cd4-speciﬁc transgenic expression of human cyclin … of a murine cd4 promoter ... jr-csf provirus...

JOURNAL OF VIROLOGY, Feb. 2006, p. 1850–1862 Vol. 80, No. 40022-538X/06/$08.00�0 doi:10.1128/JVI.80.4.1850–1862.2006Copyright © 2006, American Society for Microbiology. All Rights Reserved.

CD4-Specific Transgenic Expression of Human Cyclin T1 MarkedlyIncreases Human Immunodeficiency Virus Type 1 (HIV-1)

Production by CD4� T Lymphocytes and Myeloid Cellsin Mice Transgenic for a Provirus Encoding a

Monocyte-Tropic HIV-1 IsolateJinglin Sun,1† Timothy Soos,2,3† Vineet N. KewalRamani,2,3 Kristin Osiecki,1 Jian Hua Zheng,1

Laurie Falkin,1 Laura Santambrogio,4 Dan R. Littman,2,3 and Harris Goldstein1,5*Departments of Microbiology & Immunology,1 Pathology,4 and Pediatrics,5 Albert Einstein College of Medicine, Bronx,

New York 10461, and Molecular Pathogenesis Program2 and The Howard Hughes Medical Institute,3

Skirball Institute of Biomolecular Medicine, New York University School of Medicine,New York, New York 10016

Received 24 August 2005/Accepted 30 November 2005

Human immunodeficiency virus type 1 (HIV-1)-encoded Tat provides transcriptional activation critical forefficient HIV-1 replication by interacting with cyclin T1 and recruiting P-TEFb to efficiently elongate thenascent HIV transcript. Tat-mediated transcriptional activation in mice is precluded by species-specificstructural differences that prevent Tat interaction with mouse cyclin T1 and severely compromise HIV-1replication in mouse cells. We investigated whether transgenic mice expressing human cyclin T1 under thecontrol of a murine CD4 promoter/enhancer cassette that directs gene expression to CD4� T lymphocytes andmonocytes/macrophages (hu-cycT1 mice) would display Tat responsiveness in their CD4-expressing mousecells and selectively increase HIV-1 production in this cellular population, which is infected primarily inHIV-1-positive individuals. To this end, we crossed hu-cycT1 mice with JR-CSF transgenic mice carrying thefull-length HIV-1JR-CSF provirus under the control of the endogenous HIV-1 long terminal repeat and dem-onstrated that human cyclin T1 expression is sufficient to support Tat-mediated transactivation in primarymouse CD4 T lymphocytes and monocytes/macrophages and increases in vitro and in vivo HIV-1 productionby these stimulated cells. Increased HIV-1 production by CD4� T lymphocytes was paralleled with theirspecific 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 increasedlipopolysaccharide-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 forinvestigating the pathogenesis of various aspects of HIV-1-mediated disease and the efficacies of therapeuticinterventions.

The transcriptional activator Tat, encoded by human immu-nodeficiency virus (HIV), is essential for efficient viral replica-tion (13, 19, 54). Transcriptional activation by Tat is dependentupon its binding to the transactivation response element(TAR), an RNA stem-loop structure that is located down-stream from the transcription initiation site in the 5� longterminal repeat (LTR) (5, 18, 45). TAR displays a secondarystructure that includes a 5� bulge from position �23 to position�25 and a central loop at positions �30 to �35 (57). Althoughthe 5� bulge and central loop are both required for Tat func-tional activity, the restricted binding of Tat to the 5� bulge andnot to the central loop indicated that host cell factors wererequired to mediate the interaction between Tat and the cen-tral loop and subsequent transcriptional activation (32). Therequirement for Tat to interact with a human-host-specific cell

factor to mediate transactivation provided a rationale for thefunctional activity of Tat in human cells but not in mouse cellsand for the capacity of transferred human chromosome 12 toconfer mouse cells with the ability to support Tat transactiva-tion (29, 46). The cellular factor involved in Tat-mediatedtransactivation was identified as cellular positive transcriptionelongation factor b (P-TEFb) (40). After P-TEFb is recruitedto the nascent HIV transcript by Tat, it clears RNA polymer-ase II from the promoter by phosphorylating its carboxy ter-minal domain and thereby increasing template processivity andtranscriptional elongation (42). The kinase associated with thephosphorylation activity of P-TEFb, cyclin-dependent kinase-9(CDK-9), is required for Tat-mediated transactivation (40, 52,71). An associated cyclin protein, cyclin T1, coordinatesCDK-9 binding to Tat and is required for RNA polymerase IItranscriptional elongation (65). The sequences of human cyclinT1 (hu-cycT1) and mouse cyclin T1 are 90% homologous, andthe inability of Tat to function as a transcriptional activator inmouse cells is due to a single-amino-acid reciprocal changethat prevents the binding of mouse cyclin T1 to Tat (7, 23, 24).

* Corresponding author. Mailing address: Albert Einstein Collegeof Medicine, Forschheimer Building, Room 408, 1300 Morris ParkAvenue, Bronx, NY 10461. Phone: (718) 430-2156. Fax: (718) 430-8972. E-mail: [email protected].

† These authors contributed equally to this paper.

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Mouse cells transfected with a construct encoding human cy-clin T1 supported Tat-mediated transactivation, confirmingthat human cyclin T1 was the species-specific HIV type 1(HIV-1) cofactor and that human cyclin T1 was able to interactwith mouse CDK-9 to rescue Tat function in mouse cells (65).

Because the biological behavior of cell lines in vitro differsfrom that of primary cells in an in vivo environment, furtherdelineation of the interaction of human cyclin T1 with Tat andits role in the activation of HIV-1 replication would be greatlyfacilitated by the availability of an in vivo model system. Miceare extremely useful tools for investigating the pathogenesis ofinfectious diseases, the regulation of the immune system, andthe generation of protective immunity, but the inability ofHIV-1 to infect mouse cells has severely limited the use ofmice to investigate HIV-1 pathogenesis (38, 39). In addition tothe inefficient activity of Tat in mouse cells that is describedabove, another major block preventing infection of mouse cellsis the inability of HIV-1 to enter mouse cells due to the factthat its envelope protein, gp120, does not bind to the murinehomologues of human HIV receptors (41). The failure ofHIV-1 to penetrate mouse cells can be circumvented by con-structing mice transgenic for an infectious provirus derivedfrom HIV-1, such as the X4 laboratory isolate NL4-3 (1, 37).These transgenic mice have been used to investigate in vivoactivation of HIV-1 in monocytes by pathogens (10, 14, 21, 25),the efficacy of treatments that block proviral expression (55,56), and the role of Toll-like receptors in the pathogen-inducedactivation of the HIV LTR (2, 16). To enable study of thebehavior of primary HIV-1 isolates, we expanded upon thisapproach by developing a different mouse line that is trans-genic for HIV-1JR-CSF, a full-length HIV-1 provirus derivedfrom the well-characterized primary R5-tropic clinical isolate(9, 49). In this JR-CSF mouse transgenic line, the HIV-1JR-CSF

provirus is under the control of the endogenous HIV-1 LTR,and these mice are populated with T lymphocytes and mono-cytes that produce infectious HIV-1 (8, 47). The HIV-1 pro-duced by the JR-CSF mouse cells does not infect mouse cells,allowing our transgenic mouse system to be used to investigatefactors that modulate the postintegration phase of HIV-1 rep-lication independent of the confounding effects of secondaryinfection. A limitation of HIV transgenic mouse models is that,in contrast to what is seen for humans, where HIV-1 replica-tion occurs predominantly in CD4-expressing cells, in the HIVtransgenic mice the provirus is integrated in every cell and canbe expressed by any cell that supports HIV LTR transcription,including cells of many lineages normally not infected by HIV.Consequently, HIV transgenic mice may not recapitulate manyaspects of the pathophysiology of HIV infection. We hypoth-esized that expression of a human cyclin T1 transgene wouldmarkedly increase HIV-1 replication in the HIV transgenicmouse cells by enabling Tat-mediated transcriptional activa-tion. By targeting expression of the human cyclin T1 transgeneto CD4-expressing cells in the JR-CSF transgenic mice, wewould selectively increase HIV-1 replication in the cell popu-lation which is the primary target of HIV-1 infection in hu-mans. In this study we demonstrated that expression of humancyclin T1 under the control of a CD4 expression construct inthe JR-CSF mice markedly increased in vivo and in vitroHIV-1 production by mouse CD4� T lymphocytes and myeloidcells, was associated with selective depletion of CD4� T lym-

phocytes in the peripheral blood, and was associated with in-creased monocyte chemoattractant protein 1 (MCP-1) produc-tion by stimulated mouse myeloid cells.

MATERIALS AND METHODS

Construction of mice transgenic for human cyclin T1 and HIV-1JR-CSF. The1.15-kb cDNA fragment encoding human cyclin T1 was cloned into an engi-neered SalI site in a murine CD4 expression cassette targeting expression to CD4T lymphocytes, macrophages, and dendritic cells (34). The cassette contains themurine CD4 enhancer/promoter and the human CD4 intronic sequence, whichencompasses the macrophage enhancer and the CD4 silencer elements. Theresulting plasmid was linearized and microinjected into the pronuclei of fertilizedembryos derived from FVB � C57/B6 mouse crosses. Transgenic founders wereidentified by PCR analysis of genomic DNA extracted from tails using a primerpair specific for the amplification of human cyclin T1 DNA as described below.The JR-CSF mice were constructed and characterized as previously described(8). The animal studies performed in this report were performed under a pro-tocol reviewed and approved by the Albert Einstein College of Medicine AnimalInstitute Committee.

Detection of human cyclin T1 DNA and RNA expression. DNA was extractedfrom transgenic mouse tails using a DNeasy kit (QIAGEN, Valencia, CA).Human cyclin T1 was detected by PCR amplification (35 cycles at 94°C for 45 s,55°C for 45 s, and 72°C for 1 min) with Taq polymerase (Promega, Madison, WI)using primers 5�-TCCCAACTTCCAGTTGGTACT-3� and 5�-TCCACCAGACCGAGGATTCAG-3�, which were designed to be specific for the human cyclinT1 sequence and which yielded an amplimer of �400 bp. The specificity of theseprimers for human cyclin T1 was confirmed by demonstrating that no PCRproducts were generated after PCR amplification of control mouse DNA. AfterRNA was purified from the indicated cells by use of an RNAeasy kit (QIAGEN)and treated with DNase, human cyclin T1 mRNA with 18S rRNA mRNA as theinternal control was detected using a OneStep reverse transcription-PCR (RT-PCR) kit (QIAGEN). Briefly, RNA (1 �g) was mixed with 10 �l of 5� QIAGENOneStep RT-PCR buffer, 2 �l of dNTP mix (containing 10 �M of each dNTP),2 �l of QIAGEN OneStep RT-PCR enzyme mix, and 0.6 �M of the humancyclin T1-specific primers described above or 18S rRNA-specific primers 5�-TCAAGAACGAAAGTCGGAGG-3� and 5�-GGACATCTAAGGGCATCACA-3� (58). The RT-PCR mix was heated at 50°C for 30 min and at 95°C for 15min and then amplified for 35 cycles (94°C for 45 s, 55°C for 45 s, and 72°C for1 min) and electrophoresed through a 1.5% agarose gel containing ethidiumbromide; the amplified product was then detected under UV light.

Western blotting for human cyclin T1 detection. Whole-cell extracts of thy-mocytes and reversed-phase column-depleted splenocytes were prepared inNP-40 radioimmunoprecipitation assay buffer (150 mM NaCl, 20 mM Tris [pH7.5], 1% NP-40, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mMbenzamidine, 2 �g/ml leupeptin), sonicated for three 1-min cycles, and clarifiedby centrifugation at 14,000 rpm for 15 min at 4°C. Cellular lysate (50 �g) wasresolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels,transferred to polyvinylidene difluoride membranes, and incubated with anti-human cyclin T1 rabbit polyclonal antibody (kindly provided by Katherine Jones,The Salk Institute) and anti-mouse actin murine monoclonal antibody (SantaCruz Biotechnologies, Santa Cruz, CA). Bands were visualized after an incuba-tion of the blot with the appropriate conjugated antibody and subsequently withsubstrate.

Flow cytometric analysis. Mononuclear cells harvested from the peripheralblood of the mice were stained with a phycoerythrin-conjugated rat monoclonalantibody to mouse CD8 and a fluorescein isothiocyanate-conjugated rat mono-clonal antibody to CD4 (BD Biosciences, San Jose, CA) as described previously(8). Expression of surface proteins was assessed by flow cytometric analysis usinga FACScan cell analyzer with LYSIS-II software (BD Biosciences). Nonviablecells and unlysed red blood cells were gated out based on their forward and sidescatter profiles.

Isolation of myeloid cells from the mouse bone marrow. Bone marrow cellsextracted from mouse femurs by lavage with phosphate-buffered saline (PBS)were dispersed with vigorous pipetting, washed, resuspended in complete me-dium (RPMI medium containing 10% fetal calf serum and 2-mercaptoethanol[50 �M]), and cultured in 100-mm petri dishes at a density of 2 � 106 cells/ml.Following overnight culturing, the nonadherent cells were gently rinsed away,fresh complete medium chilled to 4°C was added, and the adherent cells wereharvested by scraping. The adherent cells were washed and were found to be�95% viable by trypan blue exclusion, and �80% of the cells expressed themonocyte marker CD11b, as detected by flow cytometric analysis.

VOL. 80, 2006 MICE TRANSGENIC FOR HUMAN CYCLIN T1 AND HIV 1851

Purification of mouse CD4� T lymphocytes, CD8� T lymphocytes, andCD11b� monocytes. CD4� T lymphocytes and CD8� T lymphocytes were puri-fied from mouse splenocytes, and CD11b� monocytes were isolated from mousebone marrow cells by use of an AutoMACS system (Miltenyi Biotec, Auburn,CA) in accordance with the manufacturer’s protocol. Mononuclear cells isolatedfrom the splenocytes or bone marrow cells by Ficoll-Hypaque density centrifu-gation were washed twice with PBS; incubated with MACS MicroBeads coupledto anti-CD4, anti-CD8, or anti-CD11b antibody; and passed through a positiveselection AutoMACS separation column by use of an AutoMACS automatedbenchtop magnetic cell sorter. The purity of the sorted cells was determined byflow cytometry and was greater than 80%.

Pseudotype HIV production luciferase assay. 293T lymphocytes were culturedin Dulbecco’s modified Eagle’s medium with 10% added fetal calf serum andcotransfected with 20 �g of an expression plasmid for vesicular stomatitis virusG protein (VSV-G) and 50 �g either of a proviral vector construct, pNL-4.3-Luc-E�R�, which contained the firefly luciferase reporter gene under the tran-scriptional control of the HIV LTR (12, 30) (obtained from the NIAID AIDSRepository, Rockville, MD), or of pHIV-SVren, which contained the Renillaluciferase reporter gene under the transcriptional control of the SV40 earlypromoter gene in the nef open reading frame (4), by use of Lipofectamine Plus(Invitrogen). After 48 h, culture supernatants were harvested and passed through0.45-�m cellulose acetate syringe filters (Gelman Sciences, Inc., Ann Arbor, MI),and virus production was quantified using an HIV-1 p24 enzyme-linked immu-nosorbent assay (ELISA) (Perkin-Elmer Life Sciences, Boston, MA). PurifiedCD4� splenic T lymphocytes (5 � 105 T lymphocytes/well) were activated in a48-well plate with anti-CD3ε (3 �g/ml) and anti-CD28 (1 �g/ml) plate-boundantibodies for 48 h and then coinfected with VSV-G-pseudotyped HIV-4.3-Luc-E�R� and HIV-SVren viruses. Bone marrow-derived macrophages were cul-tured for 1 week in complete medium with added murine macrophage colony-stimulating factor (10 ng/ml; R&D Systems, Minneapolis, MN) and thencoinfected with the HIV reporter-pseudotyped viruses described above at a celldensity of 1 � 106 macrophages per well in a 24-well plate. Whole-cell lysateswere prepared 48 h after infection using a dual luciferase reporter assay kit(Promega), and firefly and Renilla luciferase activity was measured using a Lucyluminometer (Anthos Labtec Instruments, GmbH, Austria).

Quantification of HIV production by stimulated T lymphocytes and myeloidcells. The CD4� T lymphocytes and bone marrow-derived myeloid cells (105

cells) isolated from mouse bone marrow described above were cultured in du-plicate in 96-well plates in complete medium alone in a total volume of 200 �l.CD4� T lymphocytes were stimulated with prostratin, a potent activator of HIVLTR expression in T lymphocytes (36), and monocytes were stimulated withgranulocyte-macrophage colony-stimulating factor (GM-CSF) (20 ng/ml). At theindicated time, HIV-1 production by the mouse cells was quantified by measur-ing the p24 antigen content of the culture supernatant with an HIV-1 p24 coreprofile ELISA (Perkin-Elmer Life Sciences Inc., Boston, MA) done as describedpreviously (8).

Immunohistological analysis of mouse spleens by immunofluorescence. Aftermouse spleens were harvested, they were embedded in Tissue-Tek OCT com-pound (Sakura Finetek USA, Torrance, CA), frozen in liquid nitrogen, and cutby a cryostat into 10-�m-thick sections. The sections were fixed for 10 min at 4°Cin PBS containing 4% paraformaldehyde. After the nonspecific reactivity wasblocked by incubation with goat serum and bovine serum albumin blockingsolution, the sections were incubated with rat anti-mouse CD4 or rat anti-mouseCD19 (Pharmingen) at optimized concentrations, washed, incubated with AlexaFluor 594-conjugated goat anti-rat immunoglobulin G (Invitrogen Corporation,Carlsbad, CA), washed again, and mounted on slides. The samples were analyzedwith a Leica AOBS laser scanning confocal microscope system.

Evaluation of the infectivities of JR-CSF and JR-CSF/hu-cycT1 mouse CD4�

T lymphocytes and monocytes by limiting-dilution coculture. The infectivities ofCD4� T lymphocytes and CD11b� monocytes isolated from the mice as de-scribed above were determined by culturing fourfold dilutions of cells rangingfrom 1 � 106 to 1 � 103 cells in quadruplicate in 24-well plates with a fixednumber of phytohemagglutinin-activated human donor peripheral blood mono-nuclear cells (1 � 106 cells/well) in complete medium containing interleukin 2(25 units/ml) as described previously (8). One week later, the p24 antigen contentof the culture supernatant was measured as described above. The infectivities ofthe cells are reported as tissue culture infective dose/106 cells, which was calcu-lated by determining the lowest number of added cells that initiated productiveinfection of at least half of the quadruplicate cocultures with HIV-1.

RNA extraction and RNase protection assay. The level of mRNA encodingvarious chemokines was measured using an RNase protection assay as describedpreviously (43). Briefly, total RNA was extracted from cultured myeloid cells byuse of Trizol reagent according to the manufacturer’s protocol (Invitrogen).

Radiolabeled probes specific for the C-C chemokines Ltn, RANTES, eotaxin,MIP-1�, MIP-1, MIP-2, IP-10, MCP-1, and TCA-3 and the control housekeep-ing genes encoding L32 and GADPH (panel mCK5; Pharmingen, San Diego,CA) were generated with a MAXIscript kit (Ambion, Austin, TX) according tothe manufacturer’s instructions. An RNase Protection Assay III kit (Ambion)was used to perform an RNase protection assay after the radiolabeled probeswere hybridized to cellular RNA, and complexes containing mRNA bound to theprobes were separated by electrophoresis on 5% denaturing acrylamide gels,which was followed by autoradiography. Bands representing chemokine probebound to specific chemokine mRNA were quantified by densitometry on multi-ple film exposures by use of AMBIS Quant-Probe radioanalytic imaging systemsoftware (AMBIS, Inc., San Diego, CA), and relative chemokine mRNA expres-sion was determined by calculating the ratio of the density of the indicatedchemokine mRNA to that of L32 mRNA or GADPH.

Measurement of MCP-1 and RANTES protein. MCP-1 and RANTES secretedby myeloid cells into culture supernatant were quantified by ELISA with aQuantikine immunoassay (R&D Systems, Minneapolis, MN) according to themanufacturer’s instructions. Briefly, aliquots of culture supernatant were mixedwith assay diluent, added in duplicate to wells coated with a polyclonal antibodyspecific for MCP-1 or RANTES, and incubated for 2 h at room temperature.After plates were aspirated and rinsed with wash buffer, horseradish peroxidase-conjugated polyclonal antibody specific for MCP-1 or RANTES was added to thewells and incubated for 2 h at room temperature. The plates were washed,substrate was added for 30 min, the reaction was stopped with 2 N H2SO4, andthe absorbance of each microwell was read at the 450-nm wavelength on amicroplate reader (Bio-Rad, Hercules, CA) within 30 min of stopping the reac-tion. The level of MCP-1 or RANTES in the culture supernatants was thencalculated using a standard curve generated using MCP-1 or RANTES standardsmeasured in parallel with the samples. The limit of detection of the assays was 2pg/ml.

In vivo treatment with GM-CSF. B16-F10 melanoma cells stably transducedwith a retroviral vector expressing murine GM-CSF (15) were expanded inDulbecco minimal essential medium with 10% fetal calf serum added. After thecells were harvested and washed extensively in PBS, approximately 2 � 107 cellswere subcutaneously injected into each mouse. Two to 3 weeks later, when asubcutaneous tumor with diameter of 2.5 to 3 cm had appeared, the indicatedmouse tissues were harvested and analyzed. Systemic GM-CSF production by thetumor cells was measured in vivo by quantization of mouse plasma GM-CSFlevels by use of a GM-CSF ELISA kit (R&D Systems).

Statistical analysis of the data. The Student t test for unpaired data was usedfor all statistical comparisons made. Significance was assigned to P values of0.05.

RESULTS

Myeloid cells from hu-cycT1 transgenic mice express humancyclinT1. hu-cycT1 transgenic mice were generated using amurine CD4 expression cassette encoding human cyclin T1predicted to target transgene expression to CD4 T lympho-cytes, macrophages, and dendritic cells (Fig. 1A). Transgenicfounders were identified by PCR analysis of genomic DNAextracted from their tails using a primer pair specific for theamplification of human cyclin T1 DNA. Immunoblot analysisusing an antibody specific for human cyclin T1 demonstratedexpression of the human cyclinT1 transgene in the spleens andthymuses of mice from several independent transgenic lines(Fig. 1B). Expression of the human cyclin T1 transgene in bonemarrow-derived myeloid cells from hu-cycT1 mice derivedfrom the T7 line and not in those cultured in the presence orabsence of GM-CSF from wild-type littermates was demon-strated by RT-PCR using primers specific for human cyclin T1(Fig. 1C). Taken together, these results indicated that thehu-cycT1 transgene was expressed in hu-cycT1 mouse T lym-phocytes and myeloid cells.

HIV LTR-driven expression is increased in myeloid cellsfrom hu-cycT1 transgenic mice. Expression of human cyclin T1by the mouse cells should rescue Tat function and permit

1852 SUN ET AL. J. VIROL.

Tat-mediated transactivation of the HIV-1 LTR. To examinewhether T lymphocytes and myeloid cells from the hu-cycT1transgenic mice supported Tat-mediated transactivation, puri-fied CD4� T lymphocytes and bone marrow-derived myeloidcells from four different hu-cycT1 transgenic mouse lines andcontrol littermates were infected with HIV-1 constructs con-taining a firefly luciferase reporter gene controlled by the HIVLTR or a Renilla luciferase reporter gene controlled by theSV-40 promoter pseudotyped with a VSV-G envelope (12, 30,59). After the integration of the HIV-1 provirus into the mouseCD4� T lymphocytes and myeloid cells, the firefly luciferasegene is under the control of the HIV-1 LTR; the level of LTRactivation is proportional to the level of firefly luciferase ac-tivity, with the Renilla luciferase providing a control for infec-tion efficiency. After infection with the reporter HIV, CD4� Tlymphocytes from the hu-cycT1 transgenic mouse transgeniclines displayed levels of luciferase activity up to 25-fold higherthan those displayed by CD4� T lymphocytes from wild-typelittermates (Fig. 2A). Myeloid cells from hu-cycT1 transgeniclines T4 and T7 also displayed levels of luciferase activitysixfold and fourfold higher, respectively, than those of myeloid

cells from wild-type littermates (Fig. 2B). Based on previous invitro data demonstrating that optimum Tat function occurs inthe presence of human cyclin T1, these results indicated thatexpression of human cyclin T1 in hu-cycT1 transgenic miceprovided the capacity to support Tat-mediated transactivationto primary mouse CD4� T lymphocytes and myeloid cells.

Transgenic expression of human cyclin T1 increases HIV-1production by stimulated CD4� T lymphocytes and myeloidcells. JR-CSF mice are transgenic for a full-length HIV-1 pro-virus that is expressed in the spleens, lymph nodes, thymuses,and brains of JR-CSF mice, and JR-CSF mouse T lympho-cytes, monocytes, and microglia produce infectious HIV-1 (8,47, 64). JR-CSF mouse splenocytes express Tat mRNA (datanot shown), as determined by analysis using RT-PCR as de-scribed previously (31). We crossed the hu-cycT1 transgenicmice derived from the T7 line with JR-CSF mice to obtainJR-CSF/hu-cycT1 mice that were transgenic for human cyclinT1 and the HIV-1JR-CSF provirus regulated by the endogenousHIV-1 LTR. The JR-CSF/hu-cycT1 mice were used to examinethe in vivo effect of human cyclin T1 expression on HIV-1production by primary CD4� T lymphocytes and myeloid cells.Purified CD4� T lymphocytes isolated from the spleens of

FIG. 1. Construction of human cyclin T1 mice and evaluation oftransgene expression. (A) Schematic representation of the human cy-clin 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 polyad-enylation signal. (B) Expression of the human cyclin T1 transgene inthe spleens and thymuses of mice from seven transgenic lines. Expres-sion was detected by Western blotting with a polyclonal antibodyspecific for human cyclin T1. Specificity is indicated by reactivity with3T3 cells expressing human cyclin T1 and no reactivity with thymocytesfrom wild-type mice. (C) Human cyclin T1 expression in bone marrow-derived myeloid cells from hu-cycT1 transgenic mice was evaluated byRT-PCR analysis of RNA extracted from cells cultured with or withoutGM-CSF for 2 days. Amplified products after PCR amplification usingprimers specific for human cyclin T1 mRNA or mouse 18S mRNAwere resolved and visualized on an agarose gel containing ethidiumbromide.

FIG. 2. Expression of human cyclin T1 increases LTR activation inCD4� T lymphocytes and myeloid cells. (A) CD4� T lymphocytesfrom the spleens of four hu-cycT1 transgenic lines (T1, T4, T5, and T7)or of wild-type (WT) mice were activated with plate-bound antibodiesto CD3ε and CD28 for 48 h and coinfected with VSV-G-pseudotypedpNL-4.3-Luc-E�R� and pHIV-SVren. (B) Bone marrow-derived my-eloid cells from two hu-cycT1 transgenic lines (T4 and T7) were coin-fected with the VSV-G-pseudotyped pNL-4.3-Luc-E�R� and pHIV-SVren.


JR-CSF mice and JR-CSF/hu-cycT1 mice by use of Au-toMACS were stimulated with prostratin, a unique phorbolester that potently increases HIV-1 production by T lympho-cytes carrying integrated proviral templates (36), and cellularHIV-1 production was determined by measuring the level ofp24 antigen secreted into the culture supernatant. Prostratininduced levels of HIV-1 production from JR-CSF/hu-cycT1mouse CD4� T lymphocytes that were over 50-fold higher thanthose from CD4� T lymphocytes from JR-CSF mouse litter-mates (Fig. 3A). To determine whether the murine CD4 en-hancer/promoter-regulated hu-cycT1 transgene specifically in-creased HIV-1 production by CD4� T lymphocytes, in anotherexperiment performed in duplicate, purified CD4� T lympho-cytes and CD8� T lymphocytes were obtained from the spleensof JR-CSF/hu-cycT1 mice, and prostratin-stimulated HIV-1production by these cellular populations was determined bymeasuring the level of p24 antigen secreted into the culturesupernatant. The mean level of p24 antigen produced by pros-tratin-stimulated JR-CSF/hu-cycT1 mouse CD4� T lympho-cytes (541 � 173 pg/ml) was about 10-fold higher than the level

produced by JR-CSF/hu-cycT1 mouse CD8� T lymphocytes(55 � 30 pg/ml).

We next examined the effect of human cyclin T1 expressionon HIV-1 production by bone marrow-derived myeloid cells.Because GM-CSF increases HIV-1 production by myeloid cells(20) and bone marrow-derived myeloid lineage cells from JR-CSF mice (47), we compared the levels of HIV-1 production byGM-CSF-stimulated myeloid cells from JR-CSF mice andfrom JR-CSF/hu-cycT1 mice. The level of HIV-1 produced byGM-CSF-stimulated JR-CSF/hu-cycT1 mouse myeloid cellswas almost 50-fold higher than the level of HIV-1 produced byGM-CSF-stimulated JR-CSF mouse myeloid cells (Fig. 3B).Furthermore, the infectivities of purified CD4� T lymphocytesand CD11b� myeloid cells from JR-CSF/hu-cycT1 mice were5-fold and 100-fold higher, respectively, than that of cells fromJR-CSF littermates, as measured by determining the lowestnumber of cells capable of generating a productive infection ofactivated human peripheral blood mononuclear cells (Fig. 4).Thus, expression of the hu-cycT1 transgene markedly en-hanced the HIV-1 production and infectivity of primary mousemyeloid cells and CD4� T lymphocytes from JR-CSF mice.

Expression of human cyclin T1 by JR-CSF mice is associ-ated with decreased levels of CD4� lymphocytes in the periph-eral blood and lymphoid tissues. A hallmark of HIV-1 infec-tion is the selective depletion of CD4� T lymphocytes in theperipheral blood of infected individuals. To determine theeffect of expression of the JR-CSF transgene combined withCD4-targeted expression of the human cyclin T1 gene on T-cell subpopulations, the percentages of CD4� and CD8� Tlymphocytes in the peripheral blood of control mice, JR-CSFmice, hu-cycT1 mice and JR-CSF/hu-cycT1 mice which rangedin age from 2 to 4 months were evaluated by flow cytometry(Fig. 5). Comparable levels of CD8� lymphocytes were de-tected in the peripheral blood of control mice (14% � 2%),hu-cycT1 mice (14% � 1%), JR-CSF mice (12% � 2%), andJR-CSF/hu-cycT1 mice (10% � 1%). Similar levels of CD4�

lymphocytes were also detected in peripheral blood samples

FIG. 3. Human cyclin T1 expression increases HIV-1 productionby JR-CSF CD4� T lymphocytes and myeloid cells. (A) CD4� Tlymphocytes isolated from the splenocytes of JR-CSF mice or JR-CSF/hu-cycT1 mice were cultured in duplicate in 96-well plates and stim-ulated with prostratin. After 7 days, HIV-1 production was measuredby determining the concentration of p24 antigen in the culture super-natant. (B) Purified bone marrow-derived myeloid cells from JR-CSFmice or JR-CSF/hu-cycT1 mice were cultured in duplicate in 96-wellplates in the presence of GM-CSF (20 ng/ml). After 5 days, HIV-1production was measured by determining the concentration of p24antigen in the culture supernatant. The data shown represent the mean� standard error of the mean (SEM) of HIV-1 production in threeindependent experiments with cells isolated from different mice.

FIG. 4. Human cyclin T1 expression increases the infectiousness ofJR-CSF CD4� T lymphocytes and myeloid cells. The infectivity ofpurified CD4� lymphocytes stimulated with prostratin (1 �g/ml) andthat of CD11b� monocytes stimulated with GM-CSF (20 ng/ml) weremeasured by limiting-dilution coculture. The data represent the mean� SEM of two independent experiments and are presented as tissueculture infective dose/106 thymocytes.


from control mice (47% � 2%), hu-cycT1 mice (45% � 4%),and JR-CSF mice (42% � 2%), indicating that expression ofthe hu-cycT1 gene or of the JR-CSF transgene alone did notaffect the CD4� lymphocyte population in the peripheralblood. In contrast, the level of CD4� lymphocytes in the pe-ripheral blood of JR-CSF/hu-cycT1 mice (27% � 5%) wassignificantly lower than the level of CD4� lymphocytes in theperipheral blood of JR-CSF mice (P � 0.005) or that of hu-cycT1 mice (P � 0.0006). Further depletion of peripheralblood CD4� lymphocytes was detected in older JR-CSF/hu-cycT1 mice, with JR-CSF/hu-cycT1 mice older than 1 yearhaving significantly (P � 0.006) lower numbers of CD4� lym-phocytes (11.5% � 3.4%) than JR-CSF mice older than 1 year(30% � 11%), with no significant difference (P � 0.18) be-tween the numbers of CD8� lymphocytes in these older JR-CSF/hu-cycT1 mice (14.9% � 3.5%) and in the older JR-CSFmice (12.2% � 1%). As a result of this depletion, an invertedCD4/CD8 ratio (mean, 0.57 � 0.09) was present in the periph-eral blood of all four JR-CSF/hu-cycT1 mice over 1 year of age,in contrast to the normal CD4/CD8 ratio in the four JR-CSFmice (mean, 3.07 � 0.21) examined.

The effect of combined expression of the JR-CSF transgenewith CD4-targeted expression of the human cyclin T1 gene onthe CD4 population in lymphoid tissues was examined by im-munohistochemistry and flow cytometry. Immunohistologicalanalysis of the spleens of 5-month-old JR-CSF/hu-cycT1 micedemonstrated preserved architecture of the lymphoid regions,with B cells present in the lymphoid follicles and CD4� Tlymphocytes present in the periarteriolar lymphoid sheath(Fig. 6). There was a moderate decrease in the numbers ofCD4� T lymphocytes in the marginal zones of the JR-CSF/hu-cycT1 mice from those for hu-cycT1 mouse littermates. TheCD4� lymphocyte populations in the spleens, lymph nodes,and thymuses of JR-CSF/hu-cycT1 mouse (n � 3 mice), JR-CSF mouse (n � 3 mice), and hu-cycT1 mouse (n � 3 mice)littermates were measured by flow cytometry. The fraction ofCD4� lymphocytes (17.8% � 0.3%) in the spleens of theJR-CSF/hu-cycT1 mice was significantly less (P 0.03) thanthose for JR-CSF mice (26.9% � 3.5%) and hu-cycT1 mice

(21.7% � 1.4%). Similarly, the fraction of CD4� lymphocytes(41.9% � 0.3%) in the lymph nodes of the JR-CSF/hu-cycT1mice was significantly less (P 0.037) than those for JR-CSFmice (59.3% � 3.5%) and hu-cycT1 mice (56.5% � 6.4%). Thepercentages of doubly positive and singly positive thymocytesin the thymuses from JR-CSF/hu-cycT1 mice were not signif-icantly different from those found in thymuses from hu-cycT1mice and JR-CSF mice. Taken together, these findings indicatethat expression of the hu-cycT1 gene coupled with carriage ofthe JR-CSF provirus results in the selective depletion of theCD4� lymphocyte population in the peripheral blood and sec-ondary lymphoid tissues of mice.

Expression of the hu-cycT1 transgene increases in vivoHIV-1 production in JR-CSF mice treated with GM-CSF. Wedemonstrated above that hu-cycT1 transgene expression in-creased in vitro HIV-1 production by GM-CSF-treated JR-CSF mouse myeloid cells. We next examined the in vivo effectof rescuing Tat function by transgenic expression of humancyclin T1 by comparing in vivo HIV-1 production by JR-CSFmice to that of JR-CSF/hu-cycT1 mice after chronic treatmentwith GM-CSF. To provide the mice with a continuous sourceof GM-CSF, the mice were subcutaneously injected with B16-F10-GM-CSF, a melanoma cell line stably transfected with aGM-CSF expression vector that forms a subcutaneous tumorthat continuously secretes GM-CSF. Two weeks after injectionwith the B16-F10-GM-CSF cells, there was no significant dif-ference (P 0.31) between the GM-CSF serum level of in-jected JR-CSF mice (mean, 245 � 116 pg/ml) and that ofJR-CSF/hu-cycT1 mice (mean, 306 � 10 pg/ml). In vivo HIV-1production induced by GM-CSF treatment was determined byharvesting bone marrow cells and spleens from these treatedmice and measuring their p24 antigen content levels. After invivo stimulation with GM-CSF, JR-CSF/hu-cycT1 mouse bonemarrow cells contained levels of p24 antigen 7-fold higher thanthose from JR-CSF littermate bone marrow cells (P � 0.004)(Fig. 7A), and JR-CSF/hu-cycT1 splenocytes contained levelsof p24 antigen 18-fold higher than those from JR-CSF litter-mate splenocytes (P � 0.047) (Fig. 7B). Thus, the rescue of Tatfunction in the JR-CSF mice by transgenic expression of hu-man cyclin T1 was associated with a significant increase in thein vivo HIV-1 production induced by GM-CSF.

Myeloid cells from JR-CSF/hu-cycT1 mice display increasedchemokine production after lipopolysaccharide (LPS) stimu-lation. The C-C chemokine family members, including MIP-1, MIP-1�, MCP-1, and RANTES, induce migration of acti-vated T lymphocytes and monocytes and stimulate themobilization of inflammatory cells to injured or infected loca-tions (3, 66). HIV-1 infection of monocytes may induce che-mokine production that facilitates the spread of infection byinappropriate induction of migration of T lymphocytes andinflammatory cells to the infected cells (62). We had previouslydemonstrated that MCP-1 gene expression in JR-CSF mousemicroglia and brains was more responsive to in vitro and invivo stimulation with LPS than that in microglia and brainsfrom control mice (64). Therefore, we examined whether ex-pression of the integrated HIV-1 provirus also affects chemo-kine production by stimulated JR-CSF bone marrow-derivedmouse myeloid cells and whether Tat-mediated transactivationcontributes to dysregulation of the chemokine response. Afterbone marrow-derived myeloid cells from JR-CSF mice and

FIG. 5. Depletion of CD4� T lymphocytes in the peripheral bloodof JR-CSF/hu-cycT1 mice. CD4� and CD8� T lymphocytes in theperipheral 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) weremeasured by flow cytometry. The mean percentages � SEM of CD4�

and CD8� T lymphocytes in the peripheral blood of the indicated miceare shown.


control littermates were treated with GM-CSF alone or withLPS, chemokine gene expression was evaluated by an RNaseprotection assay (Fig. 8A). GM-CSF- and LPS-stimulated JR-CSF myeloid cells produced levels of MCP-1 mRNA (Fig. 8B)over 2-fold higher and levels of MCP-1 protein (Fig. 8C) over2.4-fold higher than those of GM-CSF- and LPS-stimulatedwild-type mouse myeloid cells; they also produced 1.5-fold-higher levels of RANTES protein (Fig. 8D). We next examinedwhether the chemokine production by stimulated JR-CSFmouse myeloid cells would be further dysregulated by confer-ring support for Tat-mediated trans-activation by expression ofthe human cyclin T1 transgene (Fig. 9A). GM-CSF- and LPS-stimulated JR-CSF/hu-cycT1 myeloid cells produced levels ofMCP-1 mRNA (Fig. 9B) and of MCP-1 protein (Fig. 9C) over2-fold higher than those produced by GM-CSF and LPS-stim-ulated JR-CSF littermate myeloid cells and also produced min-imally higher levels of RANTES production (Fig. 9D). LPS-induced chemokine gene expression by monocytes from micetransgenic for human cyclin T1 alone was similar to that ob-

served for wild-type mice (data not shown). Thus, HIV-1 pro-viral expression in myeloid cells is associated with increasedproduction of MCP-1 in response to LPS stimulation that isfurther increased by the rescue of Tat function through expres-sion of the hu-cycT1 transgene.

DISCUSSION

Although the cyclin T1 subunit of the human P-TEFb com-plex is the critical factor limiting Tat-mediated transactivationin vitro in mouse cells (7, 23, 65), rodent cells engineered tosupport HIV-1 replication by stable expression of human CD4,CCR5, and cyclin T1 produced levels of HIV-1 much lowerthan those produced by human cells (6, 41). The continuedseverely compromised HIV-1 replication in these rodent cellsdespite expression of human cyclin T1 and the rescue of Tatfunction was attributed to the presence of other postintegra-tion blocks in HIV-1 replication, including compromise of theRev-mediated transport of unspliced mRNA encoding HIV-1

FIG. 6. Immunohistology of spleens from hu-cycT1 mice, JR-CSF mice, and JR-CSF/hu-cycT1 mice. The populations of CD4� lymphocytesand B cells in spleens from hu-cycT1 mice, JR-CSF mice, and JR-CSF/hu-cycT1 mice were visualized by immunofluorescence after staining withantibodies to mouse CD19 (left panels) and CD4 (right panels) by use of a Leica AOBS laser scanning confocal microscope system. The picturesshown are representative of three independent experiments.


structural proteins (6) and the inability of mouse cells to sup-port virion assembly (6, 41). The data presented in this paperextend the findings of previous in vitro studies with rodent celllines by examining the in vivo effect of transgenic expression ofhuman cyclin T1 on the rescue of Tat function and its effect onHIV-1 replication in primary murine CD4� T lymphocytes andmyeloid cells. Expression of the human cyclin T1 transgeneconferred to primary hu-cycT1 mouse monocytes/macrophagesand CD4� lymphocytes the capacity to support Tat-mediatedtransactivation of the HIV LTR. This was demonstrated by thefact that the levels of luciferase produced by hu-cycT1 primarymouse monocytes/macrophages and CD4� lymphocytes afterinfection with a VSV-G-pseudotyped HIV carrying a luciferasereporter were severalfold higher than those produced by equiv-alent cells from wild-type littermates. Furthermore, the rescueof Tat function by the hu-cycT1 transgene significantly in-creased HIV-1 production by stimulated JR-CSF mouse cells,particularly by GM-CSF-stimulated myeloid cells, indicatingthat the other HIV-1 replication blocks reported in mousefibroblast cells lines (6, 41) are less pronounced in primarymouse leukocytes. We and other investigators have reportedthat a lack of human cyclin T1 does not preclude HIV trans-genic mouse cells from producing HIV-1. For example,Doherty et al. reported that transgenic mice carrying between20 and 60 copies of the provirus encoding an X4 isolate, NL4.3,produced HIV-1 after infection with Mycobacterium avium

predominantly from monocyte/macrophage lineage cells (14).Production of HIV-1 in a Tat-independent manner by cellsfrom these transgenic mice may result from the additive effectof transcription of the many copies of the HIV provirus carriedby each transgenic mouse monocyte/macrophage. The JR-CSFmice used in the current study are transgenic for a provirusencoding the R5 isolate, HIV-1JR-CSF, and contained from twoto four copies of the provirus (8). We recently reported thatthe combination of GM-CSF and LPS stimulated JR-CSFmouse bone marrow-derived monocytes to produce levels ofp24 antigen (1,595 � 3 pg/ml) markedly higher than thoseproduced by stimulation with either GM-CSF (89 � 10 pg/ml)or LPS (44 � 1 pg/ml) alone (47). We attributed the high levelof HIV-1 production by monocytes from these mice carryinglow numbers of integrated provirus and lacking human cyclinT1 to the synergistic activation by GM-CSF and LPS of Sp1, atranscription factor reported to robustly activate the HIV-1LTR in the absence of Tat or TAR (69). These and othermechanisms may permit the production of HIV-1 by primarymouse monocyte/macrophage cells that are unable to supportTat function. The data in this paper extend those findings bydemonstrating that stimulated JR-CSF mouse CD4� T lym-phocytes and myeloid cells able to support Tat-mediated trans-activation by the expression of human cyclin T1 produce levelsof HIV-1 about 50-fold higher than those produced by JR-CSFmouse CD4� T lymphocytes and myeloid cells. The level ofHIV-1 produced by stimulated monocytes/macrophages fromJR-CSF/hu-cycT1 mice was about eightfold higher than thelevel of HIV-1 produced by stimulated T lymphocytes. In ourJR-CSF transgenic mice, production of HIV-1 by stimulated Tlymphocytes decreased in subsequent generations from thehigher levels first measured in the F1 generation (8) to thelower levels described here, possibly due to a stabilization ofthe JR-CSF transgene during breeding. Doherty et al. alsoreported for their HIV transgenic mice that HIV productionby the T lymphocytes was markedly lower than HIV produc-tion from the monocyte/macrophage lineage cells (14).

Rats exhibit a similar phenotype. After rats transgenic forthe expression of human CD4/CCR5 were infected withHIV-1, they displayed infection of T lymphocytes that wasextremely limited compared to infection of monocytes; thisextremely limited level of infection was ascribed to a T-lym-phocyte-specific posttranscriptional block to HIV-1 replication(34, 35). Because the HIV-1 provirus is integrated in the JR-CSF mouse cells, the decreased HIV-1 production by JR-CSFmouse T lymphocytes is not attributable to the postentry, pre-integration block recently described for murine T lymphocytesthat is related to the reduced efficiency of reverse transcriptionand the transfer of the preintegration complex into the nucleus(4). These results, combined with those of other groups, indi-cate that in addition to having restricted Tat function, mouseand rat T lymphocytes, like mouse 3T3 fibroblasts, harboranother block compromising the postintegration replication ofHIV-1 that can be bypassed by mouse myeloid lineage cells.This rodent-specific block to replication may be due to alteredRNA processing in mouse 3T3 cells, because the murine ho-mologue of the human splicing inhibitor, p32, insufficientlysuppresses mRNA splicing, resulting in extensive splicing ofthe 9-kb HIV mRNA transcript required for synthesis of struc-tural and enzymatic proteins (70). This posttranscriptional

FIG. 7. JR-CSF mice expressing human cyclin T1 display increasedHIV-1 production after continuous in vivo stimulation with GM-CSF.JR-CSF mice (n � 4) or JR-CSF/hu-cycT1 mice (n � 4) were im-planted with B16-F10-GM-CSF melanoma cells. Two weeks later,GM-CSF levels in the serum were measured by ELISA, and thespleens and bone marrow cells were harvested. HIV-1 production wasdetermined by measuring the HIV p24 antigen content of the lysatesfrom (A) bone marrow cells and (B) spleens and results are presentedas the mean � SEM of p24 antigen content/107 cells.


block can be overcome by providing mouse 3T3 cells withhuman p32, a more potent inhibitor of mRNA splicing thanmouse p32, which increases levels of the unspliced 9-kb HIVmRNA transcript and thereby markedly increases HIV-1 pro-duction by the 3T3 cells (70). Monocytes may not be affectedby this posttranscriptional block because their splicing machin-ery may differ from that of T lymphocytes and 3T3 cells due toexpression of lower levels of splicing factors or higher levels ofalternative splicing inhibitors. These differences may cause

less-extensive splicing of the 9-kb HIV mRNA transcript inmonocytes than in T lymphocytes and consequently result inthe production of higher levels of HIV-1 by monocytes. Thedegree of posttranscriptional blocking displayed by T lympho-cytes, 3T3 cells, and monocytes may also be related to theutilization of different mechanisms for HIV-1 assembly bythese different cell lineages (60). The primary location forHIV-1 assembly in T lymphocytes and 3T3 cells is the plasmamembrane, where a block to the assembly of HIV-1 may exist

FIG. 8. Effect of JR-CSF provirus expression on chemokine gene expression and production by stimulated mouse myeloid cells. Bonemarrow-derived myeloid cells from JR-CSF mice and control mice were cultured in 24-well plates (2 � 106 cells/well) with GM-CSF and wereeither stimulated with LPS or not stimulated. After 24 h, the cells were harvested, and RNA was extracted from the myeloid cells and analyzedfor chemokine gene expression by use of an RNase protection assay. (A) An autoradiograph of an RNase protection assay of RNA extracted frommyeloid 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-1and (D) RANTES in an aliquot of culture supernatant were measured by ELISA. The ELISA data shown represent the mean levels of MCP-1and RANTES production in four independent experiments using myeloid cells isolated from different mice, with statistical significance indicated.


in mouse cells (41). In contrast, the major location of HIV-1assembly in monocytes is in the late endosomal compartment,particularly the major histocompatibility class II compartment(50, 51, 53). Assembly in this compartment may utilize a mech-anism different from that occurring at the plasma membranethat permits HIV-1 in myeloid cells to circumvent the blockthat prevents the efficient assembly on the plasma membraneof mouse cells. We are currently examining these possibilities.

Nevertheless, stimulated CD4� lymphocytes from the JR-CSF/hu-cycT1 mice produced moderate levels of HIV-1 and levelsof HIV-1 significantly higher than those produced by CD4�

lymphocytes from the JR-CSF mice.A limitation of HIV transgenic mouse models is that in

contrast to humans, where HIV-1 replication occurs predom-inantly in CD4-expressing cells, in the HIV transgenic mice theprovirus is integrated in every cell and can be expressed by any

FIG. 9. Effect of human cyclin T1 expression on chemokine gene expression in stimulated JR-CSF mouse myeloid cells. Bone marrow-derivedmyeloid 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 eitherstimulated with LPS or not stimulated. After 24 h, the cells were harvested, and RNA was extracted from the myeloid cells and analyzed forchemokine gene expression by an RNase protection assay. (A) A representative autoradiograph of an RNase protection assay of RNA extractedfrom myeloid cells from wild-type or JR-CSF mice from three independent experiments and (B) the mean values of densitometric analysis ofautoradiographs from three separate experiments. After 72 h of stimulation, the levels of (C) MCP-1 and (D) RANTES in an aliquot of culturesupernatant were measured by ELISA. The ELISA data shown represent the mean levels of MCP-1 and RANTES production in four independentexperiments using myeloid cells isolated from different mice, with statistical significance indicated.


cell that supports HIV LTR transcription, including many celllineages normally not infected by HIV. Consequently, HIVtransgenic mice may not recapitulate many aspects of thepathophysiology of HIV infection. We hypothesized that ex-pression of a human cyclin T1 transgene would markedly in-crease HIV-1 replication in the HIV transgenic mouse cells byenabling Tat-mediated transcriptional activation. To selec-tively increase HIV-1 production by CD4-expressing cells inthe JR-CSF mice and increase the relevance of the JR-CSFmouse model to human disease, we focused expression of thehuman cyclin T1 transgene to CD4-expressing cells by using ahu-cycT1 transgene under the control of a CD4 promoter thattargets expression to CD4 T lymphocytes, monocytes, and den-dritic cells (34). Of great interest was the observation thatCD4� T lymphocytes and not CD8� T lymphocytes were de-pleted in the peripheral blood of the JR-CSF/hu-cycT1 mice,while normal levels of CD4� and CD8� T lymphocytes werepresent in the peripheral blood of JR-CSF mice and hu-cycT1mice. The extent of CD4� T lymphocyte depletion correlatedwith mouse age, with an inverted CD4/CD8 ratio (mean, 0.57� 0.09) present in the peripheral blood of all four JR-CSF/hu-cycT1 mice over 1 year of age that was in contrast to thenormal CD4/CD8 ratio seen for the four JR-CSF mice (mean,3.07 � 0.21) examined. While the JR-CSF provirus in thetransgenic mice is equally expressed by the CD4� lymphocytesand the CD8� lymphocytes, the hu-cycT1 transgene regulatedby the CD4 promoter is expressed in CD4� lymphocytes andnot in CD8� lymphocytes. It is likely that the selective deple-tion of the CD4� lymphocytes is a consequence of the in-creased production of HIV-1 by the CD4� T cells that expresshuman cyclin T1 and resembles the natural course of HIV-1infection in humans. An alternative approach that targetedHIV expression to CD4 cells in transgenic mice and that uti-lized an HIV transgene regulated by a CD4-specific promoterwas described previously. Transgenic mice were generated byuse of a construct consisting of the CD4C promoter fused to an8.8-kbp fragment of the HIV-1 pNL4-3 clone, their CD4 cellsexpressed HIV proteins, and the mice developed an AIDS-likedisease with symptoms including muscle wasting, severe atro-phy of and fibrosis in lymphoid organs, and nephritis andpneumonitis mediated by Nef expression (27, 28). This dra-matic phenotype was not seen in our transgenic mice. This maybe due to the activity of the CD4C promoter, which was muchstronger than that of the HIV LTR regulating the HIV trans-gene in our transgenic mice, resulting in the production ofhigher levels of HIV proteins deleterious to tissues in theirtransgenic mice. However, although the phenotype of the miceis dramatic, regulation of the HIV proviral transgene by theCD4C promoter is different from control by the endogenousLTR and precludes the use of these mice to investigate the invivo effect of factors that activate the HIV LTR on HIV-1transcription and viral production. In contrast, because HIVproduction in the JR-CSF/hu-cycT1 mice is regulated by theendogenous HIV LTR transactivated by Tat in a manner com-parable to that observed in HIV-1-infected individuals, theJR-CSF/hu-cycT1 mice should be a useful model to study invivo regulation of HIV-1 transcription by infected cells.

Chemokines produced by monocytes play an important rolein influencing HIV-1 replication, spread of infection, andpathogenesis of disease (17). Localized production of MCP-1

is sufficient to induce the directed migration of monocytes intoorgans in vivo, as evidenced by the organ-specific influx ofmonocytes in mice that carry an MCP-1 transgene controlledby an organ-specific promoter (22, 26). In the current study, wedemonstrated that after GM-CSF/LPS stimulation, myeloidcells from JR-CSF mice produced levels of MCP-1 that were2.4-fold higher than those produced by myeloid cells fromwild-type mice, and GM-CSF/LPS-stimulated myeloid cellsfrom JR-CSF/hu-cycT1 mice produced levels of MCP-1 thatwere 2.1-fold higher than those produced by stimulated JR-CSF mouse myeloid cells. These results indicated that carriageof the JR-CSF provirus by mouse myeloid cells is associatedwith increased MCP-1 production that is further augmentedwhen associated with rescued Tat function mediated by trans-genic expression of human cyclin T1. The 4.7-fold enhance-ment of MCP-1 production by stimulated JR-CSF/hu-cycT1mouse myeloid cells over the levels produced by stimulatedwild-type mouse myeloid cells is comparable to the two- tosevenfold enhancement of MCP-1 production reported afterinfection of human monocyte-derived macrophages withHIV-1 (44). Because Tat expression has been reported to in-duce MCP-1 production by endothelial cells (48), astrocytes(67), and dendritic cells (31), it is possible that expression ofhu-cycT1 facilitates this effect of Tat. MCP-1 is a chemokinethat plays a critical role in initiating inflammation by inducingthe migration of monocytes, memory T lymphocytes, and NKcells (61, 62). Because MCP-1 increases the production ofHIV-1 by infected cells (63), increased production of MCP-1by HIV-1-infected monocytes may amplify HIV-1 dissemina-tion by directly increasing the production of HIV-1 by infectedcells. Furthermore, increased MCP-1 production may also aug-ment the spread of HIV-1 infection by recruiting susceptiblecells, including CD4� T lymphocytes, to sites of active viralreplication, where they can become activated or rendered per-missive to infection and infected with HIV-1 as described pre-viously for MIP-1 and MIP-1� (61, 62). The increased pro-duction of chemokines by HIV-1-infected macrophages andmicroglia residing in the central nervous system may contributeto the immunopathogenesis of HIV-mediated encephalitis byinducing the transmigration of HIV-1-infected and/or unin-fected monocytes across the blood-brain barrier from the sys-temic circulation into the brain and inducing the developmentof inflammatory foci that lead to neuronal damage (33, 68).The physiological relevance of this proposed mechanism issupported by the detection of MCP-1 in the brains and cere-brospinal fluid of patients with HIV-1-associated dementia(11) and the association between elevated levels of MCP-1 incerebrospinal fluid and the development of encephalitis insimian immunodeficiency virus-infected macaques (72).

Taken together, our results demonstrate that expression ofhuman cyclin T1 markedly increases in vivo HIV-1 productionin mice and that blocks preventing efficient HIV-1 replicationin mice can be overcome by using transgenic technology toprovide human factors. This approach will be used to confirmthe role of other factors shown to compromise HIV-1 replica-tion in mouse cells identified by in vitro studies, such as hp32(70), and ultimately used to develop a mouse model displayingefficient HIV-1 replication. Differences between the biologicalbehaviors of murine and human lymphoid and myeloid cellsmay compromise the applicability of studies using murine


models for understanding the mechanism by which HIV-1causes disease in infected patients. Nevertheless, because thelymphocytes and myeloid cells present in this transgenic mousecarry the provirus of a primary R5 HIV-1 isolate under thecontrol of the endogenous HIV-1 LTR and HIV-1 replicationis specifically increased in CD4-expressing cells by targetedexpression of the hu-cycT1 transgene, it is likely that the in vivobehavior of these cells may recapitulate many aspects of HIV-1replication and cellular behavior occurring in HIV-1-infectedindividuals. In particular, we are currently investigatingwhether the selected depletion of CD4� T lymphocytes in theJR-CSF/hu-cycT1 mice is associated with the development ofan immunodeficiency that resembles that seen in AIDS. There-fore, the JR-CSF/hu-cycT1 mice should provide a new systemfor investigating the pathogenesis of various aspects of HIV-1-mediated disease and the efficacy of therapeutic interven-tions designed to prevent manifestations of HIV-1-induceddisease.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health (Na-tional Institute of Neurological Disorders and Stroke NS39201, Na-tional Institute of Allergy and Infectious Diseases Al48466, and theCenter for AIDS Research AI51519). T.S. was supported by NIAIDAIDS Training Grant T32-AI0732.

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cd4-speciﬁc transgenic expression of human cyclin … of a murine cd4 promoter ... jr-csf provirus...

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