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Journal of Virology, December 2007, p. 13938-13942, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.01745-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Addition of Deoxynucleosides Enhances Human Immunodeficiency Virus Type 1 Integration and 2LTR Formation in Resting CD4+ T Cells{triangledown}

Gabriela Plesa, Jihong Dai, Cliff Baytop, James L. Riley, Carl H. June, and Una O'Doherty*

Department of Pathology and Laboratory Medicine, Division of Transfusion Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received 9 August 2007/ Accepted 27 September 2007


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ABSTRACT
 
Resting CD4+ T cells restrict human immunodeficiency virus (HIV) infection at a step prior to integration. Despite this restriction, we showed previously that HIV integration occurs in resting CD4+ T cells in vitro, albeit at lower levels than in activated CD4+ T cells. Here we show that addition of deoxynucleosides enhanced integration and 2LTR formation in resting CD4+ T cells but that the kinetics were still significantly delayed compared to those of activated T cells. Thus, deoxynucleoside addition partially overcomes the restriction to HIV infection in resting CD4+ T cells.


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TEXT
 
Resting CD4+ T cells (rCD4 cells) appear to be a major target of human immunodeficiency virus (HIV) and simian immunodeficiency virus infections in vivo, especially in the gut during acute infection (12, 20, 42, 43, 47, 48), yet in vitro rCD4 cells resist HIV infection (8, 34, 35, 45). Understanding this paradox is important to HIV pathogenesis. Early in vitro studies showed that reverse transcription and possibly other postentry steps are restricted in rCD4 cells (3, 35, 37, 45, 46). However, extended kinetic studies showed that reverse transcription is not completely blocked in resting T cells but rather occurs with slower kinetics than in activated cells (8, 29, 33, 39). The slower kinetics of reverse transcription in resting T cells can be partially overcome by inducing the cells to enter G1b (11, 18, 40), by knocking down putative restriction factors (8, 36) and by adding deoxynucleosides (dN) (17), which are present at lower levels in resting cells than in activated cells (9, 10, 24, 26, 32).

Our previous data (1, 38) and those of Vatakis et al. (41) suggest that, despite this restriction, HIV can integrate directly into rCD4 cells, although this occurs with slower kinetics and to lower levels in resting cells than in activated cells. Given that dN increase reverse transcription in resting T cells (17), we tested the effect of dN addition on HIV integration and 2LTR formation in resting T cells. Here we show that dN increase integration and 2LTR formation in rCD4 cells.

Two populations of CD4+ T cells were prepared from PBMCs. To study the restriction of HIV infection by rCD4 cells, we prepared rCD4 cells and activated CD4+ T cells (aCD4 cells) from peripheral blood mononuclear cells (PBMCs; Fig. 1A).


Figure 1
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  		FIG. 1. (A) Preparation of primary resting CD4+ T cells from PBMCs in two steps. First, PBMCs (obtained by leukapheresis of whole blood) were depleted of other lineages (CD8, -56, -14, -36, -19) by red blood cell rosette (Rosette Sep) technology to obtain ppCD4 cells. Second, ppCD4 cells were depleted of activated cells and {gamma}{delta} T cells by staining with antibodies against the activation markers CD69, CD25, HLA-DR, and the {gamma}{delta} T-cell receptor. The resulting cells were 97% rCD4 cells. Finally, aCD4 cells were prepared by stimulating ppCD4 cells with anti-CD3/CD28 beads for 3 days. The quadrant gates were set using isotype-stained lymphocytes so that 99% of cells were in the left lower quadrant. (B) Overall experimental approach. Two populations of cells (rCD4 and aCD4) were spinoculated with HIV and cultured with or without dN. At multiple time points, reverse transcription, integration, 2LTR circles, and activation were measured. After day 3 of culture, an integrase inhibitor was added and rCD4 cells were stimulated with anti-CD3/CD28 beads plus 100 U/ml interleukin-2 for 1 day, and intracellular Gag was measured to determine the level of productively infected cells.

Partially purified CD4+ T cells (ppCD4 cells) were prepared by negative selection (Rosette Sep; Stem Cell Technologies) for other cell lineages (CD8, CD16, CD19, CD36, and CD56) (Fig. 1A). rCD4 cells were prepared from the ppCD4 cells by a second negative selection using phycoerythrin-labeled antibodies for {gamma}{delta} T-cell receptor ({gamma}{delta}TCR) and activation markers (CD69, CD25, and HLA-DR) followed by anti-phycoerythrin magnetic beads (magnetically activated cell sorting; Miltenyi). The resulting cells were 99% pure rCD4 cells (Fig. 1A). Preactivated CD4+ T cells were prepared by stimulating ppCD4 cells with anti-CD3/28-coated beads (Dynal) for 3 days (5, 6). The aCD4 cells uniformly expressed CD25 and CD69 by day 3 (see Fig. 3).


Figure 3
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  		FIG. 3. dN did not upregulate activation markers on rCD4 cells. The top plots represent expression of activation markers for ppCD4 cells before and after activation with anti-CD3/28 beads for 3 days. The bottom plots represent rCD4 cells before and after spinoculation followed by 3 days in culture in the presence of dN. Cellular activation was assessed by measuring expression levels of CD25 (black line) and CD69 (shaded histogram). The dashed line denotes the isotype control. The results shown are representative of two independent experiments.

dN enhanced integration and 2LTR formation in rCD4 cells but not aCD4 cells. To probe the restriction to postentry steps in rCD4 cells, we compared the kinetics and levels of reverse transcription, integration, and 2LTR formation in CD4+ T cells in the presence and absence of 20 µM dN (Sigma). The cells were inoculated with HIV pNL4-3 (transfection supernatant) by spinoculation (28) and then cultured with or without dN in the presence of saquinavir (1.25 µM; Roche U.S. Pharmaceutical) to inhibit viral spread. Reverse transcription was assessed using primers that detect minus-strand strong stop (RU5) and second-strand transfer (SST) (38, 39). Integration (1, 27) and 2LTR formation (4) were assessed as described previously.

Consistent with prior work (17), the kinetics and level of short reverse transcripts (RU5) were not affected by addition of dN to rCD4 or aCD4 cells (Fig. 2A). SST occurred with accelerated kinetics in rCD4 cells in the presence of dN (Fig. 2B). Eightfold more SSTs were present at 24 h in the presence of dN. In addition, the difference in levels of SSTs in rCD4 versus aCD4 cells was greater than that for RU5 at early time points as previously shown by Korin and Zack (17), but this difference diminished with time in culture (8, 29, 33, 39, 41). dN increased the kinetics and peak level of integration in rCD4 cells (Fig. 2C). At 48 h after inoculation, there were ninefold more proviruses in the presence of dN. This difference diminished over time as the level of proviruses decreased in rCD4 cells plus dN (which is associated with cell loss) while the number of proviruses in rCD4 cells alone continued to increase over time in culture. The magnitude of the dN enhancement at peak levels ranged from 4- to 10-fold between experiments/donors. Figure 2D shows the average integration levels for four different experiments. dN also enhanced the fraction of reverse transcripts that integrated (Fig. 2E). To determine the fraction, we divided the peak level of integrated DNA by the peak level of SST and averaged the results of four independent experiments. To determine the dN effect on the other end products of reverse transcription, we measured the level of 2LTR circles. dN enhanced the kinetics and level of 2LTR formation (Fig. 2F). At 48 h postinoculation, 50-fold more 2LTR circles were detected in rCD4 cells with dN. Again, this difference diminished over time, as at 72 h, only twofold more 2LTRs were detected in the presence of dN.


Figure 2
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  		FIG. 2. dN increased the level and kinetics of HIV integration and 2LTR formation in rCD4 cells, but not in aCD4 cells. Cells were spinoculated with HIV and then cultured in the presence or absence of dN. DNA was prepared at the time points indicated on the x axis. The results in panels A to C and F are from one experiment but are representative of four independent experiments. Error bars represent standard deviations from kinetic PCR replicates. (A) dN did not affect the level or kinetics of short reverse transcript accumulation in rCD4 and aCD4 cells. Short reverse transcripts were measured using RU5-specific primers. (B) dN increased the kinetics of long reverse transcript accumulation in rCD4 cells but not in aCD4 cells. Long reverse transcripts were measured using primers that detect reverse transcripts that have completed SST. (C) dN increased the kinetics and peak level of integration in rCD4 cells but not aCD4 cells. The graph shows integration levels of the same donor that are represented in panels A, B, and F. Integration was measured using the Alu-gag primer pair. (D) The graph shows an average of integration levels from four different experiments/donors. Error bars represent standard errors. (E) dN increased the proportion of reverse transcripts that integrate in rCD4 cells but not in aCD4 cells. The percentage of integrated HIV DNA was determined by dividing the peak level of integrated DNA by the peak level of long reverse transcripts as previously described (38). Error bars represent standard errors resulting from four independent experiments. (F) dN increased the peak level and kinetics of 2LTR formation in rCD4 cells but not aCD4 cells. 2LTR circles were measured as described previously (4). The values are expressed in relative units. Error bars represent standard deviations of PCR replicates.

We find that dN enhancement occurs between 10 and 50 µM (not shown), consistent with results of Korin and Zack (17). Addition of deoxynucleotide triphosphates as well as dN enhanced the level of integration (not shown). The dN effect is decreased by 50% when they are added 24 h after inoculation (not shown), consistent with other studies that estimate a half-life of approximately 1 day for preintegration complexes in resting CD4+ T cells (49).

dN did not upregulate activation markers on rCD4 cells. To test whether dN-induced enhanced integration in rCD4 cells was due to cell activation, we measured the expression of CD69 and CD25 activation markers (38) on rCD4 cells cultured in the presence of dN. We found that pre- and posttransduction levels of these markers were identical, yet CD69 and CD25 were upregulated after cell stimulation with anti-CD3/28 beads (Fig. 3). Therefore, while the CD4+ T cells were capable of upregulating activation markers, dN did not induce upregulation. In addition, pretreatment of rCD4 cells with dN for 20 h before inoculation did not enhance integration (not shown), suggesting that the dN exposure did not make the cells more susceptible to HIV.

Integrated HIV DNA can be induced to produce HIV. To test whether the transduced CD4+ T cells could be induced to produce HIV, we followed the protocol in Fig. 1B. First, we spinoculated CD4 cells with HIV, cultured the cells for 3 days with or without dN in the presence of saquinavir, and measured integration. Second, we added the integrase inhibitor L870,810 (100 nM; Merck Research Laboratories) (14) to prevent additional integration. Third, we stimulated the cells with anti-CD3/28-coated beads plus 100 U/ml interleukin-2 for 1 day (6, 19, 38) and measured the percentage of cells producing HIV by staining for intracellular Gag (Fig. 4). With different donors, we find some variation in the percentage of proviruses (i.e., integrated HIV DNA) that could be induced to produce HIV, ranging from 15 to 100% (Table 1). The variation may be because the cells die more rapidly upon production and so only when all the cells produce HIV nearly simultaneously can we capture production from all cells that contain integrated DNA. Alternatively, in some experiments, cells could contain multiple copies of integrated DNA.


Figure 4
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  		FIG. 4. Integrated DNA can be induced to produce HIV. Cells were treated as described for Fig. 1B, and intracellular Gag was measured by flow cytometry. Cells were spinoculated with HIV and cultured for 3 days in the presence or absence of reverse transcriptase inhibitor zidovudine (AZT; 100 µg/ml; AIDS Repository) or dN. An integrase inhibitor was added, and the cells were stimulated with anti-CD3/28 beads for 1 day or not stimulated. Data are representative of five independent experiments.


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  		TABLE 1. Percentages of CD4+ T cells containing integrated DNA and producing Gag in the absence and presence of dN

In conclusion, our results show that dN enhance integration and 2LTR formation in rCD4 cells although the kinetics are still delayed compared to those for activated cells. This suggests that restriction of HIV infection in resting T cells may be due, in part, to limiting dN.

The exact mechanism(s) of dN-enhanced integration (and 2LTR formation) in resting T cells remains to be elucidated. dN-enhanced integration does not appear to be due to activation of CD4+ T cells because dN treatment did not upregulate activation markers (Fig. 3). Although some signaling through dN receptors may occur (16), it is unlikely to explain the enhancement since pretreatment with dN alone did not increase the cell's susceptibility to HIV transduction. dN have the potential to enhance three steps in the HIV replication cycle: completion of reverse transcription, nuclear import of preintegration complexes, and integration. As a higher proportion of reverse transcripts integrate in the presence of dN, it appears that nuclear import or integration is enhanced to a greater extent than reverse transcription. However, we measured reverse transcripts that have completed SST and not full-length reverse transcripts. In other words, dN may enhance completion of reverse transcription to a greater extent than they increase the level of reverse transcripts that have completed SST. Thus, all three mechanisms may play a role, but the fact that dN enhance 2LTR formation and integration suggests that completion of reverse transcription or nuclear import is enhanced by dN.

Although the integration level in dN-treated resting T cells is enhanced, we are not suggesting that restriction factors (8, 13, 21) do not play a role in HIV infection. The kinetics of reverse transcription, integration, and 2LTR formation are still slower and occur at lower levels in resting T cells than in activated T cells. This suggests that a restriction factor active in resting T cells but not in aCD4 cells (2, 7, 8) is partially overcome by the addition of dN. Trim5{alpha} seems unlikely to be the restriction factor, since the restriction of HIV in rCD4 cells is apparently not saturable, as viral binding, reverse transcription, and integration decrease with dose response at a wide range of virus dilutions (1). Given that APOBEC3G/3F inhibits reverse transcription (2, 15, 23, 30, 31, 44) and that small interfering RNA knockdown of APOBEC enhances the kinetics of reverse transcription in rCD4 cells (8), APOBEC3G/3F could be the restriction factor responsible for the slower kinetics in rCD4 cells.

Two recent studies further suggest that APOBEC3G/3F may be the factor responsible for the slower kinetics in rCD4 cells (22, 25). APOBEC3G/3F inhibits 2LTR formation and integration in addition to reverse transcription in cell lines (22, 25). We find that dN increase the kinetics and levels of 2LTR formation and integration (Fig. 2). Thus, APOBEC addition and dN addition seem to have opposite effects. We suggest that increased 2LTR formation and integration in the presence of dN (and absence of APOBEC) could be secondary to enhanced levels of complete reverse transcripts. In other words, if APOBEC inhibits late steps in reverse transcription, providing more dN substrate may increase the amount of final product (complete reverse transcripts) by mass action. The exact nature of the relationship between APOBEC's inhibition of reverse transcription and dN reversal of inhibition remains to be elucidated.


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ACKNOWLEDGMENTS
 
This work was supported in part by NIH grants R01AI058862 (U.O.) and R21AI64031-A1 (U.O.). Additional support came from Merck Research Laboratories.

We thank Mike Malim, Liz Colston, and Luis Agosto for critical reading of the manuscript. The integrase inhibitor was a generous gift from Merck Research Laboratories.


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FOOTNOTES
 
* Corresponding author. Mailing address: 3620 Hamilton Walk, 265 JMB, Philadelphia, PA 19104. Phone: (215) 573-7273. Fax: (215) 573-2348. E-mail: unao{at}mail.med.upenn.edu Back

{triangledown} Published ahead of print on 10 October 2007. Back


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Journal of Virology, December 2007, p. 13938-13942, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.01745-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Vatakis, D. N., Kim, S., Kim, N., Chow, S. A., Zack, J. A. (2009). Human Immunodeficiency Virus Integration Efficiency and Site Selection in Quiescent CD4+ T Cells. J. Virol. 83: 6222-6233 [Abstract] [Full Text]  
  • Dai, J., Agosto, L. M., Baytop, C., Yu, J. J., Pace, M. J., Liszewski, M. K., O'Doherty, U. (2009). Human Immunodeficiency Virus Integrates Directly into Naive Resting CD4+ T Cells but Enters Naive Cells Less Efficiently than Memory Cells. J. Virol. 83: 4528-4537 [Abstract] [Full Text]  
  • Vatakis, D. N., Nixon, C. C., Bristol, G., Zack, J. A. (2009). Differentially Stimulated CD4+ T Cells Display Altered Human Immunodeficiency Virus Infection Kinetics: Implications for the Efficacy of Antiviral Agents. J. Virol. 83: 3374-3378 [Abstract] [Full Text]  

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