Transduction of cellular sequence by a human immunodeficiency virus type 1-derived vector

Abstract

During studies examining the rate of human immunodeficiency virus type 1 (HIV-1) mutation in a single cycle of replication, the 5' long terminal repeat of one progeny provirus was found to contain an insertion of 147 bp including an entire tRNA sequence as well as an additional 66 bp insertion of nonviral origin. Database searches revealed that 65 of 66 bp aligned with the human CpG island sequence found on chromosomes 6, 14, and 17. Therefore it seems probable that it is of human cellular sequence origin and was transduced by HIV-1. This is the first demonstration that HIV-1 can capture a cellular sequence. The site of integration of the parental provirus was mapped to chromosome 1p32.1. Sequence with homology to the transduced CpG island was not found on chromosome 1, suggesting that the transduced cellular sequence was not linked to the site of viral integration.

FIG. 1

(A) Examples of mutation screening by PCR of the 5′ LTR of progeny proviruses. The band in lane 2 is shifted up (∗) compared to the control and other progeny proviruses (lanes 1 and 3 to 19), indicating an insertion. (B) The insertion causing the change in electrophoretic mobility of the sample from lane 2 in panel A is depicted. The whole insertion is composed of an entire tRNA with its sequence denoted below the figure, including one T-to-C substitution, followed by 4 bp (TGGT) of unknown origin, 66 bp of human CpG sequence, with its sequence depicted above the diagram, including one T-to-C substitution, and another G of unknown origin. Note that the figure is not drawn to scale, as indicated by //.

FIG. 2

Products and DNA sequence obtained from inverse PCR of the producer cell provirus. (A) Lanes: 1, DNA markers; 2, template-negative PCR control; 3, second-round inverse PCR products with HindIII-digested and self-ligated genomic DNA as templates. Band B1 was amplified from the HIV-1 self-ligated product. Band B2 was anticipated to contain the HIV-1 integration site and flanking producer cell genome sequence because of its strong intensity. Both bands were cloned and sequenced. (B) Flanking human genomic sequence at the 5′ end of the HIV-1 integration site. Bold underlined letters represent the first 48 bp of the HIV-1 provirus 5′ sequence. Italic letters represent the human chromosome 1 sequence adjacent to the integration site.

FIG. 3

Proposed model for generating the mutant progeny. Thin lines represent viral RNA. Thick lines represent viral DNA. Dashed lines represent RNase H-digested RNA. Striped lines denote DNA copied from tRNA. Stippled lines indicate the captured cellular CpG island sequence. (A) The minus-strand synthesis was primed by either normal tRNA or an aberrant tRNA which has the CpG island sequence linked to it. (B) Minus-strand synthesis continued normally. (C) The plus-strand strong-stop DNA product did not terminate at the end of PBS as is typical. If there was a conventional tRNA used to prime reverse transcription, it continued to read through this entire tRNA. (D) An extra 4-bp stretch (TGGT) of unknown origin was added by RT. After that, the plus-strand strong-stop DNA growing point either continued to utilize the chimeric RNA as template or annealed to a copackaged human RNA (CpG) and copied 66 bases. (E) Then an additional base was added (see the text), and this was followed by strand transfer to the minus-strand PBS. (F) Reverse transcription was subsequently completed in typical fashion. The figure is not drawn to scale, as indicated by //. ppt, polypurine tract.