Incorporation of lysyl-tRNA synthetase into human immunodeficiency virus type 1

Abstract

During human immunodeficiency virus type 1 (HIV-1) assembly, tRNA(Lys) isoacceptors are selectively incorporated into virions and tRNA(Lys)3 is used as the primer for reverse transcription. We show herein that the tRNA(Lys)-binding protein, lysyl-tRNA synthetase (LysRS), is also selectively packaged into HIV-1. The viral precursor protein Pr55gag alone will package LysRS into Pr55gag particles, independently of tRNA(Lys). With the additional presence of the viral precursor protein Pr160gag-pol, tRNA(Lys) and LysRS are both packaged into the particle. While the predominant cytoplasmic LysRS has an apparent M(r) of 70,000, viral LysRS associated with tRNA(Lys) packaging is shorter, with an apparent M(r) of 63,000. The truncation occurs independently of viral protease and might be required to facilitate interactions involved in the selective packaging and genomic placement of primer tRNA.

FIG. 1

Detection of aminoacyl-tRNA synthetases in HIV-1. Virions were pelleted from cell culture medium and purified by centrifugation through 15% sucrose onto a 65% sucrose cushion. (A) Western blots of aminoacyl-tRNA synthetases found in the cytoplasms of HIV-1-transfected COS7 cells and in the viruses produced from these cells. Western blots of cell lysates (lane C) or viral lysates (lane V) were probed with antibody to LysRS (a), IleRS (b), or ProRS (c). Numbers at the left of each panel represent molecular weight markers. GluProRS, glutamyl-prolyl-tRNA synthetase. (B) Resistance of virus-associated proteins to the protease subtilisin. Purified virions were either left untreated (N) or treated (S) with subtilisin, and after subtilisin inactivation, viruses were lysed, and Western blots of viral lysate were probed with antibodies to CAp24 (a), gp120 (b), or LysRS (c). Purified His₆-LysRS was left untreated or treated with subtilisin (d). Lane K contains purified, His₆-tagged human LysRS, which in panel c has not been exposed to protease.

FIG. 2

Detection of LysRS in viruses purified by centrifugation through Optiprep gradients. Western blots of fractions from Optiprep gradients. (A) Blot probed with anti-CA. Lane V, viral lysate before Optiprep gradient. (B) Blot probed with anti-LysRS. Lane K, purified His₆-tagged LysRS. (C) Blot stained with Coomassie blue. Twenty times more viral lysate was used than was used for panels A and B in order to clearly detect protein by the stain. Lane M, marker proteins. (D) Blot of pellet of material from cell culture media of nontransfected COS7 cells, probed with anti-LysRS.

FIG. 3

Detection of LysRS in cell lysates and lysates of sucrose-purified viruses produced from chronically infected cell lines. Western blots are probed with anti-LysRS. Cell lysates are from uninfected (−) or infected (+) cells. Numbers at the left represent molecular weight markers. LysRS, purified His₆-tagged LysRS.

FIG. 4

Detection of LysRS in lysates of sucrose-purified viruses produced from COS7 cells transfected with wild-type and mutant HIV-1 DNA. (A) Western blot of viral lysates probed with anti-LysRS. LysRS, purified His₆-tagged LysRS. wt, wild type. PR(−), viral protease-negative. P31L, substitution mutation in the region between the two Cys-His boxes in the nucleocapsid. Dr2, insertion mutation in the connection domain of reverse transcriptase. Gag, Pr55^gag particles which do not contain Pr160^gag-pol. COS7, cytoplasmic lysate. (B) Western blot of viral lysate probed with anti-LysRS. Lanes: 2, wt: 3, P31L; 4 and 5, viruses from cells cotransfected with P31L DNA and DNA coding for either wild-type Pr160^gag-pol (4) or wild-type Pr55^gag (5).