Characterization of association of human mitochondrial lysyl-tRNA synthetase with HIV-1 Pol and tRNA3Lys

Abstract

Background: An important step in human immunodeficiency virus type 1 (HIV-1) replication is the packaging of tRNA₃^Lys from the host cell, which plays the role of primer RNA in the process of initiation of reverse transcription. The viral GagPol polyprotein precursor, and the human mitochondrial lysyl-tRNA synthetase (mLysRS) from the host cell, have been proposed to be involved in the packaging process. More specifically, the catalytic domain of mLysRS is supposed to interact with the transframe (TF or p6*) and integrase (IN) domains of the Pol region of the GagPol polyprotein.

Results: In this work, we report a quantitative characterization of the protein:protein interactions between mLysRS and its viral partners, the Pol polyprotein, and the isolated integrase and transframe domains of Pol. A dissociation constant of 1.3 ± 0.2 nM was determined for the Pol:mLysRS interaction, which exemplifies the robustness of this association. The protease and reverse transcriptase domains of GagPol are dispensable in this association, but the TF and IN domains have to be connected by a linker polypeptide to recapitulate a high affinity partner for mLysRS. The binding of the viral proteins to mLysRS does not dramatically enhance the binding affinity of mLysRS for tRNA₃^Lys.

Conclusions: These data support the conclusion that the complex formed between GagPol, mLysRS and tRNA₃^Lys, which involves direct interactions between the IN and TF domains of Pol with mLysRS, is more robust than suggested by the previous models supposed to be involved in the packaging of tRNA₃^Lys into HIV-1 particles.

Keywords: Binding affinity; HIV-1; Integrase; Mitochondrial lysyl-tRNA synthetase; Transframe (TF or p6*); tRNA3 Lys packaging.

Fig. 1

Analysis of the purified proteins by SDS-PAGE. The Pol sub-domain of the GagPol polyprotein (Pol), the integrase domain of Pol (IN), and the mitochondrial species of human LysRS (mLysRS), expressed in insect cells and purified as described in “Materials and Methods”, were analyzed by SDS-polyacrylamide gel electrophoresis. Size markers (M) were run in parallel. Molecular masses are indicated in kDa

Fig. 2

Association of mLysRS with Pol and p38. The binding affinities of mLysRS to Pol (a) or p38 (b) were determined in an HTRF assay using 1.5 nM (a) or 0.5 nM (b) of HA-tagged mLysRS and increasing concentrations of His-tagged Pol or p38, all expressed as dimer concentrations. Experimental values (symbols) were fit (curves) to a binding equation assuming that one dimer of Pol or p38 binds one dimer of mLysRS. The binding constants and the associated standard deviations result from three independent measurements

Fig. 3

Characterization of TF expressed in E. coli. The transframe (TF, also known as p6*) domain of Pol was expressed in E. coli with a C-terminal His-tag. a Purified TF was subjected to size exclusion chromatography on a Yarra SEC-2000 column as described under “Materials and Methods”. Elution of TF was followed at 280 nm, and confirmed by Western blotting with anti-TF antibodies. The apparent molecular mass of TF was deduced from its relative elution volume K_av (inset). b CD spectra of TF at 20 °C and 90 °C

Fig. 4

Association of mLysRS with IN and TF. The binding affinities of mLysRS to IN or TF were determined in the HTRF assay using 1.5 nM of HA-tagged mLysRS and increasing concentrations of His-tagged interacting proteins, as described in Fig. 2. The binding constant of mLysRS to TF could not be determined (nd)

Fig. 5

Association of the TF-Sx-IN surrogates of Pol with mLysRS. The fusion proteins with spacers made of 2 (S2), 7 (S7), 12 (S12), 17 (S17) or 22 (S22) amino acids were analyzed by SDS-PAGE and their binding affinities for mLysRS were determined in the HTRF assay, as described in Fig. 2. The binding constants and the associated standard deviations result from three independent measurements

Fig. 6

Analysis of tRNA-binding by fluorescence polarization. The binding affinity of mLysRS for tRNA₃^Lys was measured using 50 nM of Cy-3 labeled tRNA₃^Lys, in the absence or in the presence of 0.5 μM of TF-S12-IN. TF-S12-IN alone did not bind tRNA₃^Lys. The deduced binding constants and the associated standard deviations result from at least three independent measurements

Fig. 7

Association of Pol with mLysRS in the presence of tRNA₃^Lys. The binding affinity of Pol to mLysRS was determined in the absence or in the presence of 1 μM tRNA₃^Lys, in the HTRF assay as described in Fig. 2. The binding constants and the associated standard deviations result from three independent measurements

Fig. 8

Comparison of mLysRS and mLysRS∆C for binding to IN and to tRNA₃^Lys. mLysRS and mLysRS∆C were expressed in E. coli with a C-terminal HA-tag. The HTRF assay, conducted as described in the legend of Fig. 4, was used to compare their binding constants to IN (a), and fluorescence polarization, conducted as described in the legend of Fig. 6, was used to compare their affinities for Cy-3 labeled tRNA₃^Lys (b). The binding constants and the associated standard deviations result from three independent measurements