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
. 2004 Jun;78(12):6122-33.
doi: 10.1128/JVI.78.12.6122-6133.2004.

Resting CD4+ T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals carry integrated HIV-1 genomes within actively transcribed host genes

Yefei Han  1 , Kara Lassen, Daphne Monie, Ahmad R Sedaghat, Shino Shimoji, Xiao Liu, Theodore C Pierson, Joseph B Margolick, Robert F Siliciano, Janet D Siliciano
Affiliations

Resting CD4+ T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals carry integrated HIV-1 genomes within actively transcribed host genes

Yefei Han et al. J Virol. 2004 Jun.

Abstract

Resting CD4+ T-cell populations from human immunodeficiency virus type 1 (HIV-1)-infected individuals include cells with integrated HIV-1 DNA. In individuals showing suppression of viremia during highly active antiretroviral therapy (HAART), resting CD4+ T-cell populations do not produce virus without cellular activation. To determine whether the nonproductive nature of the infection in resting CD4+ T cells is due to retroviral integration into chromosomal regions that are repressive for transcription, we used inverse PCR to characterize the HIV-1 integration sites in vivo in resting CD4+ T cells from patients on HAART. Of 74 integration sites from 16 patients, 93% resided within transcription units, usually within introns. Integration was random with respect to transcriptional orientation relative to the host gene and with respect to position within the host gene. Of integration sites within well-characterized genes, 91% (51 of 56) were in genes that were actively expressed in resting CD4+ T cells, as directly demonstrated by reverse transcriptase PCR (RT-PCR). These results predict that HIV-1 sequences may be included in the primary transcripts of host genes as part of rapidly degraded introns. RT-PCR experiments confirmed the presence of HIV-1 sequences within transcripts initiating upstream of the HIV-1 transcription start site. Taken together, these results demonstrate that HIV-1 genomes reside within actively transcribed host genes in resting CD4+ T cells in vivo.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Purification of resting CD4+ T cells from patients on HAART. Flow cytometric analysis of unfractionated peripheral blood mononuclear cells (left panel) and highly purified resting CD4+ T lymphocytes (right panel) stained with phycoerythrin-conjugated anti-CD4 and fluorescein isothiocyanate-conjugated anti-HLA-DR antibodies. Numbers indicate the percentages of cells in each quadrant.
FIG. 2.
FIG. 2.
Detection of HIV-1 integration sites in resting CD4+ T lymphocytes from patients on HAART by inverse PCR. (A) Inverse PCR strategy for cloning HIV-1 integration sites. Genomic DNA from highly purified resting CD4+ T cells was digested with PstI, which cuts frequently in genomic DNA but only once in most HIV-1 isolates, at nt 1419 in gag. Digested DNA was subjected to intramolecular ligation and nested PCR amplification as described in the text. Boxed nucleotides represent the 5′ end of the LTR. (B) Inverse PCR products from a representative patient. (Top panel) Results of 12 independent inverse PCRs for the same patient. Sequence analysis demonstrated that each band represented a unique integration event from a single infected resting CD4+ T cell. (Bottom panel) Control reactions carried out either without ligase or without DNA.
FIG. 3.
FIG. 3.
Distribution of HIV-1 integration events along the lengths of targeted genes. Results are shown both for genes that are part of the RefSeq database (51) (black bars [bottom portion of bars]) of well-characterized genes and for genes predicted on the basis of sequenced human mRNAs or by sequence analysis (gray bars [top portion of bars]). Each transcript, regardless of its length, was divided into 20 equal parts, and the position of HIV-1 integration within the transcript was plotted by using these relative length units.
FIG. 4.
FIG. 4.
Analysis of host gene expression in resting CD4+ T cells. All transcriptional units in which an integration site was identified were analyzed by RT-PCR (Table 1). Representative examples are shown. In all three panels, GAPDH served as a positive control for RT-PCR. RT-PCRs were carried out with (+) or without (−) RT. (A) Patterns of expression of GAPDH and LOX by resting CD4+ T cells, by activated CD4+ T cells, and by chondrocytes. In all instances, RT-PCR signals were dependent on the presence of RT. (B) Expression of TOP2A by mitogen-activated but not resting CD4+ T cells. RT-PCR signals were dependent on the presence of RT. (C) Analysis of NFATc3 and FKBP51 expression in resting and activated CD4+ T cells and in the thymus. RT-PCR signals were dependent on the presence of RT. (D) Patterns of expression of genes targeted for integration in resting CD4+ T cells and in the memory subset of resting CD4+ T cells.
FIG. 5.
FIG. 5.
Detection of transcripts containing HIV-1 sequences in resting CD4+ T lymphocytes from patients on HAART. (A) RT-PCR strategy for detecting HIV-1 transcripts initiating at the HIV-1 LTR and transcripts initiating upstream of the transcription start site. The positions of the two forward PCR primers (HIV-START5′ and HIV-UP5′) relative to the transcription start site are indicated. (B) Sensitivity of heminested PCR analysis with, as a template, 10-fold serial dilutions of a plasmid (pLAI) containing the entire HIV-1 genome. The forward primer was either HIV-START5′ or HIV-UP5′. For the first reaction (rxn), the reverse primer was HIV-RT. For the nested reaction, the reverse primer was HIV-3′. dH2O, distilled H2O. (C) Analysis of transcripts from two independent aliquots (a and b) of 106 sorted, resting CD4+ T cells from a representative patient. (Left panel) Transcripts were amplified in quadruplicate reactions with forward primer HIV-START5′. (Middle panel) Transcripts were amplified in quadruplicate reactions with forward primer HIV-UP5′. (Right panel) Control reactions lacking RT were performed with the indicated forward primers. GAPDH served as a positive control for RNA isolation and RT-PCRs for each aliquot.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adams, M., L. Sharmeen, J. Kimpton, J. M. Romeo, J. V. Garcia, B. M. Peterlin, M. Groudine, and M. Emerman. 1994. Cellular latency in human immunodeficiency virus-infected individuals with high CD4 levels can be detected by the presence of promoter-proximal transcripts. Proc. Natl. Acad. Sci. USA 91:3862-3866. - PMC - PubMed
    1. Baughman, G., G. J. Wiederrecht, F. Chang, M. M. Martin, and S. Bourgeois. 1997. Tissue distribution and abundance of human FKBP51, an [sic] FK506-binding protein that can mediate calcineurin inhibition. Biochem. Biophys. Res. Commun. 232:437-443. - PubMed
    1. Blankson, J. N., D. Persaud, and R. F. Siliciano. 2002. The challenge of viral reservoirs in HIV-1 infection. Annu. Rev. Med. 53:557-593. - PubMed
    1. Boeke, J. D., and S. E. Devine. 1998. Yeast retrotransposons: finding a nice quiet neighborhood. Cell 93:1087-1089. - PubMed
    1. Bohnlein, E., J. W. Lowenthal, M. Siekevitz, D. W. Ballard, B. R. Franza, and W. C. Greene. 1988. The same inducible nuclear proteins regulates mitogen activation of both the interleukin-2 receptor-alpha gene and type 1 HIV. Cell 53:827-836. - PubMed

Publication types