Abstract
Retroviral nucleocapsid (NC) proteins are molecular chaperones that facilitate nucleic acid (NA) remodeling events critical in viral replication processes such as reverse transcription. Surprisingly, the NC protein from human T-cell leukemia virus type 1 (HTLV-1) is an extremely poor NA chaperone. Using bulk and single molecule methods, we find that removal of the anionic C-terminal domain (CTD) of HTLV-1 NC results in a protein with chaperone properties comparable with that of other retroviral NCs. Increasing the ionic strength of the solution also improves the chaperone activity of full-length HTLV-1 NC. To determine how the CTD negatively modulates the chaperone activity of HTLV-1 NC, we quantified the thermodynamics and kinetics of wild-type and mutant HTLV-1 NC/NA interactions. The wild-type protein exhibits very slow dissociation kinetics, and removal of the CTD or mutations that eliminate acidic residues dramatically increase the protein/DNA interaction kinetics. Taken together, these results suggest that the anionic CTD interacts with the cationic N-terminal domain intramolecularly when HTLV-1 NC is not bound to nucleic acids, and similar interactions occur between neighboring molecules when NC is NA-bound. The intramolecular N-terminal domain-CTD attraction slows down the association of the HTLV-1 NC with NA, whereas the intermolecular interaction leads to multimerization of HTLV-1 NC on the NA. The latter inhibits both NA/NC aggregation and rapid protein dissociation from single-stranded DNA. These features make HTLV-1 NC a poor NA chaperone, despite its robust duplex destabilizing capability.
Highlights
Nucleic acid (NA), whereas the intermolecular interaction leads to multimerization of HTLV-1 nucleocapsid proteins (NC) on the NA
Using bulk and single molecule methods, we find that removal of the anionic C-terminal domain (CTD) of HTLV-1 NC results in a protein with chaperone properties comparable with that of other retroviral NCs
The wild-type protein exhibits very slow dissociation kinetics, and removal of the CTD or mutations that eliminate acidic residues dramatically increase the protein/DNA interaction kinetics. These results suggest that the anionic CTD interacts with the cationic N-terminal domain intramolecularly when HTLV-1 NC is not bound to nucleic acids, and similar interactions occur between neighboring molecules when NC is NA-bound
Summary
Protein and NA Preparation—The genes encoding HTLV-1 [33, 34] and -2 (GenBankTM accession number NC_001488) NC were PCR-amplified from full-length HTLV-1 (pCS-HTLV-1) and -2 proviral plasmids (generous gifts from David Derse, NCI-Frederick, National Institutes of Health) and cloned into pET32a, generating plasmids pDR2559 (for HTLV-1 NC) and pDR2560 (for HTLV-2 NC). Solutions containing 15 nM refolded 32P-labeled mini-TAR RNA and 90 nM refolded miniTAR DNA in reaction buffer (20 mM HEPES, pH 7.5, 20 mM NaCl, 5 mM dithiothreitol, and 0.2 mM MgCl2) were incubated for 5 min at 37 °C. Sedimentation/Aggregation Assays—Solutions containing refolded 32P-labeled mini-TAR RNA (15 nM) and mini-TAR DNA (45 nM) were incubated with varying concentrations of WT and mutant HTLV-1 NC for 30 min at room temperature in reaction buffer (20 mM HEPES, pH 7.5, 20 mM NaCl, 5 mM dithiothreitol, and 0.2 mM MgCl2). Folded TAR DNA at a final concentration of 100 nM was incubated with and without NC in a buffer containing 20 mM HEPES, pH 7.5, 50 mM NaCl, 10 M TCEP, 5 mM -mercaptoethanol, and 1 M zinc acetate for 30 min. We prepared three additional HTLV-1 NC mutants that lack (either through substitution with Ala or deletion) one or more C-terminal acidic residues
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