, the cleavage product of virion precursor protein pVI from which the N-terminal 33 amino acids have been removed. The pressure exerted by the tightly packed genome on the envelope of the virion creates considerable friction, freezing the DNA in place and rendering DNA-bound proteins likewise immobile. Given this situation inside the virion, it is not clear how a bimolecular interaction between AVP and pVI can occur that leads to cleavage of pVI and activation of the enzyme by released pVIc. Given this situation inside the virion, it is not clear how a bimolecular interaction between AVP and pVI can occur that leads to cleavage of pVI and activation of the enzyme by released pVIc Without this occurring, the virus particle cannot become infectious. This mechanism for promoting bimolecular interactions is a new paradigm for how substrates interact with “non-nucleic acid enzymes” and a new paradigm for virion maturation
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