Abstract
Human cytomegalovirus (HCMV) genome replication is a complex and still not completely understood process mediated by the highly coordinated interaction of host and viral products. Among the latter, six different proteins form the viral replication complex: a single-stranded DNA binding protein, a trimeric primase/helicase complex and a two subunit DNA polymerase holoenzyme, which in turn contains a catalytic subunit, pUL54, and a dimeric processivity factor ppUL44. Being absolutely required for viral replication and representing potential therapeutic targets, both the ppUL44–pUL54 interaction and ppUL44 homodimerization have been largely characterized from structural, functional and biochemical points of view. We applied fluorescence and bioluminescence resonance energy transfer (FRET and BRET) assays to investigate such processes in living cells. Both interactions occur with similar affinities and can take place both in the nucleus and in the cytoplasm. Importantly, single amino acid substitutions in different ppUL44 domains selectively affect its dimerization or ability to interact with pUL54. Intriguingly, substitutions preventing DNA binding of ppUL44 influence the BRETmax of protein–protein interactions, implying that binding to dsDNA induces conformational changes both in the ppUL44 homodimer and in the DNA polymerase holoenzyme. We also compared transiently and stably ppUL44-expressing cells in BRET inhibition assays. Transient expression of the BRET donor allowed inhibition of both ppUL44 dimerization and formation of the DNA polymerase holoenzyme, upon overexpression of FLAG-tagged ppUL44 as a competitor. Our approach could be useful both to monitor the dynamics of assembly of the HCMV DNA polymerase holoenzyme and for antiviral drug discovery.
Highlights
We developed a number of cell lines stably expressing several ppUL44 and pUL54 -YFP and -Renilla luciferase (RLuc) fusion proteins, and combinations thereof, to compare the utility of different expression strategies in BRET inhibition assays, upon expression of the BRET
Since BRET saturation experiments allowed to estimate the affinity of ppUL44 dimerization, we aimed at setting up similar assays for the ppUL44/pUL54 interaction, being able to compare the affinity of both processivity factor multimerization and DNA
The fact that a nuclear localization defective ppUL44 can both homodimerize and bind to pUL54 as efficiently as the wild-type protein suggests that dsDNA binding does not influence ppUL44 dimerization nor pUL54 biding
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Substitution of either basic gap loop residues, or of residues at the dimerization interface such as L86 and L87 [4], cause a dramatic alteration of ppUL44 subcellular localization and reduce DNA binding in cells, completely abolishing the ability of ppUL44 to trans-complement oriLyt-dependent. In this study we developed quantitative assays to monitor both processes in living cells, taking advantage of fluorescence and bioluminescence resonance energy transfer (FRET and BRET) methods We applied such methods to a series of ppUL44 substitution derivatives selectively impaired in key properties such as the ability to bind to either dsDNA or pUL54, to self-interact, as well as being translocated to the nucleus. Our results further confirm that both ppUL44 dimerization and its interaction with pUL54 rely on different key residues of ppUL44 Both events can take place either in the nucleus or in the cytosol, and occur in cells with similar affinity.
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