Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection

Abstract

To better understand the basis for human immunodeficiency virus type 1 (HIV-1) persistence and latency, the form in which viral DNA exists in the peripheral T lymphocyte reservoir of infected individuals was investigated. In asymptomatic individuals, HIV-1 was harbored predominantly as full-length, unintegrated complementary DNA. These extrachromosomal DNA forms retained the ability to integrate upon T cell activation in vitro. In patients with acquired immunodeficiency syndrome (AIDS), there was an increase in integrated relative to extrachromosomal DNA forms. By analysis of DNA from patient lymphocyte subpopulations depleted of human lymphocyte antigen-Dr receptor-positive cells, quiescent T cells were identified as the source of extrachromosomal HIV-1 DNA. Thus quiescent T lymphocytes may be a major and inducible HIV-1 reservoir in infected individuals.

Fig. 1.

Discrimination between extrachromosomal and integrated HIV-1 DNA forms. (A) Doubling dilutions (from 128 to 2) of 8E5 cells (containing one defective provirus per cell) were mixed with 400 uninfected lymphocytes. After isolation of total cellular DNA, fractionated high (H) and low (L) molecular weight DNA (12) were analyzed by PCR with primers specific for HIV-1 pol (25). (B) HIV-1 PCR analysis of high and low molecular weight DNA fractions from 2000 MT-4 cells (human CD4⁺ T cell line) infected with an integration minus HIV-1 mutant (6, 14). (C) Four 8E5 lymphocytes were mixed with 2000 uninfected T lymphocytes and distributed in five replicate fractions (1 to 5). Total cellular DNA was isolated, and HIV-1 and tubulin DNA were amplified by 30 and 20 cycles of PCR, respectively. PCR product sizes are in base pairs.

Fig. 2.

Arrangement of HIV-1 DNA before and after T cell activation in vitro. Replicate macrophage-depleted cell samples (400 cells per sample) were prepared from lymphocyte cultures of patient B4 (asymptomatic) before (A) and after (B) activation of the culture in vitro with PHA. Presence of HIV-1 DNA in high and low molecular weight fractions was determined by PCR with primers specific for HTV-1 pol or primers directed to a region spanning the 5′ LTR-gag terminus (25). The separation of genomic DNA in high and low molecular weight fractions was confirmed using α-tubulin–specific primers.

Fig. 3.

Ligation-mediated PCR analysis of 3′ LTR termini in enriched quiescent lymphocyte populations. (A) The ligation PCR protocol was essentially as described (21). A representation of the HIV-1 3′ LTR and locations of the U3-R, and R-U5 junctions (26) are shown at the top of the figure. Total cellular DNA from HLA-Dr–depleted quiescent lymphocytes of HIV-1–infected individuals is denatured by alkali (1) and annealed to an HIV-1 R-specific LTR primer (2) complementary to the (−) strand of the HIV-1 LTR. DNA polymerase extension from the R primer (2) results in formation of double-stranded blunt-end 3′ LTR terminus, the end of which is defined by the U5 terminus. The newly created blunt-end 3′ U5 LTR terminus provides a substrate for ligation of a universal linker (3). The composition of the universal linker is as described (21). Annealing and extension from the R-specific LTR primer results in a blunt-end LTR terminus only in linear extrachromosomal HIV-1 DNA forms. A second polymerase extension step (4) from a nested (−) strand R-specific primer results in formation of a new (+) strand that is complementary to the LTR (−) strand and that incorporates the sequence of the longer oligomer component of the universal linker. The resultant double-stranded products provide suitable substrates for PCR (5) by means of the nested R-specific LTR primer and the longer oligomer component of the universal linker. The expected PCR product size of 155 bp includes 130 bp of HIV-1 LTR [extending from nucleotide 9591 (26) at the HIV-1 R-specific primer binding site to the 3′ GCAGT terminus of U5 at nucleotide 9720] and 25 bp of incorporated common linker sequence. (B through D) PBLs from two HIV-1–infected individuals with ARC (B28 and B29) were depleted of macrophages and activated T cells by magnetic affinity sorting with HLA-Dr antibody–conjugated magnetic particles (27). Total cellular DNA from the HLA-Dr⁻ lymphocyte population was subject to direct PCR (B) with primers to the HIV-1 5′ LTR-gag junction (upper panel) and to the human α-tubulin gene (lower panel) or to a ligation-mediated PCR approach (C and D), as outlined in (A), using a single round (upper panel) or double round (lower panel) of PCR. In (D), samples were processed exactly as outlined in (A), in that the substrate for ligation of the universal linker was provided after DNA denaturation and polymerase extension from an annealed HIV-1 R-specific primer. In a second series of reactions (C), lymphocyte DNA was ligated directly to the universal linker, essentially bypassing the DNA denaturation and primer extension steps [steps 1 and 2 in (A)]. 8E5 is a human CD4⁺ T cell line that contains one integrated noninfectious HIV-1 provirus per cell and completely lacks extrachromosomal HIV-1 DNA (13). ΔIN represents DNA from CD4⁺ lymphocytes infected with an integration minus mutant of HIV-1 (6, 14).