Abstract
The primer tRNA for reverse transcription in HIV-1, tRNALys3, is selectively packaged into the virus during its assembly, and annealed to the viral genomic RNA. The ribonucleoprotein complex that is involved in the packaging and annealing of tRNALys into HIV-1 consists of Gag, GagPol, tRNALys, lysyl-tRNA synthetase (LysRS), and viral genomic RNA. Gag targets tRNALys for viral packaging through Gag's interaction with LysRS, a tRNALys-binding protein, while reverse transcriptase (RT) sequences within GagPol (the thumb domain) bind to tRNALys. The further annealing of tRNALys3 to viral RNA requires nucleocapsid (NC) sequences in Gag, but not the NC sequences GagPol. In this report, we further show that while the RT connection domain in GagPol is not required for tRNALys3 packaging into the virus, it is required for tRNALys3 annealing to the viral RNA genome.
Highlights
During assembly of HIV-1, the major tRNALys isoacceptors in mammalian cells, tRNALys1,2 and tRNALys3, are selectively incorporated into the virus [1]. tRNALys3 is the primer for initiating minus-strand cDNA synthesis, and its annealing to the 18 nucleotide primer binding site (PBS) region in the 5' part of the viral genome via the 3' 18 nucleotides in tRNALys3 complementary to the PBS, is a key step in viral replication [2]
We present data indicating that the reverse transcriptase (RT) connection domain, while nonessential for tRNALys3 incorporation into virions, is required for tRNALys3 annealing to the viral RNA genome
Total viral RNA was isolated from these virions, and dot blots of this RNA were annealed with probes specific for either viral genomic RNA or tRNALys3, to determine the tRNALys3/genomic RNA in each viral variant
Summary
During assembly of HIV-1, the major tRNALys isoacceptors in mammalian cells, tRNALys and tRNALys, are selectively incorporated into the virus [1]. tRNALys is the primer for initiating minus-strand cDNA synthesis, and its annealing to the 18 nucleotide primer binding site (PBS) region in the 5' part of the viral genome via the 3' 18 nucleotides in tRNALys complementary to the PBS, is a key step in viral replication [2]. Other regions upstream and downstream of the PBS may anneal with additional sequences in the tRNA [3,4] Both tRNALys and sites of annealing in viral RNA contain double stranded regions which may require denaturation for annealing to proceed efficiently. While, tRNALys is annealed efficiently in protease-negative HIV-1 (about 80% that found in wild-type virions), optimal placement on the viral genome to achieve efficient initiation of reverse transcription requires exposure of the viral genome to mature nucleocapsid protein [15]. In these protease-negative viruses, mutations in NC sequences within Gag inhibit (page number not for citation purposes)
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