C-terminal Domain Modulates the Nucleic Acid Chaperone Activity of Human T-cell Leukemia Virus Type 1 Nucleocapsid Protein via an Electrostatic Mechanism

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 singlestranded DNA. These features make HTLV-1 NC a poor NA chaperone, despite its robust duplex destabilizing capability. Nucleic acid (NA)5 chaperones are proteins that facilitate NA remodeling and annealing (1). Retroviral nucleocapsid proteins (NC) are essential NA chaperones (2-4) that facilitate many steps in the retroviral life cycle, including dimerization of the RNA genome (5-10), reverse transcription (11-14), annealing of the tRNA primer to the primer-binding site (7,(15)(16)(17)(18)(19)(20)(21)(22), and integration of viral DNA into the host genome (23-27). Previous work has shown that NCs from different retroviruses display a wide range of NA chaperone activities (28). A modelannealing reaction involving complementary trans-activation response element (TAR) RNA and DNA hairpins derived from the R region of the HIV-1 genome was used to characterize NCs from human immunodeficiency virus, type 1 (HIV-1), Rous sarcoma virus, murine leukemia virus, and human T-cell leukemia virus, type 1 (HTLV-1) (28). Surprisingly, the annealing activity of these NCs varies by 5 orders of magnitude; HIV-1 NC was the most efficient chaperone and HTLV-1 NC was the least efficient. A single molecule (SM) Förster resonance energy transfer (FRET) study confirmed the observation that HTLV-1 NC is a very poor NA chaperone (29).To determine the physical origin of these differences in retroviral NC chaperone activity, we measured the NA aggregation and duplex destabilization activity for each protein (28). Both of these capabilities, together with rap...

C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA

“…The intrinsically disordered CTDs of HTLV-1 NC (50), E. coli ssDNA binding protein (51), and mammalian high-mobility group B1 (60) proteins inhibit binding of their N termini to their respective nucleic acid substrates. Furthermore, the CTD of HTLV-1 NC also accelerates RNA dissociation (50), which may suggest a general mechanism for RNA chaperone turnover.…”

Single-molecule stretching studies of RNA chaperones

“…71,76 Similar experiments on HTLV-1 NC indicate that this protein has a slow off rate on the same order as T4 gp32, with a time constant of about 50 s. The same time decay experiment was also performed on a C-terminal truncation mutant, HTLV-1 NCΔC29, in which 29 C-terminal residues are removed. 96 Interestingly, truncating the C-terminus increases the off rate ~10-fold, with a time constant of about 5 s, similar to the off rate of T7 gp2.5. Thus, a model in which the C-terminal domain of HTLV-1 NC regulates its NA interactions (reminiscent of similar models describing T4 gp32 and T7 gp2.5), is proposed to explain this result.…”

“…95 At the same time, these NC proteins' ability to destabilize DNA duplex appeared to be rather similar for all tested NC proteins, including the worst chaperone-HTLV-1 NC. [95][96][97] Although some defects in the ability of mutant NC proteins to facilitate NA annealing were consistently observed, 67,92,95,96,98,99 these defects are not nearly as strong as the up to 6 orders of magnitude decrease in viral infectivity measured in vivo for the viruses with the same mutations in NC. [100][101][102] While simple sedimentation assays suggest that these mutant NC proteins are still capable of aggregating NA, 41,51,95,96 it is possible that these "slow" NC proteins may interfere with reverse transcription by partially losing their ability to tightly aggregate NA within the capsid, thereby leading to early capsid uncoating prior to the completion of the RTion.…”

“…95,96 This NC protein is also known to be unable to aggregate NA. The slow off rate of HTLV-1 NC protein from ssDNA can be quantitatively characterized by measuring the relaxation time of the force, as the DNA length is held constant at some point during the DNA release after its force-induced melting.…”

“…103 Incidentally, the anionic CTD of HTLV-1 NC appears to be rather similar in amino acid composition and charge to the p6 domain of the HIV-1 NC precursor protein HIV-1 NCp15. 96 Moreover, both proteins share the property of slow ssDNA interaction kinetics, 41,96 the inability to facilitate NA aggregation, 21,96,104 and poor NA annealing activity. 96 The biological role of this coincidence is still unknown.…”