Department of Medicine, Division of Hematology and Oncology, University of California, San Francisco, CA 94143, USA
Correspondence
Jay A. Levy
jalevy{at}itsa.ucsf.edu
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ABSTRACT |
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Present address: Division of Infectious Diseases, Maxygen Inc., 200 Penobscot Drive, Redwood City, CA 94063, USA.
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INTRODUCTION |
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NSI and SI viruses differ in their use of chemokine coreceptors for entrance into host cells. NSI viruses utilize the -chemokine cell receptor 5 (CCR5) and are called R5 isolates, whereas SI viruses utilize the CXCR4 coreceptor (Berger, 1997
) and are called X4 isolates (Berkowitz et al., 1998
). Mutations in variable regions 2 and 3 (V2 and V3) of the viral gp120 envelope appear to correlate with syncytium induction in T-cell lines, chemokine coreceptor usage and the R5 and X4 tropisms in macrophages and T-cell lines, respectively (Cheng-Mayer et al., 1988
; Choe et al., 1996
; Distler et al., 1995
; Shioda et al., 1991
, 1992
). The mutations contained within the V3 region of the envelope impart a net (positive) charge that may facilitate a stronger interaction with the chemokine coreceptor CXCR4 (Berger, 1997
; Distler et al., 1995
; Korber et al., 1994
).
Although both R5 and X4 HIV strains can be transmitted, R5 viruses predominate in the infected person shortly after infection and during the asymptomatic phase (Cornelissen et al., 1995; Wade et al., 1998
; Zhu et al., 1993
). The reason for this dominance of R5 viruses is not known. Recent studies have indicated that dendritic cells are infected productively with R5 isolates to a greater extent than with X4 isolates (Vanham et al., 2000a
, b
) and may mediate HIV transmission through the mucosal lining (Meng et al., 2002
). However, other studies suggested that dendritic cells may transmit HIV-1 independently of viral coreceptor usage (Hladik et al., 1999
).
Previous studies in our laboratory showed that R5 viruses induce greater proliferation of CD4+ cells and CD71 expression than X4 viruses (Greco et al., 1999). The present studies were conducted to extend these findings and to further explore the effect of R5 and X4 HIV infection on proliferation and activation of CD4+ T lymphocytes. Additional phenotypic markers that are important in T-cell activation were studied, including CD25 [interleukin 2 (IL2)
-chain receptor, a lymphocyte marker of activation and T-cell memory-cell marker], CD69 (an early indicator of lymphocyte activation), CD71 (transferrin receptor, a later indicator of lymphocyte activation and proliferation) and HLA-DR (a class II antigen and late marker of lymphocyte activation and proliferation). Together with [3H]thymidine incorporation and Ki67, which are indicators of DNA synthesis and T-cell proliferation, these activation markers were used to characterize the changes in T cells in response to HIV-1 infection in vitro.
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METHODS |
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Molecular characterization of viruses.
Viral RNA was isolated from frozen aliquots of a bulk virus culture by using TRIzol/RNeasy according to the manufacturer's instructions (Qiagen). RT-PCR was used to amplify the envelope region encoding the V3 region of HIV-1 by using the primers CCTCAGCCATTACACAGGAATGTCCAAAG for the forward reaction and CCTTGGTGGGTGCTACTCCTAATGGTTCA for the reverse direction [a gift from Dr Eric Delwart, University of California, San Francisco (UCSF), CA, USA]. Cycling was performed according to the following protocol: 96 °C for 90 s; 35 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min; 72 °C for 7 min; 4 °C thereafter. DNA sequencing was performed by the Biomolecular Resource Center, UCSF, and chromatograms were analysed by using ABI Prism (Applied Biosystems) and Macvector (Macworld) software programs.
Virus cultures.
PBMCs were recovered from whole blood by gradient centrifugation using Ficoll-Hypaque (Sigma) (Castro et al., 1988). CD4+ T cells were then isolated immediately by using CD4+ immunomagnetic beads, according to the manufacturer's instructions (Dynal) (Mackewicz et al., 1991
). The purified CD4+ cells were treated with 10 µg polybrene ml1 (hexamethabromide; Sigma) for 30 min prior to inoculation with 100 TCID50 virus per 106 CD4+ cells (an m.o.i. of 104). We found that purified CD4+ cells showed the most substantial differences between R5 and X4 viruses and the effect of the viruses on host-cell activation and proliferation. In contrast, results using PBMCs were difficult to interpret because the percentage changes were smaller and not significant.
Following 1 h incubation, the CD4+ cells were washed twice and resuspended at 2x106 cells ml1 in RPMI 1640 medium supplemented with 10 % heat-inactivated (56 °C, 30 min) fetal bovine serum, 100 U recombinant human IL2 ml1 (Boehringer Mannheim), 2 mM L-glutamine, 100 U penicilllin ml1 and 100 µg streptomycin ml1. Virus replication was monitored by using the RT assay (Hoffman et al., 1985). From many years of experience using this RT procedure, we have found that values of <5000 c.p.m. are negative (Mackewicz et al., 1995
). Uninfected and unstimulated CD4+ T cells were used as negative controls; phytohaemagglutinin (3 µg ml1) was used for positive controls of cellular activation. Cell cultures were passaged in fresh RPMI 1640 medium every 48 h and cell viability was determined by trypan blue exclusion. Cell cultures with <70 % cell viability were discarded. The control and HIV-infected cell cultures were maintained for up to 2 weeks and CD4+ cells from the same donor were used for each parallel experiment, using subject-matched or laboratory strains of R5 and X4 viruses.
Cell proliferation.
To determine the degree of cellular proliferation, CD4+ cells from control and HIV-infected cultures were resuspended in complete medium that did not contain IL2 and plated in triplicate at 105 cells per well in 96-well plates. [3H]Thymidine [10 µCi (370 kBq); NEN Sciences] was then added to each well and the tissue-culture plates were incubated for 8 h. The plates were then harvested and the cells were obtained by using an MBI cell harvester (Skatron/Molecular Devices) and the incorporation of [3H]thymidine was measured on a -scintillation counter (LKB 1205; Wallac/Perkin-Elmer). [3H]Thymidine incorporation (c.p.m.x1000) was determined for each sample.
Flow cytometry.
To characterize the differential expression of proliferation and activation markers on HIV-infected CD4+ T cells, two-colour flow cytometry was employed by using a FACSort with CellQuest software (BD Biosciences). Briefly, a monoclonal antibody (mAb) specific for intracellular Ki67 (Beckman Coulter) was used to measure cellular proliferation following permeabilization and fixation (BioErgonomics). Permea-Sure was used as a positive control for cellular permeabilization and intracellular staining (Biosource International). Cell-surface antigens were characterized by using phycoerythrin-conjugated mAbs specific for CD25, CD69, CD71 and HLA-DR (Becton Dickinson/Pharmingen). Acquisition of events was selected only on viable cells. To control for HIV-induced CD4 downmodulation, the proportion of cells expressing the cell-surface activation markers was defined as percentage positive in both CD4+ and CD4 cell populations. The percentage of positive CD4+ cells and mean fluorescence intensity were used to enumerate the mean number of molecules expressed in a given cell population.
Statistical analysis.
The data from the cellular proliferation were analysed by using Student's t-test. The results of the immunophenotypic markers were analysed by using non-parametric methods, as the distribution of the data was non-Gaussian; the significance of differences between these two groups was calculated with the MannWhitney U-test by using the StatView software program (Abacus Concepts).
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RESULTS |
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To determine whether this CD4 downregulation was a result of direct HIV infection, we determined the levels of HIV p24 expression within the CD4+ and CD4-downmodulated populations by using intracellular staining and fluorescence-associated cell-sorting analysis. After 7 days in culture, approximately 50 % of the CD4+ cell population showed infection by R5 and X4 HIV isolates and 95 % of the cells with CD4 downmodulation were infected (Fig. 4). These results, observed in several experiments, did not show any substantial difference in the percentage of CD4+ cells infected with R5 and X4 viruses.
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DISCUSSION |
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Several studies indicate that activation of memory CD4+ lymphocytes that co-express CCR5 may help to increase HIV replication and facilitate a predominance of HIV bearing an R5 phenotype (Annunziato et al., 2000; Greco et al., 1999
; Kreisberg et al., 2001
; Vicenzi et al., 1999
). R5 virus preferentially infects CD62L CD4+ lymphocytes bearing CD25 and the memory marker CD45RO and can remain latent until the infected cell is stimulated with IL2 or tumour necrosis factor alpha (Blaak et al., 2000
; Gondois-Rey et al., 2002
; Poli & Fauci, 1993
). In addition, the triggering of cellular activation by binding to the CCR5 coreceptor by R5 viruses may enhance kinase activity of secondary messengers that increase transcription-factor binding to the HIV-1 promoter in the long terminal-repeat region (Cicala et al., 1999
; Popik & Pitha, 2000a
, b
). Moreover, binding of inactivated X4 HIV-1 causes increased apoptosis and upregulation of FasL/Fas, but not CD25 or CD69 (Esser et al., 2001
; Lawson et al., 2004
). Thus, the inherent affinity of R5 HIV-1 for resting memory CD4+ lymphocytes that co-express CCR5 may be a mechanism for enhancing R5 viral fitness and facilitating depletion of the memory subset of CD4+ lymphocytes. Our findings suggest that R5 virus isolates may persist longer in culture by enabling the infected CD4+ lymphocyte to maintain a higher threshold of activation and proliferation that promotes HIV-1 replication.
Activation of CD4+ lymphocytes by R5 viruses may also help to explain different effects of antiviral drugs on this lymphocyte subpopulation. For example, zidovudine appears to be preferentially phosphorylated into its antiviral form in activated lymphocytes (Gao et al., 1993, 1994
; Shirasaka et al., 1995
) and is most effective against R5 variants (Koot et al., 1993
; van't Wout et al., 1996
). In contrast, didanosine is equally effective against both R5 and X4 variants and is biologically active in both resting and activated CD4+ T lymphocytes (van't Wout et al., 1997
).
In summary, we have found that infection of CD4+ T lymphocytes by R5 and X4 viruses is associated with decreased cellular proliferation and cell-surface expression of phenotypic markers of activation. Importantly, diminished cellular proliferation and activation were more substantial with CD4+ cells infected with X4 virus isolates than with R5 virus isolates. Therefore, long-term growth of X4 viruses in vitro is reduced. These differences in cellular proliferation and activation may be initiated by signal-transduction pathways that are activated via chemokine coreceptor binding (e.g. CCR5) by R5 virus isolates. In addition, other domains of the HIV-1 envelope may contribute to these differential effects of R5 and X4 viral subtypes. Some of these characteristics may result in the survival of R5-infected CD4+ T lymphocytes, to produce more R5 viral progeny than X4-infected CD4+ T lymphocytes. Our findings could also provide some explanation for the preferred replication of R5 viruses after virus transmission.
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ACKNOWLEDGEMENTS |
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Received 7 October 2004;
accepted 5 January 2005.
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