Background: Memory T cells are preferentially infected and serve as a major viral reservoir.
Results: The cortical actin of memory and naive cells is distinct, and this difference affects cell susceptibility to HIV.
Conclusion: Cortical actin is an early determinant of cellular susceptibility to HIV for memory and naive T cells.
Significance: HIV-mediated actin dynamics are critical for HIV infection and pathogenesis.
Keywords: Actin, Cell Signaling, Chemotaxis, Cytoskeleton, Gene Regulation, HIV-1, Immunology, Microbiology, Signal Transduction, T Cell
Abstract
Human memory and naive CD4 T cells can mainly be identified by the reciprocal expression of the CD45RO or CD45RA isoforms. In HIV-1 infection, blood CD45RO memory CD4 T cells are preferentially infected and serve as a major viral reservoir. The molecular mechanism dictating this differential susceptibility to HIV-1 remains largely obscure. Here, we report that the different susceptibility of memory and naive T cells to HIV is not determined by restriction factors such as Apobec3G or BST2. However, we observed a phenotypic distinction between human CD45RO and CD45RA resting CD4 T cells in their cortical actin density and actin dynamics. CD45RO CD4 T cells possess a higher cortical actin density and can be distinguished as CD45RO+Actinhigh. In contrast, CD45RA T cells are phenotypically CD45RA+Actinlow. In addition, the cortical actin in CD45RO memory CD4 T cells is more dynamic and can respond to low dosages of chemotactic induction by SDF-1, whereas that of naive cells cannot, despite a similar level of the chemokine receptor CXCR4 present on both cells. We further demonstrate that this difference in the cortical actin contributes to their differential susceptibility to HIV-1; resting memory but not naive T cells are highly responsive to HIV-mediated actin dynamics that promote higher levels of viral entry and early DNA synthesis in resting memory CD4 T cells. Furthermore, transient induction of actin dynamics in resting naive T cells rescues HIV latent infection following CD3/CD28 stimulation. These results suggest a key role of chemotactic actin activity in facilitating HIV-1 latent infection of these T cell subsets.
Introduction
The persistence of viral reservoirs in HIV-infected patients precludes an effective cure to the disease (1, 2). One of the major viral reservoirs in the body is a small pool of latently infected CD45RO memory CD4 T cells (3, 4). In infected patients, CD45RO memory CD4 T cells are preferentially infected and harbor more integrated proviral DNA than CD45RA naive T cells (4–7). These findings are recapitulated by multiple in vitro studies showing that purified CD45RO memory CD4 T cells support higher levels of HIV-1 replication than CD45RA T cells (5, 7–10).
In the human immune system, the reciprocal expression of the CD45RO or CD45RA isoforms can largely identify the memory and naive resting CD4 T cell subsets (11–13). CD45RO and CD45RA T cells exhibit multiple phenotypic differences (13). CD45RO memory cells have a broader cytokine expression profile (14) and appear to be metabolically more active (15). In addition, CD45RO memory cells have a less stringent requirement for T cell activation, whereas CD45RA naive T cells require professional antigen-presenting cells such as dendritic cells for strong costimulation (16). Nevertheless, in HIV-1 infection, the higher susceptibility of memory CD4 T cells to the virus, as observed in cell culture conditions, seems to be intrinsic and not caused by T cell activation, cytokine stimulation, or transcriptional enhancement by factors such as NF-κB and AP-1 (8). This intrinsic difference was also not affected by mixing CD45RO memory and CD45RA naive T cells (8) nor by adding exogenous deoxynucleoside to overcome the metabolic limitation (17). Thus, the cellular and molecular mechanism dictating this differential susceptibility to HIV was not understood.
Besides the aforementioned T cell functionality, CD45RO memory and CD45RA naive T cells also differ significantly in their migratory behavior; memory T cells possess higher motility and chemotactic mobility than naive T cells (18, 19). T cell migration is mainly driven by cortical actin polymerization mediated by actin regulators such as Arp2/3 and cofilin, which are downstream effectors of chemokine receptor signaling (20). The cortical actin is also a major determinant of T cell susceptibility to viral pathogens such as HIV-1 (21–26). During HIV infection of resting CD4 T cells, the virus utilizes the envelope protein to interact with the chemokine coreceptor to trigger cofilin activation and actin dynamics, mimicking the chemotactic process to facilitate viral entry, DNA synthesis, and intracellular migration (22–24, 27). Given the difference in migratory potential between memory and naive T cells, we speculated that there may be a difference in their actin dynamics that could affect their susceptibility to HIV-1 infection. In this study, we report for the first time that there is a phenotypic distinction between human CD45RO and CD45RA resting CD4 T cells in their cortical actin density (CD45RO+Actinhigh versus CD45RA+Actinlow). In addition, the cortical actin in CD45RO memory CD4 T cells is more dynamic and can respond to low levels of chemotactic induction, whereas that of CD45RA naive cells cannot. This higher cortical actin activity predisposes memory cells to HIV-1 infection; memory but not naive T cells are highly responsive to HIV-mediated actin dynamics that promote higher viral entry, DNA synthesis in memory cells. These results demonstrate that the cortical actin is an early determinant regulating the differential susceptibility of memory and naive T cells to HIV-1 infection.
EXPERIMENTAL PROCEDURES
Isolation of Resting CD4 T Cells from Peripheral Blood
All protocols involving human subjects were reviewed and approved by the George Mason University institutional review board. Resting CD4 T cells were purified from peripheral blood by two rounds of negative selection as described previously (28). Briefly, for the first round of depletion, we used monoclonal antibodies against human CD14, CD56, HLA-DR, DP, and DQ (BD Biosciences). For the second round of depletion, we used monoclonal antibodies against human CD8, CD11b, and CD19 (BD Biosciences). Antibody-bound cells were depleted by using Dynabeads Pan Mouse IgG (Invitrogen). For further negative selection of the memory and naive CD4 T cell subsets, monoclonal antibody against either CD45RA (0.02 μl per million cells) or CD45RO (0.1 μl per million cells) (BD Biosciences) was added during the second round of depletion. Purified cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), penicillin (50 units/ml) (Invitrogen), and streptomycin (50 μg/ml) (Invitrogen). Cells were rested overnight before infection or treatment.
Virus Preparation and Infection of T Cells
Virus stocks of HIV-1NL4-3 (29) were prepared by transfection of HeLa cells with cloned proviral DNA as described (28). HIV-1(VSV-G) was prepared as described previously (25). Levels of p24 in the viral supernatant were measured in triplicate on the same ELISA plates using the Alliance p24 antigen ELISA kit (PerkinElmer Life Sciences). Viral titer (TCID50) was determined on the Rev-dependent GFP indicator cell Rev-CEM (30, 31).
For infection of resting memory or naive CD4 T cells, unless otherwise specified, 103.5 to 104.5 TCID50 units of HIV-1 were used to infect 106 cells. For infection, CD4 T cells were incubated with the virus for 2 h, washed three times, and then resuspended into fresh medium (106 per ml) and incubated for 1–5 days without stimulation. Cells were activated with anti-CD3/CD28 magnetic beads at four beads/cell. On certain occasions, resting naive CD4 T cells were also pre-stimulated with okadaic acid (OA)4 (Calbiochem) to observe enhancement of viral replication. Because of the cytotoxicity of the drug and donor variation, OA treatment needs to be titrated on each individual donor. Cells were pretreated with OA (from 250 nm to 1 μm) for 10 min and then infected with HIV-1 in the continuous presence of OA. Cells were washed three times, incubated for 1 day in the absence of OA, and then stimulated with anti-CD3/CD28 beads. Naive cells were also pre-stimulated with latrunculin A (Lat-A) (Sigma) to observe inhibition or enhancement of viral replication. Because of donor variation, Lat-A treatment also needs to be titrated on each individual donor. For observing Lat-A inhibition, cells were pretreated with Lat-A for 5 min and then infected with HIV-1 for 2 h in the continuous presence of Lat-A. Cells were washed three times, cultured in the absence of Lat-A for 5 days, and then stimulated with anti-CD3/CD28 beads (four beads/cell). For observing Lat-A-mediated enhancement of viral replication, cells were pretreated with Lat-A (from 25 to 500 nm) for 5 min. Cells were washed twice, infected with HIV for 2 h in the absence of the drug, washed three times, incubated for 1 day in the absence of Lat-A, and then stimulated with anti-CD3/CD28 beads (four beads/cell).
Surface Staining of CD4, CXCR4, and CD45RA/CD45RO
Cells were stained with FITC-labeled monoclonal antibody against human CD4 (clone PRA-T4) or CXCR4 (clone 12G5) (BD Biosciences). On occasion, cells were also double-stained with FITC-labeled anti-CD45RA and PE-cy5-labeled anti-CD45RO antibodies (BD Biosciences). Cells were stained on ice in PBS + 0.1% BSA for 30 min, washed with cold PBS, 0.5% BSA, and then analyzed on a FACSCalibur (BD Biosciences).
Chemotaxis Assay
Half a million cells were resuspended into 100 μl of RPMI 1640 medium and then added to the upper chamber of a 24-well transwell plate (Corning). The lower chamber was filled with 600 μl of medium premixed with SDF-1. The plate was incubated at 37 °C for 2 h, and then the upper chamber was removed, and cells in the lower chamber were counted and analyzed by flow cytometry.
Viral Entry Assays
The BlaM-Vpr-based viral entry assay was performed as described previously (22, 32). We also used a Nef-luciferase-based entry assay as described (33). Briefly, cells (1 × 106) were infected with 200 ng of Nef-luciferase containing viruses at 37 °C for 2 h and then washed three times with medium. Cells were resuspended in 0.1 ml of luciferase assay buffer (Promega), and luciferase activity was measured in live cells using a GloMax-multidetection system (Promega).
FITC-Phalloidin Staining of F-actin and Flow Cytometry
F-actin staining using FITC-phalloidin (Sigma) was carried out as described previously (22). Briefly, each staining was carried out using 1–2 × 106 cells. Cells were pelleted, fixed, and permeabilized with CytoPerm/Cytofix buffer (BD Biosciences) for 20 min at room temperature and washed with cold Perm/Wash buffer (BD Biosciences) twice, followed by staining with 5 μl of 0.3 mm FITC-labeled phalloidin (Sigma) for 30 min on ice in the dark. Cells were washed twice with cold Perm/Wash buffer, then were resuspended in 1% paraformaldehyde, and analyzed on a FACSCalibur (BD Biosciences). Cells were also co-stained with FITC-labeled phalloidin and PE-cy5-labeled anti-CD45RA or anti-CD45RO antibodies (BD Biosciences) for surface expression. Briefly, cells were stained with PE-cy5-labeled anti-CD45RA or anti-CD45RO antibodies on ice, washed twice, fixed with Cytofix/Cytoperm (BD Biosciences), and then stained with FITC-labeled phalloidin.
Real Time PCR Amplification of Viral DNA
Viral total DNA was measured by real time PCR as described previously (22). Briefly, viruses used for measuring viral DNA synthesis were assembled only in HeLa cells by plasmid transfection (22). Viral supernatant was harvested at 48 h post-transfection and filtered through a 0.45-μm nitrocellulose membrane and then treated with Benzonase (Novagen) (125 units/ml) at 37 °C for 15 min. Quantitative real time PCR analyses of viral DNA were carried out using the Bio-Rad iQ5 real time PCR detection system, utilizing the forward primer 5′-LTR-U5, the reverse primer 3′-gag, and the probe FAM-U5/gag (22). Pre-quantified, full-length proviral plasmid pNL4-3 was used as the DNA standard. Viral DNA and 2-LTR circles in shRNA lentiviral vector-transduced cells (shNTC and shLIMK-007) were measured as described previously (23). For measuring 2-LTR circles (34), the DNA was amplified by real time PCR with primers and probe MH536, MH535, and MH603 (35, 36).
Cytoplasmic and Nuclear DNA Fractionation
The fractionation of viral DNA in memory and naive T cells was conducted as described previously (22). Briefly, 2 million cells were pelleted at 270 × g for 5 min in a microcentrifuge at 4 °C, washed once with ice-cold PBS, resuspended into ice-cold cell lysis buffer (10 mm Tris-Cl, pH 7.5, 140 mm NaCl, 5 mm KCl, 1% EDTA, 1% Nonidet P-40), incubated on ice for 5 to 10 min, and then centrifuged at 270 × g for 5 min at 4 °C to pellet the nuclei. The supernatant was removed and centrifuged at 13,000 × g for 5 min to remove nuclear contamination. The supernatant was then mixed with an equal volume of 8 m guanidine thiocyanate. The nuclear pellet was washed once with ice-cold cell lysis buffer and then dissolved in DNA extraction lysis buffer (SV Total RNA Isolation System, Promega). Both cytoplasmic and nuclear DNA was extracted using the SV Total RNA Isolation System (Promega) with a modified protocol for DNA extraction (Promega). To control for possible leakage of cytoplasmic contents into the nucleus, a small amount (∼250 pg) of pre-quantified plasmid pMSCVneo (Clontech) was add to the cell lysis buffer prior to fractionation. The nuclear β-actin pseudogene was also used for fractionation control for possible leakage of the nuclear contents into the cytoplasmic fraction. HIV DNA was amplified with primer HIV/tat-rev-5′ (5′-GGTTAGACCAGATCTGAGCCTG-3′) and LTR-gag2 (5′-TTAATACCGACGCTCTCGCACC-3′) using SuperTaq Plus polymerase (Ambion) and 35 cycles at 94 °C for 20 s and 68 °C for 10 min. pMSCVneo was PCR-amplified with primer pMSCVneo-Forward-1904 (5′-GCTGTTCTCCTCTTCCTCATC-3′) and pMSCVneo-Reverse-2733 (5′-CAGAAGAACTCGTCAAGAAGG-3′) using SuperTaq Plus polymerase (Ambion) and 28 cycles at 94 °C for 20 s and 68 °C for 1 min. The β-actin pseudogene was amplified using the actin primer from Quantum RNA β-actin internal standards (Ambion).
Cell Cycle Analysis by 7-Aminoactinomycin D and Pyronin Y Staining
Resting naive CD4 T cells (106) were treated with anti-CXCR4 magnetic beads or anti-CD3/CD28 magnetic beads (two beads/cell) (Invitrogen) overnight. Beads were removed from the cells by gentle pipetting. Cells were suspended in 1 ml of 0.03% saponin in PBS and then incubated in 20 μm 7-aminoactinomycin D (Sigma) for 30 min at room temperature in the dark. Cells were kept on ice for at least 5 min; pyronin Y (Sigma) was added to a final concentration of 5 μm, and the cells were then incubated for 10 min on ice. Stained cells were directly analyzed by flow cytometry on a FACSCalibur (BD Biosciences).
Western Blot for LIMK, Apobec3G, and BST2
Normally, 1 million cells were lysed in NuPAGE LDS Sample Buffer (Invitrogen), separated by SDS-PAGE, and then transferred onto nitrocellulose membranes (Invitrogen). The membranes were washed in TBST for 3 min and then blocked for 30 min at room temperature with Starting Block blocking buffer (Pierce). For probing with different antibodies, blots were incubated with rabbit anti-LIMK1 antibodies (Cell Signaling), with rabbit anti-Apobec3G antibodies (from AIDS Research and Reference Reagent Program, National Institutes of Health, catalog no. 10082) (1:1000 dilution), or with a mouse anti-BST2 antibody (Abnova) (1:500). These antibodies were diluted in 2.5% milk/TBST and rocked overnight at 4 °C. The blots were washed three times for 15 min, then incubated with either anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (KPL) (1:5000) for 1 h, and then developed with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce). For loading control, the same blots were also stripped and reprobed with antibodies against GAPDH (Abcam) (1:1000). Images were captured with a CCD camera (FluorChem 9900 Imaging Systems) (Alpha Innotech).
Conjugation of Antibodies to Magnetic Beads
Monoclonal antibodies against human CXCR4 (clone 12G5), CD3 (clone UCHT1), and CD28 (clone CD28.2) were from Pharmingen. For conjugation, 10 μg of antibodies were conjugated with 4 × 108 Dynabeads Pan Mouse IgG (Invitrogen) for 30 min at room temperature. Free antibodies were washed away with PBS + 0.5% BSA, and the magnetic beads were resuspended in 1 ml of PBS + 0.5% BSA.
Confocal Microscopy
Stained cells were imaged using a Zeiss laser-scanning microscope, LSM 510 META, with a 40 NA 1.3 or 60 NA 1.4 oil DIC Plan-NeoFluar objective. Samples were excited with the 488 nm laser line for FITC. Images were processed and analyzed with LSM 510 META software.
RESULTS
Preferential Infection of CD45RO Memory CD4 T Cells by HIV-1
Previous studies have demonstrated that CD45RO memory CD4 T cells support higher levels of HIV-1 replication than CD45RA T cells (5, 7–10). To determine whether these observations can be recapitulated in our in vitro CD4 T cell latent infection system (28), we purified resting memory (CD45RO+/CD45RA−, purity 95.4 ± 2.3%) and naive (CD45RA+/CD45RO−, purity 97.6 ± 1.2%) CD4 T cells from the peripheral blood by negative depletion (supplemental Fig. 1), and we then identically infected these two T cell subsets with HIV-1. Infected cells were rested for 5 days, during which productive viral replication did not occur in either T cell subset. However, viral replication could be induced by CD3/CD28 stimulation (28). We stimulated infected resting T cells at day 5 with anti-CD3/CD28 antibody-conjugated magnetic beads and observed viral replication only in the CD45RO+/CD45RA− memory cells but not the CD45RA+/CD45RO− naive CD4 T cells (Fig. 1, A and B). We repeated the experiment in multiple donors, and among the 17 donors examined, we observed little or no viral replication in the CD45RA naive T cells (Fig. 1, A and B; supplemental Fig. 2, A and B), in great contrast to the high levels of viral replication observed in the CD45RO memory T cells from all donors (Fig. 1, A and B; supplemental Fig. 2, A and B). These results are consistent with previous studies demonstrating the preferential infection of CD45RO memory CD4 T cells by HIV-1 (5, 7–9).
FIGURE 1.
Preferential infection of CD45RO memory CD4 T cells by HIV-1. A and B, resting memory (CD45RO+/CD45RA−) and naive (CD45RA+/CD45RO−) CD4 T cells were purified from the peripheral blood through two rounds of negative depletion and then identically infected with HIV-1 for 2 h. Cells were washed, cultured for 5 days, and then activated with magnetic beads coated with anti-CD3/CD28 antibodies (anti-CD3/CD28 beads). Viral replication was monitored by extracellular p24 release following CD3/CD28 activation. C and D, resting memory and naive T cells from three additional donors were purified and lysed for Western blot to measure the expression of Apobec3G (C) or BST-2 (D) as described under “Experimental Procedures,” using an equal number of memory and naive T cells. For loading control, the blots were also probed with an antibody against GAPDH. E, resting memory or naive T cells from an additional donor were identically separated and pre-activated with anti-CD3/CD28 beads for 1 h, and then infected with a VSV-G pseudotyped HIV-1, HIV-1(VSV-G). Viral extracellular p24 release was monitored. F, resting memory and naive CD4 T cells were also similarly infected with the VSV-G pseudotyped HIV-1, washed, and then immediately activated with anti-CD3/CD28 beads. Viral extracellular p24 release following CD3/CD28 activation was monitored. G, resting memory and naive T cells from five donors (G0510, G1028A, G1028B, H0409, and H0610) were infected with HIV-1 for 2 h, washed, cultured for 5 days, and then activated with anti-CD3/CD28 beads. Viral p24 release was monitored. For comparison, resting memory and naive T cells from three additional donors (H0224A, H0224B, and K0419) were infected with HIV-1(VSV-G), washed, and then activated with anti-CD3/CD28 beads. Shown is the relative p24 release at 4 days post-stimulation.
In our system, the differential susceptibility of memory and naive T cells to HIV-1 infection could result from the possible selective presence of HIV restriction factors in naive T cells; however, when examined for known HIV restriction factors such as Apobec3G and BST2 (37–41), we did not observe differences between memory and naive T cells in BST2 expression, whereas levels of Apobec3G were actually slightly higher in the memory T cell subset (Fig. 1, C and D). These results suggest that it is unlikely that these restriction factors are responsible for the higher restrictiveness of naive T cells to HIV infection. In addition, when memory and naive T cells were similarly infected with a VSV-G pseudotyped HIV-1, either before or after CD3/CD28 stimulation, memory and naive T cells supported similar low levels of viral p24 release (Fig. 1, E–G; supplemental Fig. 3) (25). The VSV-G pseudotyping mediates viral entry via endocytosis, which bypasses the CD4/CXC4 receptor and the cortical actin, and directly delivers viral core into the cytoplasm. Thus, similar levels of p24 from HIV-1(VSV-G) infection of memory and naive T cells implied a comparable intracellular environment for the virus. Therefore, it is unlikely that there are certain cytoplasmic restriction factors uniquely present only in resting naive T cells to restrict HIV infection selectively.
Resting CD4 T cells, either memory or naive, are intrinsically resistant to HIV infection (28, 42). It has been suggested that lower levels of intracellular dNTP may limit viral DNA synthesis in resting T cells (42, 43). However, this dNTP limitation may be present both in resting memory and resting naive CD4 T cells and is not unique to naive cells, as the addition of exogenous deoxynucleoside did not overcome their difference in mediating HIV infection (17). Nevertheless, it is still possible that the varied cellular states or different levels of T cell activation following CD3/CD28 stimulation may affect memory and naive T cells differently (44). Thus, we examined markers of T cell activation following CD3/CD28 stimulation both in memory and naive T cells. As shown in Fig. 2, resting memory and naive CD4 T cells were identically stimulated with anti-CD3/CD28 beads (four beads/cell) for 1 day, and the up-regulation of CD25 and CD69 was measured. We did not observe defects or lower levels of T cell activation in naive T cells in response to anti-CD3/CD28 bead stimulation, as judged by CD25 and CD69 up-regulation. Our result appears to be consistent with a previous report showing that memory and naive T cells proliferated equally for at least 14 days following CD3/CD28 stimulation (44). This result, combined with the result of equal p24 release in HIV-1(VSV-G) infection (Fig. 1E), suggests that differential T cell activation may not be the major reason for the different susceptibility of memory and naive T cells to HIV-1 infection, at least in our system.
FIGURE 2.
Measurement of the CD25 and CD69 up-regulation following CD3/CD28 stimulation of memory and naive T cells. Resting memory and naive CD4 T cells were separated and stimulated identically with anti-CD3/CD28 beads (four beads/cell) for 24 h. Up-regulation of the CD25 (A) and CD69 (B) receptors following stimulation was measured by surface staining with PE-labeled monoclonal antibodies against either CD25 or CD69. Shown are the results from flow cytometry, demonstrating that naive CD4 T cells have no defect in response to anti-CD3/CD28 bead stimulation, as judged by CD25 and CD69 expression. CD25 and CD69 were stained either before (Resting) or after CD3/CD28 activation (CD3/CD28 stimulation). Cell surface staining with corresponding isotype antibodies is also shown (isotype staining).
Resting Naive CD4 T Cells Are More Restrictive in Supporting HIV Early Processes in the Absence of T Cell Activation
To further determine what exact steps of the viral life cycle were affected in resting naive T cells, we followed viral latent infection, specifically, viral entry and early DNA synthesis, which occurs within the first 2 h in the absence of T cell activation (22, 23). The Vpr-β-lactamase-based entry assay (32) showed a roughly 50% reduction of viral entry in naive T cells (Fig. 3A and supplemental Fig. 4), consistent with a previous report demonstrating lower entry of HIV into resting naive T cells (17). Nevertheless, HIV Nef-luciferase- and p24-based entry assays (33) showed minimal difference (supplemental Fig. 5, A and B). The variation between these assays may have resulted from the use of different dosages of the virus; the Vpr-β-lactamase-based assay requires the use of concentrated viruses, whereas the other two assays were performed at a much lower viral dosage. The results were reminiscent of the cofilin kinase LIMK1 knockdown T cell, shLIMK-007, which showed a reduced entry only at high viral dosages (23). Nevertheless, even with a 50% reduction in viral entry into naive cells, the decrease does not explain the dramatic difference observed in HIV infection of memory and naive T cells (17). Therefore, we examined the subsequent steps of viral infection, namely early DNA synthesis. We observed a reduction in viral DNA synthesis in naive T cells (Fig. 3B) (5, 7). In contrast, when the VSV-G pseudotyped HIV-1 was used to infect memory and naive T cells, we did not see a significant difference in viral DNA synthesis (p = 0.49) (Fig. 3B). These data suggested that the difference in viral DNA synthesis likely resulted from CD4/CXCR4-mediated entry and subsequent post-entry processes (25). The synthesis of viral DNA has been suggested to occur mainly on the actin cytoskeleton (22, 45). Thus, we also performed subcellular fractionation of viral DNA (supplemental Fig. 6) and found that the levels of viral DNA in the cytoplasm of naive T cells were diminished in comparison with memory CD4 T cells (Fig. 3C). Indeed, in memory T cells, the synthesis of cytosolic viral DNA was sensitive to the inhibition of the reverse transcriptase inhibitor azidothymidine, whereas in naive T cells, the residual cytosolic viral DNA detected was not sensitive to azidothymidine inhibition (Fig. 3C). In conclusion, these results demonstrated that resting naive T cells are largely defective in mediating early steps of HIV-1 infection, resulting in lower viral entry and diminished viral DNA synthesis. Importantly, this differential susceptibility of naive cells to HIV-1 infection exists naturally in resting T cells in the absence of T cell activation and any other stimulation. Our results are also consistent with previous results showing a 4–10-fold reduction in HIV DNA in naive T cells in comparison with memory T cells (5). In addition, the phenotype of lower viral DNA synthesis in naive T cells also resembles the phenotype observed in the LIMK1 knockdown cell shLIMK-007, which carries a lower level of the cortical actin and actin dynamics (Fig. 3, D–F) (23).
FIGURE 3.
Early steps in HIV-1 latent infection of memory (CD45RO+/CD45RA−) and naive (CD45RA+/CD45RO−) CD4 T cells. A, resting memory and naive T cells were infected with a Vpr-β-lactamase (Vpr-BlaM)-tagged HIV-1 (1750 ng of p24 per million cells) for 2 h. Viral entry into memory and naive T cells was measured with flow cytometry for Vpr-BlaM-mediated cleavage of CCF2. B, synthesis of viral DNA at 2 h following infection with HIV-1 or HIV-1(VSV-G) was measured by real time PCR. C, resting memory and naive T cells were also pre-treated with 50 μm azidothymidine (AZT) overnight and then infected with HIV-1 for 2 h. Following infection and washing, total cytoplasmic DNA was isolated and amplified with real time PCR for HIV-1 DNA. ND, not detectable. D–F, for comparison, the LIMK knockdown cell, shLIMK-007, and the control cell, shNTC, were measured with Western blot for LIMK expression with an anti-LIMK antibody. The blot was also probed with an anti-GAPDH antibody for loading control. The relative LIMK level normalized by GAPDH was plotted at the bottom (from three independent Western blots) (D). The syntheses of HIV-1 DNA (E) and of 2-LTR circles (F) following HIV-1 infection of the LIMK knockdown cell, shLIMK-007, and the control cell, shNTC, were measured by real time PCR.
Phenotypic Distinction between CD45RO and CD45RA CD4 T Cells in the Cortical Actin
Given that HIV-1(VSV-G), which enters cells through endocytosis, synthesized similar levels of viral DNA (Fig. 3B) and p24 in memory and naive T cells (Fig. 1, E–G), we speculated that, in wild-type HIV-1 infection, the factor discriminating memory and naive T cells is likely at the level of CD4/CXCR4 receptors and/or the cortical actin. Thus, we measured the surface density of both CD4 and CXCR4 in resting memory and naive CD4 T cells, but we did not observe differences in these receptors (Fig. 4, A–C), consistent with a previous report (17). This led us to speculate that the cortical actin may be a factor affecting HIV infection of memory and naive CD4 T cells. Indeed, recent studies have demonstrated that the cortical actin is involved in HIV entry, DNA synthesis, and nuclear migration and is required for the establishment of HIV latent infection of resting CD4 T cells (22–26). To substantiate this speculation, we examined the cortical actin density in resting CD4 T cells from 68 healthy donors. In our initial assessment of mixed resting CD4 T cell population, we frequently observed two different peaks of F-actin staining, which we designated Actinhigh and Actinlow CD4 T cells (Fig. 4, D and E). The ratio of these two cell populations varied from donor to donor, but in some donors, it reached ∼1. Because the CD45RO/CD45RA subsets constitute the two major subpopulations of CD4 T cells in adult blood, we performed costaining of F-actin with surface CD45RO or CD45RA and found a direct correlation of these CD45 isoforms with a higher or lower F-actin content, respectively (Fig. 4, F–H).
FIGURE 4.
Phenotypic distinction in the cortical actin between the CD45RO and CD45RA resting CD4 T cell subsets. A–C, resting CD4 T cells were purified from the peripheral blood by negative depletion. Expression of the CXCR4 receptor on T cell subsets was measured by costaining cells with a FITC-labeled anti-CXCR4 antibody and a PE-cy5-labeled anti-CD45RA antibody (B). Expression of the CD4 receptor on these T cell subsets was similarly measured by staining cells with a FITC-labeled anti-CD4 antibody (C). D and E, resting CD4 T cells were similarly purified from the peripheral blood through negative depletion. Shown is the T cell purity following purification, as analyzed by surface CD4 staining and flow cytometry (D). Resting CD4 T cells were also stained with FITC-labeled phalloidin to measure actin filaments (F-actin). Shown are the histograms of F-actin staining from three representative donors (E). The two peaks of F-actin staining are labeled with red arrows. F and G, resting CD4 T cells were also co-stained with FITC-labeled phalloidin and a PE-cy5-labeled anti-CD45RA antibody (F) or a PE-cy5-labeled anti-CD45RO antibody (G) to measure F-actin in these T cell subsets. H, cells from three additional donors were costained with FITC-labeled phalloidin and a PE-cy5-labeled anti-CD45RA antibody to measure F-actin in these T cell subsets.
Further separation of cells into the CD45RO+ and CD45RA+ subsets confirmed that they are indeed distinct in their cortical actin density (Fig. 5, A–E; supplemental Fig. 7, A and B). Thus, we designate them CD45RO+Actinhigh and CD45RA+Actinlow resting CD4 T cells. The lower actin phenotype in naive T cell also resembles that of the LIMK1 knockdown T cell, shLIMK-007 (Fig. 5F) (23). In shLIMK1–007, the decrease in the cortical actin density leads to a decrease in both T cell mobility and the susceptibility to HIV infection (Fig. 3, E–G) (23). Thus, we sought to determine whether there were similar differences in the chemotactic properties between memory and naive T cells that may affect their susceptibility to HIV infection.
FIGURE 5.
Cortical actin phenotypically distinguishes the memory (CD45RO+/CD45RA−) and naive (CD45RA+/CD45RO−) CD4+ T cell subsets. A, purified resting CD4 T cells were further separated through negative depletion into memory (CD45RO+/CD45RA−) and naive (CD45RA+/CD45RO−) subsets. Shown are cells co-stained with a FITC-labeled anti-CD45RA antibody and a PE-cy5-labeled anti-CD45RO antibody before (Total CD4 T cells) and after separation. B, separated memory (CD45RO+/CD45RA−) and naive (CD45RA+/CD45RO−) CD4 T cells were stained with FITC-labeled phalloidin for F-actin. Resting CD4 T cells before separation (Total CD4) were also identically stained for comparison. Cells were analyzed with a flow cytometer, and the histograms of F-actin staining are shown. C, memory (CD45RO+/CD45RA−) and naive (CD45RA+/CD45RO−) CD4 T cells were also examined with confocal microscopy. Images were acquired under identical conditions. D, relative intensity of F-actin staining was measured along the red lines and plotted. E, relative F-actin intensities in memory and naive CD4 T cells from 8 donors were quantified by FITC-labeled phalloidin staining and flow cytometry. F, for comparison, the F-actin in the LIMK1 knockdown T cell, shLIMK-007, and the control cell, shNTC, were identically stained with FITC-labeled phalloidin and analyzed with a flow cytometer.
CD45RO Memory CD4 T Cells Is More Sensitive to Chemotactic Induction
Stromal-derived factor-1 (SDF-1) is the natural ligand of CXCR4 that mediates CD4 T cell chemotaxis (46). The basal levels of SDF-1 in plasma have been found to be at ∼0.6 ng/ml (47), whereas local SDF-1 concentrations in tissues could be high and in the range of ng/ml to μg/ml (48, 49). In vitro, SDF-1 attracts blood T cells optimally at around 50–100 ng/ml (50). Using these conditions, we measured SDF-1-mediated chemotaxis of resting CD4 T cells in a transwell assay (Fig. 6A). Following cell migration, the composition of T cell subsets was also analyzed by CD45RO/CD45RA co-staining (Fig. 6B). We observed an SDF-1 dosage-dependent cell migration (Fig. 6A). At the highest SDF-1 dosage used (50 ng/ml), among the migratory CD4 T cells, 65.9% were CD45RO memory and 27.9% were CD45RA naive T cells (Fig. 6B). The ratio of memory to naive cells (2.4:1) approaches the natural ratio of these cells (2.3:1) in the peripheral blood of the donor (Fig. 6B, Control). This result suggested that both memory and naive T cells responded similarly to SDF-1 at the high dosage. However, when the SDF-1 concentration was 2 ng/ml, the ratio of memory to naive T cells increased to 7.8 to 1 (Fig. 6B). At the lowest SDF-1 dosage (0.08 ng/ml), the ratio was even higher, reaching 9 to 1, and ∼90% of the migratory T cells were memory T cells (Fig. 6B). These results demonstrate that although high dosages of SDF-1 attract both memory and naive T cells, low dosages of SDF-1 attract mostly memory but not naive T cells, suggesting higher sensitivity of memory T cells to chemotactic induction.
FIGURE 6.
CD45RO memory CD4 T cells are more sensitive to chemotactic induction. A, resting CD4 T cells were purified by negative depletion and then assayed in SDF-1-induced chemotaxis in transwell plates. The percentage of migratory cells was measured. B, composition of the migratory T cells was further analyzed by costaining of cells with a FITC-labeled anti-CD45RA antibody and a PE-cy5-labeled anti-CD45RO antibody. The natural composition of the memory and naive CD4 T cell subsets in the peripheral blood of the donor was also identically measured (Control). C–F, differential actin dynamics between memory and naive T cells in response to chemotactic induction. Resting CD4 T cells were separated through negative depletion into memory (CD45RO+/CD45RA−) and naive (CD45RA+/CD45RO−) CD4 T cell subsets. Cells were then stimulated with 50 ng/ml (C) or 0.1 ng/ml (E) SDF-1 for various times. Actin dynamics following SDF-1 stimulation were measured with FITC-labeled phalloidin staining and flow cytometry. D and F show the plots of relative F-actin intensity in the course of SDF-1 stimulation.
The selective migration of memory cells at low SDF-1 dosages cannot be explained by differential expression of the chemokine receptor nor can it be attributed to possible defects in the signaling pathways of naive T cells. First, CXCR4 is present on both memory and naive CD4 T cells, and the receptor density is also similar (Fig. 4B) (17). Second, both memory and naive cells migrated equally at the high SDF-1 dosage (Fig. 6B, 50 ng/ml), suggesting that the signaling pathways leading to cell migration are intact and functional in both memory and naive T cells. However, the signaling threshold for triggering actin activity and cell mobility might be different. Thus, we further compared the actin dynamics in memory and naive cells following stimulation with either a high or a low dosage of SDF-1 (Fig. 6, C–F).
Resting memory (CD45RO+/CD45RA−) and naive (CD45RA+/CD45RO−) CD4 T cells were purified by negative depletion. These two cell subsets were then identically treated with SDF-1 for a time course to measure SDF-1-induced actin activity. We observed similar actin dynamics at the high SDF-1 dosage (50 ng/ml), when both memory and naive cells were attracted to migrate (Fig. 6, C and D). However, at the low SDF-1 dosage (0.1 ng/ml), when only memory T cells were attracted to migrate, we observed a transient actin polymerization only in memory T cells but minimal actin activity in naive cells (Fig. 6, E and F). These results confirmed that naive T cells have a higher threshold for triggering actin activity and are less sensitive to chemotaxis induction. For memory T cells, the higher sensitivity and more robust response of the actin cytoskeleton to chemotaxis induction correlate with their higher cortical actin content (Figs. 4 and 5). Memory T cells are also highly susceptible to HIV infection, which is known to rely on chemokine coreceptor signaling and the actin cytoskeleton for initiating infection (22–24, 27). Collectively, our results support the hypothesis that a lower cortical actin density and less dynamic actin in naive T cells lead to lower viral entry and DNA synthesis (22, 23, 27). Our results are also consistent with a previous demonstration that the actin inhibitor jasplakinolide diminishes viral DNA synthesis in resting CD4 T cells (22, 24).
Cortical Actin Regulates the Susceptibility of Memory and Naive T cells to HIV-1
To demonstrate the direct involvement of the cortical actin in the differential infection of memory and naive T cells, we identically infected these T cell subsets and compared the actin dynamics. We observed a high level of actin activity in memory T cells, but a low level in naive cells, upon stimulation with HIV-1 (Fig. 7, A and B; supplemental Fig. 8). These results demonstrate that naive T cells are intrinsically less responsive to HIV-mediated actin dynamics than memory T cells. This correlates with the lesser susceptibility of naive T cells to viral infection (Fig. 1, A and B). Consistently, the higher ability of memory T cells to HIV infection can be greatly reduced when memory T cells were treated for 2 h during infection with an actin modulator, latrunculin A (Lat-A) (Fig. 7C), which is known to specifically induce actin depolymerization through reversible binding to actin monomers (22, 51). To conclusively prove the role of the cortical actin in limiting HIV infection of naive T cells, we attempted to rescue viral replication in naive T cells by increasing the cortical actin dynamics through a pharmacological drug, OA, which has recently been shown to trigger the activation of the cofilin kinase LIMK1 in resting CD4 T cells (23). Brief treatment of resting naive T cells with OA triggered actin polymerization (Fig. 7D). Consistently, similar brief treatment of naive T cells with 250 nm OA prior to infection rescued HIV-1 replication in naive T cells following CD3/CD28 stimulation (Fig. 7E and supplemental Fig. 9). These data support a critical role of early actin dynamics in the initiation of HIV-1 latent infection. To further confirm these results, we used Lat-A to transiently induce actin activity through reversible actin depolymerization. We found that brief Lat-A treatment (5 min) of naive T cells was able to disturb the cortical actin and artificially trigger actin activity (Fig. 7F). Remarkably, this brief 5-min treatment of naive T cells with Lat-A prior to infection effectively rescued HIV replication in naive T cells following CD3/CD28 stimulation (Fig. 7G and supplemental Fig. 10). The fact that a single actin modulator promoted HIV-1 latent infection of naive T cells suggests that the static cortical actin in naive cells is a major factor limiting HIV latent infection of naive T cells.
FIGURE 7.
Stimulation of actin dynamics rescues HIV-1 latent infection of naive T cells. A and B, differential actin dynamics between resting memory and naive CD4 T cells in response to HIV-1 infection. Resting memory (CD45RO+/CD45RA−) and naive (CD45RA+/CD45RO−) CD4 T cells were isolated by negative depletion and then identically stimulated with HIV-1 for various times. HIV-1-mediated actin dynamics were measured by FITC-labeled phalloidin staining. C, dosage-dependent inhibition of HIV-1 infection of memory CD4 T cells by Lat-A. Resting memory CD4 T cells were pre-treated with Lat-A for 5 min and then infected with HIV-1 for 2 h in the continuous presence of Lat-A. Following infection, cells were washed, cultured in the absence of Lat-A for 5 days, and then activated with anti-CD3/CD28 beads. Viral replication following stimulation was measured by extracellular p24 release. As a control, naive CD4 T cells from the same donor were purified and identically infected. D, resting naive (CD45RA+/CD45RO−) CD4 T cells were stimulated with 250 nm OA for various times, and the induced actin dynamics were measured by FITC-labeled phalloidin staining. E, resting naive (CD45RA+/CD45RO−) CD4 T cells were mock-treated or treated with 250 nm OA for 10 min and then infected with HIV-1 (110 ng p24 per million cells) in the presence of OA for 50 min. Cells were washed, cultured for 1 day in the absence of OA, and activated with anti-CD3/CD28 beads. F, resting naive (CD45RA+/CD45RO−) CD4 T cells were briefly stimulated with 250 nm Lat-A for 5 min, washed, and then cultured for various times in the absence of the drug. Lat-A-triggered actin dynamics were measured by FITC-phalloidin staining. G, resting naive (CD45RA+/CD45RO−) CD4 T cells were mock-treated or treated with 250 nm Lat-A for 5 min, washed, and then infected with HIV-1 (110 ng of p24 per million cells) in the absence of Lat-A for 2 h. Cells were washed, cultured for 1 day, and activated with anti-CD3/CD28 beads. Viral replication was measured by extracellular p24 release following infection. H, resting naive (CD45RA+/CD45RO−) CD4 T cells were cultured or pre-stimulated with anti-CXCR4 magnetic beads (two beads/cell) overnight and then analyzed for cell cycle progression using 7-aminoactinomycin D (7-ADD) and pyronin Y (PY) staining. Anti-CD3/CD28 magnetic bead-stimulated naive T cells were used as a control. I, resting naive CD4 T cells from two donors were pre-stimulated with anti-CXCR4 magnetic beads overnight, infected with HIV-1 for 2 h, and then washed three times. Viral DNA synthesis was analyzed by real time PCR. J, for one of the donors (K0423), cells were also stimulated with anti-CD3/CD28 beads (two beads/cell) following HIV-1 infection and washing. Viral replication was measured by extracellular p24 release.
To provide further complementary evidence, we also pre-stimulated resting naive T cells with an anti-CXCR4 antibody conjugated to magnetic beads. We speculated that this stimulation may complement the inability of HIV-1 to trigger chemotactic signaling and actin dynamics through CXCR4 in naive T cells (Fig. 7A). Pre-stimulation of CXCR4 did not activate resting naive T cells (Fig. 7H), but it triggered chemotactic signaling that led to the activation of cofilin and the rearrangement of actin (22). This low level stimulation (two beads/cell) of resting CD4 T cells also did not block viral entry (22). When resting naive T cells were pre-stimulated with this anti-CXCR4 antibody and infected with HIV-1, we observed a dramatic 50-fold enhancement of viral DNA synthesis at 2 h post-infection (Fig. 7I and supplemental Fig. 11A). This increase in viral DNA synthesis in pre-stimulated naive T cells also correlated with an enhancement of viral replication following CD3/CD28 activation (Fig. 7J and supplemental Fig. 11B). In conclusion, these results are consistent with our hypothesis that the lower early activities in viral infection of naive T cells largely result from the lower actin dynamics initiated from HIV-CXCR4 interaction. This lack of actin activity can be complemented by stimulating CXCR4-mediated chemotactic signaling and actin dynamics.
DISCUSSION
Here, we have identified a phenotypic distinction between human memory and naive T cells in the cortical actin and their actin dynamics, the primary driving force of T cell mobility. Resting memory CD4 T cells possess a higher cortical actin density than resting naive T cells. This cortical actin difference also correlates with their varied actin dynamics in response to chemotactic induction. We further demonstrate that this difference in the cortical actin also directly affects memory and naive CD4 T cells in their susceptibility to HIV-1. Thus, the cortical actin is an early determinant regulating the differential susceptibility of memory and naive T cells to HIV-1 latent infection. Indeed, in HIV-infected patients, memory T cells, particularly the central memory T cells, are preferentially infected and serve as a major viral reservoir (4). Memory T cells harbor more integrated proviral DNA than naive T cells (4–7, 52). These in vivo observations are consistent with our in vitro results presented in this study.
Although perturbation of actin dynamics with OA, Lat-A, or anti-CXCR4 beads directly promoted HIV latent infection of naive T cells (Fig. 7), the increase in actin dynamics did not replace the requirement for CD3/CD28 stimulation to initiate productive viral replication. This is expected, given that both actin modulators and the stimulation of CXCR4 (Fig. 7H) do not activate resting naive T cells (22, 23); actin dynamics mainly impact viral early processes such as entry, DNA synthesis, nuclear migration, and integration (22, 23, 45).
Besides the cortical actin, memory and naive T cells exhibit multiple other phenotypic differences, such as different requirements for T cell activation and differential expression of adhesion molecules (13). Although these differences do not seem to be responsible for the restrictive nature of naive T cells in our system, we do not exclude their potential importance for the in vivo transmission of HIV-1 (53, 54). It is possible that in our system the potent stimulation of resting T cells with CD3/CD28 beads may equalize memory and naive cells in activation, as it was suggested previously that memory and naive cells proliferated equally for at least 14 days following CD3/CD28 stimulation (44) (Fig. 2). However, mitogenic stimulation with phytohemagglutinin (PHA) selectively promoted higher proliferation and greater calcium flux in the naive T cell subset, diminishing the differences between memory and naive T cells in supporting HIV replication (8, 9, 55). In our system, cells were only stimulated with the CD3/CD28 beads after infection to give a similar activation background for the virus to replicate. With this analogous setting, the dominant role of the cortical actin in the early steps of viral infection was borne out. In contrast to memory cells, naive cells supported less viral entry (Fig. 3A) (17) and minimal levels of viral DNA synthesis (Fig. 3, B and C) (5, 7). These events occurred within the first 2 h of infection in the absence of T cell activation (Fig. 3, A–C), excluding effects of T cell activation on viral receptors and early post entry processes (44). Nevertheless, we do not exclude the possibility that multiple factors, including T cell activation, may affect HIV-1 infection of memory and naive T cells in vivo, although our data clearly demonstrate that the cortical actin is a critical cellular component differentiating HIV-1 infection of these cells at the earliest time of infection. Our results are consistent with previous demonstrations that the differential susceptibility of memory and naive T cells is intrinsic and not caused by T cell activation, cytokine stimulation, transcriptional enhancement, or dNTP levels in these cells (8, 17).
A previous report has also demonstrated that memory CD4 T cells supported higher viral replication than naive T cells when stimulated with anti-CD3/CD28 antibodies (9). However, this report is in contrast to our and others' results (5, 7, 17) in suggesting that the differential susceptibility between memory and naive T cells may not be attributed to viral early steps such as entry and viral DNA synthesis (9). Several possibilities may explain this discrepancy. First, the cells in the report by Spina et al. (9) were positively selected through fluorescence-activated sorting. Thus, at least one cell population had anti-CD45 antibodies attached to the surface during infection, whereas our cells were negatively selected and had no antibodies attached. It is possible that viral entry may be affected by the presence of antibodies on the surface. Second, in the report by Spina et al. (9), cells were infected immediately after sorting, whereas our cells were rested overnight and then infected. Recently, it has been shown that mechanical shear stress activates actin dynamics that drastically enhance viral entry and DNA synthesis (26). It is possible that electronic and mechanical stress during cell sorting may have transiently altered cell susceptibility to HIV, diminishing the differences in viral entry and DNA synthesis in the initial infection. Third, cells in the report by Spina et al. (9) were infected for 16–18 h, whereas cells in our experiments were only infected for 2 h. There may be a lot more nonproductive viral entry in this study (9). In addition, we also aggressively treated virion particles with DNase I (benzonase, 125 units/ml) before infection to diminish contaminating plasmid DNA.
In this study, we observed distinctive actin phenotypes in memory versus naive T cells. This difference likely results from a previous antigenic response that leaves a distinctive imprint on memory T cells; the engagement of T cell receptor with the MHC-peptide complex triggers T cell activation that requires actin remodeling to facilitate the formation of the immunological synapse (56). This process could have profoundly remodeled the actin cytoskeleton and its regulatory pathways in memory T cells to predispose them to a faster and greater response in the event of antigenic re-exposure. It is tempting to speculate that the cortex region in memory T cells may not simply possess more actin filaments but may also be pre-loaded with more or different sets of actin regulators and signaling molecules for higher sensitivity. It is possible that at the initiation of an infection, low levels of local chemokines may attract mostly memory T cells because of this higher sensitivity. At later stages, or when memory T cells fail to control the infection, inflammatory chemokines accumulate to high levels, and naive cells are then attracted. The possible advantage of such a scheme may be related to effective immunosurveillance or induction of T cell tolerance (57). Interestingly, in certain autoimmune diseases, prolonged inflammation does trigger the accumulation of large numbers of naive T cells in the affected tissue (58).
The demonstration of a sequestering role of the cortical actin in the differential infection of memory and naive T cells may provide a mechanistic understanding of viral infection and pathogenesis in these T cell subsets. The natural course of HIV infection almost always starts with the robust replication of the CCR5-utilizing viruses, which can quickly infect and kill a large number of target T cells such as the active memory CD4 T cells present in the gastrointestinal tract (59–61). It is possible that the higher chemotactic actin activity in memory T cells, as demonstrated in this study, may be partly responsible for the high susceptibility of these cells to the CCR5-utilizing viruses. The late emergence of the CXCR4-utilizing viruses (62, 63) in about 50% of patients may also be a reflection of the restrictive nature of the CXCR4-positive target cells, as a large number of these cells are naive CD4 T cells (64), which are less sensitive to HIV-mediated chemotactic signaling (Fig. 7A).
Given the restrictive nature of the cortical actin in resting T cells, our results emphasize the potential importance of T cell homing and inflammatory chemokines in HIV infection (24, 65–69), as some of these chemokines are directly involved in the trafficking of memory and naive T cells in vivo and are known to trigger actin dynamics to facilitate viral infection of resting T cells (24, 70).
Supplementary Material
Acknowledgments
We are grateful to the George Mason University Student Health Center for blood donation and the National Institutes of Health AIDS Research and Reference Reagent Program for reagents. We also thank W. Ou of the Food and Drug Administration and D. A. Stephany and K. L. Holmes of NIADS, National Institutes of Health, for fusion assay; and J. Guernsey for editorial assistance.
This work was supported, in whole or in part, by National Institutes of Health USPHS Grant 1R01AI081568 from NIAID (to Y. W.).

This article contains supplemental Figs. 1–11.
- OA
- okadaic acid
- PE
- phycoerythrin
- Lat-A
- latrunculin A.
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