Association of human mitochondrial lysyl-tRNA synthetase with HIV-1 GagPol does not require other viral proteins

Abstract

In human, the cytoplasmic (cLysRS) and mitochondrial (mLysRS) species of lysyl-tRNA synthetase are encoded by a single gene. Following HIV-1 infection, mLysRS is selectively taken up into viral particles along with the three tRNA^Lys isoacceptors. The GagPol polyprotein precursor is involved in this process. With the aim to reconstitute in vitro the HIV-1 tRNA₃^Lys packaging complex, we first searched for the putative involvement of another viral protein in the selective viral hijacking of mLysRS only. After screening all the viral proteins, we observed that Vpr and Rev have the potential to interact with mLysRS, but that this association does not take place at the level of the assembly of mLysRS into the packaging complex. We also show that tRNA₃^Lys can form a ternary complex with the two purified proteins mLysRS and the Pol domain of GagPol, which mimicks its packaging complex.

Keywords: HIV-1; Lysyl-tRNA synthetase; Packaging complex; tRNA3Lys.

Fig. 1

Two-hybrid analysis of LysRS:pHIV-1 interaction. (A) The complete genome of HIV-1 is shown. The polyproteins Gag, GagPol and Env, and proteins Vif, Vpr, Tat, Rev, Vpu and Nef are encoded on the three reading frames of the viral genome. The viral (pHIV-1) proteins were expressed fused to LexA in pEG202. (B) The two-hybrid protein:protein interaction assay between pHIV-1, and cLysRS, mLysRS, or pmLysRS, was conducted in EGY48 strain. The three LysRS species for the cytoplasmic (cLysRS), mitochondrial (mLysRS), and premitochondrial (pmLysRS) enzymes were expressed fused to the B42 transcription activator, under the control of a galactose-inducible promoter in pJG4-5. As a control, all the strains grew on a galactose medium in the presence of leucine (YNBGal + L). When the strains were plated on a galactose medium in the absence of leucine (YNBGal), a protein:protein interaction resulted in the expression of the LEU2 gene, as observed following coexpression of any of the three LysRS species with Nef, Vpr, Rev or Tat. If the expression of LEU2 requires interaction between LysRS and pHIV-1, then strains should not grow in the absence of leucine on a glucose medium (YNB) where LysRS is not expressed. All the strains grew on YNB in the presence of leucine (not shown). (C) Expression and stability of the fusion proteins produced in yeast. Left: Western blot analysis of the fusion proteins expressed in yeast from the pJG4-5 plasmid. The cLysRS, mLysRS or pmLysRS species (80 kDa) were expressed fused to the HA11 hemagglutinin epitope. Right: Western blot analysis of the fusion proteins expressed in yeast from the pEG202 plasmid. The Vif (45.3 kDa), Vpu (31.8 kDa), Tat (32.5 kDa), Rev (35.7 kDa), Vpr (34.4 kDa), Nef (46 kDa) and Env (119.7 kDa) fusion proteins were expressed fused to the LexA DNA-binding domain.

Fig. 2

Two-hybrid analysis of the LysRS domains interacting with pHIV-1. (A) The cytoplasmic- (cESD), mitochondrial- (mESD), or premitochondrial- (pmESD) eukaryote-specific domains, the anticodon-binding domain (ABD), and the catalytic domain (CAT) of LysRS were expressed fused to the B42 transcription activator, under the control of a galactose-inducible promoter in pJG4-5. The two-hybrid protein:protein interaction assay between the LysRS constructs and the pHIV-1 constructs expressed fused to LexA in pEG202, was conducted in EGY48 strain. Expression of the CAT domain alone was as efficient as the expression of the three full-length LysRS species to restore growth in the presence of Vpr, Rev, or Tat. No growth was observed on YNB plates, except for the strain expressing the Nef construct. These data also show that none of the HA-LysRS fusions activate transcription of the reporter gene in the absence of a suitable B42-fusion protein. For clarity, only the YNBGal selection plate is shown. (B) Expression of the fusion proteins in yeast was analyzed by Western blot. The cESD (19.3 kDa), mESD (21.1 kDa), pmESD (22.7 kDa), ABD (31.1 kDa) or CAT (53.1 kDa) domains of LysRS, were expressed fused to the HA11 hemagglutinin epitope.

Fig. 3

Co-immunoprecipitation of mLysRS with HIV-1 proteins. Anti-FLAG agarose beads carrying FLAG peptide (FLAG) or the FLAG-Rev, FLAG-Vpr, FLAG-Tat or FLAG-Nef fusion proteins were incubated with the mitochondrial species of LysRS (Input). After removal of the unbound fraction, beads were washed and then incubated with FLAG peptide in excess, and the eluted fraction was recovered (FLAG elution). mLysRS present in these fractions was analyzed by Western blotting with anti-ΔNLysRS antibodies. The ‘Input’ and ‘Unbound’ fractions were diluted 10×.

Fig. 4

Rev and Vpr do not copurify with the mLysRS:Pol complex. The mLysRS species, Pol or IN with a C-terminal His-Tag (Pol, IN), Vpr and Rev were expressed independently in insect cells. Extracts were mixed as indicated, and subjected to NiNTA-agarose pull-down. The fractions obtained before (A: Input) or after (B: Output) NiNTA pull-down were analyzed by Western blotting with anti-IN (for Pol and IN), anti-Vpr, anti-Rev, or (C) with anti-LysRS antibodies.

Fig. 5

Association of mLysRS with Pol in the absence or in the presence of tRNA₃^Lys. Pol with a C-terminal His-Tag and mLysRS were expressed independently in insect cells. Individual extracts or mixed extracts (mLysRS + Pol) were incubated in the absence (−tRNA₃^Lys) or in the presence (+tRNA₃^Lys) of tRNA₃^Lys and subjected to NiNTA-agarose pull-down. (A) The initial fractions (Input) and the fractions obtained after NiNTA pull-down (Output) were analyzed by Western blotting with anti-LysRS or anti-integrase antibodies. (B) The presence of tRNA₃^Lys in the corresponding fractions was determined by Northern blotting with a digoxigenin-labeled oligonucleotide complementary to tRNA₃^Lys (K3). With Pol or mLysRS alone, the background level of tRNA₃^Lys recovered corresponds either to nonspecific binding of tRNA on NiNTA beads, or to the binding of tRNA on proteins from the total extracts that bind nonspecifically to the beads.