Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Nov;13(11):1237-45.
doi: 10.1128/CVI.00184-06. Epub 2006 Sep 20.

Human immunodeficiency virus persistence and production in T-cell development

Kevin B Gurney  1 , Christel H Uittenbogaart
Affiliations

Human immunodeficiency virus persistence and production in T-cell development

Kevin B Gurney et al. Clin Vaccine Immunol. 2006 Nov.

Abstract

Human immunodeficiency virus type 1 (HIV-1) replication depends on CD4 and coreceptor expression as well as host factors associated with the activation state of the cell. To determine the impact of the activation stage of thymocytes on the HIV-1 life cycle, we investigated R5 and X4 HIV-1 entry, reverse transcription, and expression in discrete thymocyte subsets at different stages of T-cell development. Early after infection, preferential entry and replication of R5 HIV-1 were predominantly detected in mature CD3(+/hi) CD27(+) thymocytes. Thus, R5 HIV-1 targets the stage of development where thymocytes acquire functional responsiveness, which has important implications for HIV pathogenesis. In contrast, X4 HIV-1 expression and replication were primarily found in immature CD3(-/+/low) CD27(-) CD69(-) thymocytes. HIV-1 proviral burden and virus expression in thymocyte subsets correlated with the expression of the highest levels of the respective coreceptor. R5 and X4 HIV-1 entered and completed reverse transcription in all subsets tested, indicating that the activation state of thymocytes and coreceptor expression are sufficient to support full reverse transcription throughout development. Although R5 HIV-1 is expressed mainly in mature CD3(+/hi) CD27(+) thymocytes, 5.3% of HIV-1-infected immature thymocytes express R5 HIV-1, indicating that potentially latent viral DNA can be established early in T-cell development.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Thymocyte development scheme. (A) Combinations of antibodies were used to identify thymocytes at five distinct stages of T-cell development. Stage I, CD69− thymocytes; stage I-P (a subset of stage I in which thymocytes express CD71), CD69− thymocytes; stage II, CD69+ cells that have not yet acquired CD27 expression; stage III, CD69+ CD27+ thymocytes that have not yet acquired CD45RA expression; stage IV, CD27+ thymocytes expressing CD45RA. Within stage I, the first circle represents the CD34+ thymic immigrant. The second circle represents the immature CD4 single-positive thymocyte. The third and fourth circles represent CD3− CD4+ CD8+ double-positive (DP) thymocytes and CD3+/low DP thymocytes, respectively. The fifth circle (stages II/III) represents CD69+ positively selected thymocytes in transition to the next stage of either CD4 single-positive or CD8 single-positive thymocytes. The last stage (stage IV) represents the CD3+/hi CD45RA+ thymic emigrant. (B) Cell cycle analysis was determined for total and CD3− CD71+ thymocytes by a combination of cell surface staining with antibodies to CD3 and CD71 and 7-AAD. Histograms were drawn for DNA content (7-AAD) in ungated thymocytes (left panel) and CD3− CD71+ gated thymocytes (right panel). The proportions of the thymocytes in the G0/G1 stages and S/G2/M stages of the cell cycle were calculated by Modfit software.
FIG. 2.
FIG. 2.
X4 HIV-1 replicates in immature thymocytes, while R5 HIV-1 replicates in mature thymocytes in HIV-1-infected SCID-hu mice. Thymocytes were obtained from HIV-1-infected implants in SCID-hu mice at 19 days (NL4-3) and 26 days (JR-CSF) postinfection. The cells were surface stained with antibodies to CD3, CD71, CD69, CD27, and CD45RA combined with intracellular staining for GagHIV-1 proteins (KC57-FITC). The phenotype of GagHIV-1-expressing cells was determined by gating on KC57+ cells. The percentages of positive cells in each quadrant are indicated. Isotype controls were used to set the cursors. Results for one representative staining of two thymic implants at these time points are shown.
FIG. 3.
FIG. 3.
R5 and X4 HIV-1 proviral burdens in thymocyte subsets at different stages of maturation. Thymocyte subsets were obtained from HIV-1-infected implants from SCID-hu mice by immunomagnetic bead selection. Quantitative TaqMan PCR for HIV-1 proviral burden relative to that of the human beta-globin gene was performed and calculated based on interpolation from standard curves. The level of HIV-1 per 100 cell equivalents for each implant in each subset is shown (open circles) (five to six data points in each subset). Asterisks indicate statistically significant differences. (A) Proviral burdens at 4 and 7 weeks postinfection (R5 HIV-1JR-CSF-infected implants) and (B) at 19 days postinfection (X4 HIV-1NL4-3).
FIG. 4.
FIG. 4.
HIV-1 coreceptor expression in thymocytes at five developmental stages. CCR5 and CXCR4 expression at stages I-P to IV of thymocyte development was determined by four-color flow cytometric analysis using combinations of monoclonal antibodies to CCR5-PE, CXCR4-PE, CD3-FITC and -APC, CD45RA-APC, CD27-FITC, CD69-TC, and CD71-FITC in five separate experiments as described in Materials and Methods. Asterisks indicate statistically significant differences. As only small numbers of thymocytes express CCR5 and almost all thymocytes express CXCR4, CCR5 data are expressed in percentages and CXCR4 data as mean fluorescence intensities. (A) The mean percent CCR5+ cells in each subset was as follows: for subset I-P, 0.95% ± 0.6%; for subset I, 0.08% ± 0.07%; for subset II, 0.16% ± 0.06%; for subset III, 2.5% ± 0.8%; and for subset IV, 1% ± 0.9%. (B) Since CXCR4 is expressed in each of these subsets, we measured the mean fluorescent intensity (or geometric mean) of CXCR4 for each subset relative to that for subset I-P, which consistently expressed the highest levels of CXCR4 in all five experiments. Subset I-P, 100%; subset I, 32% ± 9%; subset II, 14% ± 9%; subset III, 23% ± 17%; and subset IV, 39% ± 28%.
FIG. 5.
FIG. 5.
HIV-1 entry in thymocyte subsets at different stages of maturation. Postnatal thymocytes were separated into individual subsets by magnetic beads and infected in vitro. Eighteen hours postinfection, quantitative PCR was performed to measure the number of initial reverse transcription products (R-U5), and the cell numbers were determined by beta-globin quantitative PCR. Values were calculated based on interpolation from a standard curve, and HIV-1 copy numbers were normalized to 1,000-cell equivalents from each subset after infection by R5 HIV-1JR-CSF (A), R5 HIV-1NFN-SX (B), and X4 HIV-1NL4-3 (C). The mean viral-copy numbers and standard deviations for X4 and R5 HIV-1 DNA based on triplicate measurements in one representative experiment out of three are shown. Asterisks indicate statistically significant differences calculated based on data from three independent experiments.
FIG. 6.
FIG. 6.
Levels of HIV-1 reverse transcription are similar in thymocyte subsets at different stages of maturation. Postnatal thymocytes were separated into individual subsets by magnetic beads and infected in vitro. Eighteen hours postinfection, quantitative PCR was performed to measure the numbers of initial and full reverse transcription products (R-U5) and full reverse transcripts only (LTR-gag). The percent completion of reverse transcription for each subset was calculated by dividing the number of full-length transcripts by the number of total transcripts (initial and full). Values were calculated based on interpolation from a standard curve. (A) R5 HIV-1NFN-SX-infected thymocytes. (B) X4 HIV-1NL4-3-infected thymocytes. The means and standard deviations for triplicate measurements from one representative experiment out of three are shown.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adachi, A., H. E. Gendelman, S. Koenig, T. Folks, R. Willey, A. Rabson, and M. A. Martin. 1986. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 59:284-291. - PMC - PubMed
    1. Aldrovandi, G. M., G. Feuer, L. Gao, B. Jamieson, M. Kristeva, I. S. Y. Chen, and J. A. Zack. 1993. The SCID-hu mouse as a model for HIV-1 infection. Nature 363:732-736. - PubMed
    1. Baba, M., O. Nishimura, N. Kanzaki, M. Okamoto, H. Sawada, Y. Iizawa, M. Shiraishi, Y. Aramaki, K. Okonogi, Y. Ogawa, K. Meguro, and M. Fujino. 1999. A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc. Natl. Acad. Sci. USA 96:5698-5703. - PMC - PubMed
    1. Bakri, Y., C. Schiffer, V. Zennou, P. Charneau, E. Kahn, A. Benjouad, J. C. Gluckman, and B. Canque. 2001. The maturation of dendritic cells results in postintegration inhibition of HIV-1 replication. J. Immunol. 166:3780-3788. - PubMed
    1. Berkowitz, R. D., S. Alexander, C. Bare, V. Linquist-Stepps, M. Bogan, M. E. Moreno, L. Gibson, E. D. Wieder, J. Kosek, C. A. Stoddart, and J. M. McCune. 1998. CCR5- and CXCR4-utilizing strains of human immunodeficiency virus type 1 exhibit differential tropism and pathogenesis in vivo. J. Virol. 72:10108-10117. - PMC - PubMed

Publication types