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 Feb;80(4):1850-62.
doi: 10.1128/JVI.80.4.1850-1862.2006.

CD4-specific transgenic expression of human cyclin T1 markedly increases human immunodeficiency virus type 1 (HIV-1) production by CD4+ T lymphocytes and myeloid cells in mice transgenic for a provirus encoding a monocyte-tropic HIV-1 isolate

Jinglin Sun  1 , Timothy Soos, Vineet N Kewalramani, Kristin Osiecki, Jian Hua Zheng, Laurie Falkin, Laura Santambrogio, Dan R Littman, Harris Goldstein
Affiliations

CD4-specific transgenic expression of human cyclin T1 markedly increases human immunodeficiency virus type 1 (HIV-1) production by CD4+ T lymphocytes and myeloid cells in mice transgenic for a provirus encoding a monocyte-tropic HIV-1 isolate

Jinglin Sun et al. J Virol. 2006 Feb.

Abstract

Human immunodeficiency virus type 1 (HIV-1)-encoded Tat provides transcriptional activation critical for efficient HIV-1 replication by interacting with cyclin T1 and recruiting P-TEFb to efficiently elongate the nascent HIV transcript. Tat-mediated transcriptional activation in mice is precluded by species-specific structural differences that prevent Tat interaction with mouse cyclin T1 and severely compromise HIV-1 replication in mouse cells. We investigated whether transgenic mice expressing human cyclin T1 under the control of a murine CD4 promoter/enhancer cassette that directs gene expression to CD4(+) T lymphocytes and monocytes/macrophages (hu-cycT1 mice) would display Tat responsiveness in their CD4-expressing mouse cells and selectively increase HIV-1 production in this cellular population, which is infected primarily in HIV-1-positive individuals. To this end, we crossed hu-cycT1 mice with JR-CSF transgenic mice carrying the full-length HIV-1(JR-CSF) provirus under the control of the endogenous HIV-1 long terminal repeat and demonstrated that human cyclin T1 expression is sufficient to support Tat-mediated transactivation in primary mouse CD4 T lymphocytes and monocytes/macrophages and increases in vitro and in vivo HIV-1 production by these stimulated cells. Increased HIV-1 production by CD4(+) T lymphocytes was paralleled with their specific depletion in the peripheral blood of the JR-CSF/hu-cycT1 mice, which increased over time. In addition, increased HIV-1 transgene expression due to human cyclin T1 expression was associated with increased lipopolysaccharide-stimulated monocyte chemoattractant protein 1 production by JR-CSF mouse monocytes/macrophages in vitro. Therefore, the JR-CSF/hu-cycT1 mice should provide an improved mouse system for investigating the pathogenesis of various aspects of HIV-1-mediated disease and the efficacies of therapeutic interventions.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Construction of human cyclin T1 mice and evaluation of transgene expression. (A) Schematic representation of the human cyclin T1 transgene construct. E4/P4, murine CD4 enhancer/promoter; N, Xb, Xh, Cl, Sc, B, and S, restriction enzyme sites for NotI, XbaI, Xho1, ClaI, SacI, BamHI and SalI, respectively; SVpA, SV40 polyadenylation signal. (B) Expression of the human cyclin T1 transgene in the spleens and thymuses of mice from seven transgenic lines. Expression was detected by Western blotting with a polyclonal antibody specific for human cyclin T1. Specificity is indicated by reactivity with 3T3 cells expressing human cyclin T1 and no reactivity with thymocytes from wild-type mice. (C) Human cyclin T1 expression in bone marrow-derived myeloid cells from hu-cycT1 transgenic mice was evaluated by RT-PCR analysis of RNA extracted from cells cultured with or without GM-CSF for 2 days. Amplified products after PCR amplification using primers specific for human cyclin T1 mRNA or mouse 18S mRNA were resolved and visualized on an agarose gel containing ethidium bromide.
FIG. 2.
FIG. 2.
Expression of human cyclin T1 increases LTR activation in CD4+ T lymphocytes and myeloid cells. (A) CD4+ T lymphocytes from the spleens of four hu-cycT1 transgenic lines (T1, T4, T5, and T7) or of wild-type (WT) mice were activated with plate-bound antibodies to CD3ɛ and CD28 for 48 h and coinfected with VSV-G-pseudotyped pNL-4.3-Luc-E−R− and pHIV-SVren. (B) Bone marrow-derived myeloid cells from two hu-cycT1 transgenic lines (T4 and T7) were coinfected with the VSV-G-pseudotyped pNL-4.3-Luc-E−R− and pHIV-SVren.
FIG. 3.
FIG. 3.
Human cyclin T1 expression increases HIV-1 production by JR-CSF CD4+ T lymphocytes and myeloid cells. (A) CD4+ T lymphocytes isolated from the splenocytes of JR-CSF mice or JR-CSF/hu-cycT1 mice were cultured in duplicate in 96-well plates and stimulated with prostratin. After 7 days, HIV-1 production was measured by determining the concentration of p24 antigen in the culture supernatant. (B) Purified bone marrow-derived myeloid cells from JR-CSF mice or JR-CSF/hu-cycT1 mice were cultured in duplicate in 96-well plates in the presence of GM-CSF (20 ng/ml). After 5 days, HIV-1 production was measured by determining the concentration of p24 antigen in the culture supernatant. The data shown represent the mean ± standard error of the mean (SEM) of HIV-1 production in three independent experiments with cells isolated from different mice.
FIG. 4.
FIG. 4.
Human cyclin T1 expression increases the infectiousness of JR-CSF CD4+ T lymphocytes and myeloid cells. The infectivity of purified CD4+ lymphocytes stimulated with prostratin (1 μg/ml) and that of CD11b+ monocytes stimulated with GM-CSF (20 ng/ml) were measured by limiting-dilution coculture. The data represent the mean ± SEM of two independent experiments and are presented as tissue culture infective dose/106 thymocytes.
FIG. 5.
FIG. 5.
Depletion of CD4+ T lymphocytes in the peripheral blood of JR-CSF/hu-cycT1 mice. CD4+ and CD8+ T lymphocytes in the peripheral blood of BALB/c mice (n = 7), hu-cycT1 mice (n = 7), JR-CSF mice (n = 7), and JR-CSF/hu-cycT1 mice (n = 7) were measured by flow cytometry. The mean percentages ± SEM of CD4+ and CD8+ T lymphocytes in the peripheral blood of the indicated mice are shown.
FIG. 6.
FIG. 6.
Immunohistology of spleens from hu-cycT1 mice, JR-CSF mice, and JR-CSF/hu-cycT1 mice. The populations of CD4+ lymphocytes and B cells in spleens from hu-cycT1 mice, JR-CSF mice, and JR-CSF/hu-cycT1 mice were visualized by immunofluorescence after staining with antibodies to mouse CD19 (left panels) and CD4 (right panels) by use of a Leica AOBS laser scanning confocal microscope system. The pictures shown are representative of three independent experiments.
FIG. 7.
FIG. 7.
JR-CSF mice expressing human cyclin T1 display increased HIV-1 production after continuous in vivo stimulation with GM-CSF. JR-CSF mice (n = 4) or JR-CSF/hu-cycT1 mice (n = 4) were implanted with B16-F10-GM-CSF melanoma cells. Two weeks later, GM-CSF levels in the serum were measured by ELISA, and the spleens and bone marrow cells were harvested. HIV-1 production was determined by measuring the HIV p24 antigen content of the lysates from (A) bone marrow cells and (B) spleens and results are presented as the mean ± SEM of p24 antigen content/107 cells.
FIG. 8.
FIG. 8.
Effect of JR-CSF provirus expression on chemokine gene expression and production by stimulated mouse myeloid cells. Bone marrow-derived myeloid cells from JR-CSF mice and control mice were cultured in 24-well plates (2 × 106 cells/well) with GM-CSF and were either stimulated with LPS or not stimulated. After 24 h, the cells were harvested, and RNA was extracted from the myeloid cells and analyzed for chemokine gene expression by use of an RNase protection assay. (A) An autoradiograph of an RNase protection assay of RNA extracted from myeloid cells from a wild-type or a JR-CSF mouse and (B) densitometric analysis of the gel. After 72 h of stimulation, the levels of (C) MCP-1 and (D) RANTES in an aliquot of culture supernatant were measured by ELISA. The ELISA data shown represent the mean levels of MCP-1 and RANTES production in four independent experiments using myeloid cells isolated from different mice, with statistical significance indicated.
FIG. 9.
FIG. 9.
Effect of human cyclin T1 expression on chemokine gene expression in stimulated JR-CSF mouse myeloid cells. Bone marrow-derived myeloid cells from JR-CSF mice and JR-CSF/hu-cycT1 mice were cultured in 24-well plates (2 × 106 cells/well) with GM-CSF and either stimulated with LPS or not stimulated. After 24 h, the cells were harvested, and RNA was extracted from the myeloid cells and analyzed for chemokine gene expression by an RNase protection assay. (A) A representative autoradiograph of an RNase protection assay of RNA extracted from myeloid cells from wild-type or JR-CSF mice from three independent experiments and (B) the mean values of densitometric analysis of autoradiographs from three separate experiments. After 72 h of stimulation, the levels of (C) MCP-1 and (D) RANTES in an aliquot of culture supernatant were measured by ELISA. The ELISA data shown represent the mean levels of MCP-1 and RANTES production in four independent experiments using myeloid cells isolated from different mice, with statistical significance indicated.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abramczuk, J. W., D. S. Pezen, J. M. Leonard, E. Monell-Torrens, J. H. Belcher, M. A. Martin, and A. L. Notkins. 1992. Transgenic mice carrying intact HIV provirus: biological effects and organization of a transgene. J. Acquir. Immune Defic. Syndr. 5:196-203. - PubMed
    1. Bafica, A., C. A. Scanga, M. L. Schito, S. Hieny, and A. Sher. 2003. Cutting edge: in vivo induction of integrated HIV-1 expression by mycobacteria is critically dependent on Toll-like receptor 2. J. Immunol. 171:1123-1127. - PubMed
    1. Baggiolini, M., B. Dewald, and B. Moser. 1997. Human chemokines: an update. Annu. Rev. Immunol. 15:675-705. - PubMed
    1. Baumann, J. G., D. Unutmaz, M. D. Miller, S. K. Breun, S. M. Grill, J. Mirro, D. R. Littman, A. Rein, and V. N. KewalRamani. 2004. Murine T cells potently restrict human immunodeficiency virus infection. J. Virol. 78:12537-12547. - PMC - PubMed
    1. Berkhout, B., R. H. Silverman, and K. T. Jeang. 1989. Tat trans-activates the human immunodeficiency virus through a nascent RNA target. Cell 59:273-282. - PubMed

Publication types

MeSH terms

-