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Journal of Virology, July 2000, p. 6418-6424, Vol. 74, No. 14
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

CCR5 Signal Transduction in Macrophages by Human Immunodeficiency Virus and Simian Immunodeficiency Virus Envelopes

James Arthos,1,* Andrea Rubbert,1,dagger Ronald L. Rabin,2 Claudia Cicala,1 Elizabeth Machado,1 Kathryne Wildt,1 Meredith Hanbach,1 Tavis D. Steenbeke,1 Ruth Swofford,2 Joshua M. Farber,2 and Anthony S. Fauci1

Laboratory of Immunoregulation1 and Laboratory of Clinical Investigation,2 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892

Received 28 December 1999/Accepted 14 April 2000


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1beta . Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1alpha , viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Macrophages are a target of human immunodeficiency virus (HIV) infection in vivo (15, 44). However, only a subset of primary isolates and molecular clones are capable of replicating in macrophages. The molecular determinants of macrophage tropism (M-tropism) lie within the viral envelope (6, 10, 23, 30, 48), suggesting that envelope-receptor interactions determine this restriction. HIV entry into macrophages requires the engagement of CD4 and one of the two principal coreceptors, either CCR5 or CXCR4 (1, 11, 19). Because the majority of primary M-tropic HIV isolates utilize CCR5 rather than CXCR4 (11), a simple paradigm emerged soon after the discovery of the fusion coreceptors, in which CCR5-specific viral isolates were equated with M-tropism (11, 16, 18, 28). The underlying basis for this restriction was unclear, however, because macrophages can express readily detectable concentrations of CXCR4. Moreover, many CXCR4-utilizing isolates enter macrophages; however, they encounter a block at subsequent steps in the replication cycle (39). In fact, more extensive investigations identified primary CXCR4-utilizing isolates that can replicate in macrophages (40), as well as CCR5-utilizing isolates that fail to replicate in macrophages (9). In most instances, the failure of T-cell-tropic viruses to replicate in macrophages occurs at steps postentry, even though the envelope protein is the principal HIV determinant of cell tropism (29, 39). Schmidtmayerova et al. examined a panel of T-tropic isolates and found that they enter macrophages but fail to complete either reverse transcription or nuclear translocation (39). Similarly, Mori et al. found that the simian immunodeficiency virus (SIV) T-tropic molecular clone Mac239, which utilizes CCR5 to enter macrophages, fails to complete reverse transcription (29). They further demonstrated that this phenotype is the result of amino acid substitutions encoded within the viral envelope (30). To better understand how envelope-receptor interactions might restrict viral replication in macrophages, we considered a previous study in which we found that one CXCR4-utilizing, tissue culture-adapted HIV-1 molecular clone, NL4-3, which is normally restricted from growing in macrophages, is capable of overcoming that restriction when macrophages are stimulated with bacterial cell wall products (31). In light of the role of the envelope in macrophage tropism, this observation suggested that a stimulus provided by the viral envelope might also facilitate viral replication in macrophages. This hypothesis is consistent with one first put forward by Chackerian et al., which holds that CCR5 participates in early events in the postentry steps in viral replication, including reverse transcription and translocation of the viral core to the nucleus (7).

The viral envelope can stimulate primary T cells via interactions with CD4 and either CCR5 or CXCR4. In T cells, engagement of CD4 by the envelope results in phosphorylation of the tyrosine kinase Lck (45). Envelope-CCR5 signal transduction has also been reported to occur in primary T cells (12, 46). We have previously demonstrated that engagement of CCR5 by the envelope leads to increases in intracellular calcium concentrations ([Ca]i) (46). In addition, we have reported that M-tropic envelopes mediate the phosphorylation of CCR5 and its association with activated focal adhesion kinase (12). These responses parallel those elicited by treatment of primary cells with CCR5-specific CC chemokines (2, 33, 36, 38), indicating that envelope-CCR5 engagement and CC chemokine-CCR5 engagement activate overlapping signal transduction pathways. In contrast to T cells, little is known about envelope-mediated signal transduction in macrophages. Although CD4, CCR5, and CXCR4 are expressed, Lck is not. Moreover, no envelope-mediated signal transduction through either CCR5 or CXCR4 has been reported. In this study, we investigated whether the HIV envelope delivers signals to macrophages through CCR5. In addition, we have addressed the potential role of envelope-CCR5 signal transduction in providing a stimulus that promotes postentry steps in the viral life cycle. We compare the chemokine receptor signal-transducing properties of M-tropic and T-tropic envelope proteins derived from both HIV and SIV in monocyte-derived macrophages (MDMs). We found that envelopes from viruses that replicate in macrophages signal through CCR5 while those that fail to replicate in macrophages signal inefficiently or not at all through the CCR5 coreceptor. We further show that for one CCR5-utilizing isolate that signals inefficiently and fails to replicate in MDMs, the addition of exogenous CC chemokines to a culture provides the necessary stimulus to allow viral replication to occur.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Reagents and cells. RPMI 1640 (Bio-Whittaker, Walkersville, Md.) was supplemented with 10% heat-inactivated fetal calf serum (HyClone Laboratories, Ogden, Utah), 10% heat-inactivated human serum (Sigma, St. Louis, Mo.), and granulocyte-macrophage colony-stimulating factor (0.1 µg/ml) (Peprotech, Frederick, Md.). Macrophages were obtained by bead depletion of normal human blood obtained by apheresis. Monocytes were obtained by negative selection with immunomagnetic beads (StemCell Technologies, Vancouver, Canada) specific to T and B cells as specified by the manufacturer. Monocytes were cultured in six-well plates and fed every 2 days with medium supplemented with granulocyte-macrophage colony-stimulating factor. The cells were allowed to differentiate for a minimum of 8 days prior to use. All envelopes have been expressed and purified as previously described (32, 43) and are available through the AIDS Reference and Reagent Program of the Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. (www.aidsreagent.org). Envelope proteins were assessed for bioactivity by precipitation with sCD4 (data not shown). In addition, envelope proteins were visualized on silver-stained polyacrylamide gels to determine their purity and integrity. MIP-1alpha and MIP1-beta were obtained from Peprotech. All recombinant proteins were determined to be endotoxin free (less than 0.5 U/ml) prior to use by the Limulus amoebocyte lysate (LAL) method (Bio-Whittaker).

Calcium mobilization. Calcium mobilization assays were carried out as previously described (36). Briefly, >14-day-old macrophages were harvested by gentle scraping and resuspended in Hanks balanced salt solution with calcium and magnesium-10 mM HEPES. The fluorescent probe indo-1/acetoxymethylester (final concentration, 10 µM) (Molelcular Probes, Eugene, Oreg.) and pleuronic acid (final concentration, 300 µg/ml) (Molelcular Probes) were added, and the cell suspension was incubated at 30°C for 45 min with occasional gentle mixing. Cells were washed twice in fetal bovine serum (FBS). Aliquots of 106 cells in 1 ml were warmed at 37°C prior to treatment and analysis. The cells were analyzed on a FACSVantage flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif.) equipped with a Time Zero injection module (Cytek, Fremont, Calif.). Indo-1 fluorescence was measured at wavelengths of 390/20 nm (bound) and 530/20 nm (free). Fluorescence data were collected for 30 s, and then cells were injected with a buffer sham. At 60 s, envelope or chemokine was delivered in a volume of 60 µl. Concentrations of envelope or chemokine ranged from 0.2 to 200 nM. Multiple preparations of individual envelopes were tested to minimize the effects of variation between preparations. Data analysis was carried out using FLOWJO software (Treestar, Stanford, Calif.). Cells that fluoresced at a level of >15% of basal fluorescence were considered positive.

Viral strains. HIV-1 92MW959 was obtained through the AIDS Research and Reference Reagent Program of the Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health. HIV-1 Ba-L was obtained from Advanced Biotechnologies, Columbia, Md. Viral stocks were expanded by one-time passage on phytohemagglutinin-stimulated peripheral blood mononuclear cells obtained from normal donors. Supernatants were cleared of cells by centrifugation, subjected to end-point titer determination on phytohemagglutinin blasts, and stored at -70°C until use. Virus supernatants were treated with RNase-free DNase (50 U/ml; Boehringer Mannheim) for 30 min at 25°C to remove contaminating DNA prior to use.

Virus entry and reverse transcription DNA-PCR. Cells were incubated at 100,000 cells per well with virus supernatants at a multiplicity of infection of 0.01 to 0.001 for 4 h, washed three times to remove unbound virus, and frozen at -70°C. DNA PCR was performed as previously described (42). The oligonucleotide primers specific for R/U5 amplification (M667 and AA55) were used to assess viral entry. Signal intensities were measured on a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.) and compared with these of standards derived simultaneously from ACH-2 cells, a T-cell line containing a single proviral copy per cell.

Virus infection. Macrophages were inoculated with cell-free HIV isolates at a multiplicity of infection between 0.001 and 0.01/cell. Cultures were fed every 2 days. p24 antigen was measured from filtered culture supernatants by enzyme-linked immunosorbent assay (ELISA) (Dupont, Wilmington, Del.).


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

M-tropic envelopes mobilize calcium through CCR5 on MDMs. CC chemokine-mediated signal transduction through CCR5 results in the mobilization of intracellular stores of calcium (35). This mobilization occurs rapidly after treatment (within approximately 30 s), and the duration is short, lasting less than 60 s. We previously demonstrated that CCR5-specific envelope proteins mobilize calcium in T lymphocytes through CCR5 in a manner analogous to MIP1-beta stimulation of CCR5 (46). To address the capacity of HIV envelopes to signal through the CCR5 receptor on macrophages, we used a similar strategy involving a flow-cytometry based calcium mobilization assay that is more quantitative than standard fluorometric calcium mobilization assays (36). We assayed the capacity of recombinant envelopes derived from the CCR5-utilizing virus JR-FL and the CXCR4-utilizing virus NL4-3 to mobilize calcium. We also used two SIV recombinant gp120s derived from PBj 1.9 and SIV Mac239. Of note, although both PBj 1.9 and Mac239 utilize CCR5 and enter macrophages (29), only PBj 1.9 replicates in macrophage cultures (3, 17, 20). Treatment of MDMs with JR-FL envelope at 20 nM resulted in a rapid but transient mobilization of calcium (Fig. 1A). Cells were sensitive to envelope concentrations as low as 0.2 nM and typically gave a maximal response at concentrations close to 200 nM (Fig. 1H). Because treatment with 20 nM JR-FL appeared to fall within the dynamic range of the assay, further treatment with additional envelopes was carried out at this concentration (Fig. 1A to G). The kinetics of this response were similar to those observed upon treatment of MDMs with MIP-1beta (Fig. 1B). In contrast, neither JR-FL envelope nor MIP1-beta produced intracellular calcium mobilization in MDMs derived from a CCR5 Delta 32 homozygote (Fig. 1C and data not shown). These CCR5 Delta 32 MDMs did, however, flux calcium in response to SDF-1 (Fig. 1D). Although we observed interdonor differences in sensitivity to both envelope and CC chemokines, MDMs were typically responsive to the JR-FL envelope in a dose-dependent manner (Fig. 1H) from 0.2 to 20 nM. We interpret this result to indicate that there is a spectrum of responsiveness to the envelope within the population of treated MDMs. Donor macrophages responded to the envelope at concentrations as low as 0.2 nM, a level of sensitivity similar to that observed when MDMs are treated with MIP1-beta (data not shown). However, at equimolar concentrations, the envelope never induced as many cells as did MIP1-beta (data not shown). This may indicate that MIP1-beta signals more efficiently through CCR5 or, alternatively, that relatively low CD4 receptor levels on MDMs may limit envelope interactions with CCR5.


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  		FIG. 1.   Flow cytometric intracellular calcium analysis of MDMs. CCR5 wild-type MDMs (A, B, E, F, and G) or CCR5-Delta 32 MDMs (C and D) loaded with indo-1 were stimulated with 20 nM JR-FL gp140 (A and C), 20 nM MIP-1beta (B), 20 nM SDF-1 (D), 20 nM NL4-3 gp140 (E), 20 nM PBj gp120 (F), or 20 nM Mac239 gp120 (G). Titration of JR-FL gp140 against CCR5 wild-type MDMs (H) was carried out in triplicate. Data shown are representative of at least three independent experiments using different donor MDMs.

We next treated MDMs with NL4-3 envelope at 20 nM and observed no increase in [Ca]i (Fig. 1E). The same donor MDMs did respond to SDF-1 (data not shown). When MDMs were treated with PBj 1.9 gp120, they responded by mobilizing calcium (Fig. 1F). No increase in [Ca]i was observed in response to Mac239 gp120 (Fig. 1G). Finally, we treated MDMs with Mac239 and NL4-3 envelopes at 200 nM, the concentration at which we observed a near-maximal response to the JR-FL envelope, and observed no detectable response (Fig. 1H). In summary, a subset of HIV envelopes signal through CCR5 on macrophages and mobilize calcium in a manner similar in both kinetics and magnitude to mobilization by MIP1-beta . The envelopes that we assayed differed in their capacity to flux intracellular calcium in that two M-tropic envelopes, JR-FL and PBj, mobilized calcium while two T-tropic envelopes did not. We cannot preclude the possibility that at higher concentrations, NL4-3 and Mac239 envelopes could mediate a calcium response; however, any such response would be necessarily less efficient than those mediated by JR-FL or PBj envelopes.

92MW959 enters macrophages but fails to replicate. To further investigate the role of envelope-CCR5 signaling in macrophages, we tested the HIV isolate 92MW959 (21). 92MW959 is a clade C isolate that utilizes CCR5 exclusively (A. Rubbert, unpublished observation) but fails to replicate in macrophages (Fig. 2A). We first asked whether the restriction to replication of 92MW959 in MDMs results from a failure of this virus to enter macrophages. Either 92MW959 or the M-tropic isolate Bal was allowed to adsorb onto macrophages for 4 h at 37°C. The cells were washed, and DNA was isolated and subsequently analyzed by PCR for virus entry by using a primer pair designed to amplify the R/U5 region of the long terminal repeat (42, 51). 92MW959 entered MDMs, as indicated by the presence of the short long terminal repeat PCR product (Fig. 2B). This result indicates that the failure of 92MW959 to replicate in MDMs results from a block that occurs postentry. Similar observations have been reported for other primary isolates and T-cell-line-adapted isolates of HIV and SIV (9, 29, 39).


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  		FIG. 2.   Macrophage infection by HIV-1 Bal and 92MW959. (A) p24 antigen in MDM culture supernatants measured over a 12-day culture for 92MW959 and a control M-tropic isolate, HIV-1 Bal. d, day. (B) Cellular DNA PCR analysis of the entry of 92MW959 and Bal into MDMs as measured by using R/U5-specific primers (M667 and AA55).

The 92MW959 envelope signals inefficiently through CCR5. We next compared the calcium-mobilizing properties of the 92MW959 envelope and the JR-FL envelope and found that at 20 nM, JR-FL was more than threefold as efficient as 92MW959 (Fig. 3). At 2 nM, JR-FL increased the [Ca]i in more than 17% of the MDM population while the response to 92MW959 was barely detectable (Fig. 3). At 200 nM, where the response of MDMs to JR-FL envelope appears to plateau, the difference between the response of MDMs to 92MW959 and to JR-FL was greatly diminished, consistent with the saturable nature of this response. Thus, in MDMs, an envelope derived from the 92MW959 envelope signals through CCR5 significantly less efficiently than does the JR-FL envelope.


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  		FIG. 3.   Comparison of calcium mobilized in MDMs from a single donor by JR-FL gp140 and 92MW959 gp140. MDMs were treated with JR-FL gp140 or 92MW959 at a concentration of 200, 20, or 2 nM. Data shown are representative of at least three independent experiments.

CC chemokines promote the replication of 92MW959 and a CXCR4-utilizing virus in macrophages. We next determined whether the block to 92MW959 replication in MDMs could be overcome by treatment with a CC chemokine. MDMs mobilize calcium in response to both MIP-1beta (Fig. 1B), and Mip-1alpha (Fig. 4A insert). Cells were exposed to 92MW959 in the presence of 0.1 µg of MIP1-alpha per ml. In the absence of exogenously added MIP-1alpha , HIV p24 was not detected in the culture supernatant. With the addition of MIP-1alpha , increasing concentrations of p24 antigen were detected in the culture supernatant throughout the course of the experiment (Fig. 4A), demonstrating that the addition of an exogenous stimulus that signals through CCR5 removed the restriction to replication of 92MW959 in MDM cultures. The levels of replication were low and probably reflect the dichotomous activities of MIP-1alpha , which can block the entry of virus into cells at the same time as it promotes viral replication downstream of viral entry. In this regard, Kelly et al. have shown that the sequence in which MDMs are treated with CC chemokines can dramatically affect the potency with which these chemokines promote viral replication (24). We next asked whether CC chemokine treatment of MDMs could promote the replication of CXCR4-utilizing viruses in MDMs. Addition of CC chemokines to cultures inoculated with the tissue culture-adapted virus NL4-3 did not result in measurable viral replication (data not shown). However, when we inoculated MDMs with the primary CXCR4-utilizing isolate, 2005 (40), which has been reported to replicate at low levels in MDMs, we observed a significant increase in viral replication in the presence of MIP-1beta (Fig. 4B).


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  		FIG. 4.   (A) Replication of 92MW959 in the presence or absence of exogenous MIP-1alpha (0.1 µg/ml). A representative Ca+ mobilization of MDMs by MIP-1alpha is shown in the insert. (B) Replication of 2005 in the presence or absence of exogenous MIP-1beta . Viral replication was assessed by measurement of p24 antigen in culture supernatants. Data shown are representative of at least three independent experiments.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

In the present study, we have demonstrated that CCR5-specific HIV envelopes transduce signals in macrophages through CCR5. In addition, envelopes differ in their capacity to transduce these signals. Since the region of the envelope that interacts directly with CCR5 is the third variable domain (V3) (13, 37, 41, 43), the differences that we observed in the magnitude of signals transduced by different envelopes are probably the result of sequence differences within V3 that lead to qualitatively distinct interactions with CCR5. Other factors may also influence envelope-CCR5 signaling. For example, CCR5 engagement by the envelope is dependent on CD4 binding (43, 49). Thus, envelope-mediated signal transduction may also be influenced by the binding kinetics of envelope-CD4 interactions.

Increases in [Ca]i are not a necessary consequence of envelope-CCR5 interactions, nor are they necessary for virus-cell fusion. We found that the envelope derived from the T-tropic SIV Mac239, which successfully utilizes CCR5 as a fusion receptor (8), does not induce measurable increases in [Ca]i. In this regard, Edinger et al. have reported that closely related SIV envelopes, which utilize CCR5 but differ with respect to M versus T tropism, bind to different extracellular loops of CCR5 (18). We have observed that the envelope derived from PBj 1.9 competes with the anti-CCR5 monoclonal antibody 2D7 while Mac239 envelope does not (J. Arthos and A. Rubbert, unpublished observations). In light of these data and the findings presented in this study, we suggest that a highly specific binding of CCR5 by envelope is required for increased [Ca]i while a more general interaction is sufficient for virus-membrane fusion. A precise description of the structural properties of the envelope that influence calcium mobilization through CCR5 will require additional study.

Our data are consistent with a model of M-tropism in which early events in HIV replication in macrophages are facilitated by envelope-CCR5 signaling. The two M-tropic envelopes, JR-FL and PBj 1.9 increase [Ca]i, while the T-tropic envelopes we analyzed, NL4-3 and Mac239, do not increase [Ca]i or do so at a level below the sensitivity of our assay. Viral isolates corresponding to each of these envelopes enter macrophages, but only the two that transduce signals through CCR5 replicate (3, 26). Because the number of envelopes we were able to assay is limited, the correlation we have observed may not hold true for all envelopes. A more complete determination of the range of signaling mediated by different envelopes will require the production of a larger panel of recombinant envelopes. 92MW959, which is similar to Mac239, is a CCR5-utilizing isolate that fails to replicate in MDMs. Analogous to many isolates that are restricted from replication in macrophages, this isolate enters MDMs but does not complete reverse transcription. We found that an envelope derived from 92MW959 mobilizes calcium inefficiently through CCR5 relative to an envelope derived from the M-tropic isolate JR-FL, suggesting that a threshold level of signaling through CCR5 is necessary for progression of the replication cycle. We therefore asked whether the inclusion of MIP1-alpha , which signals through CCR5 and mobilizes calcium, in the culture could overcome the block to replication. In this regard, when macrophages were exposed to 92MW959 in the presence of MIP-1alpha , viral replication was observed (Fig. 4A). We also attempted to induce the replication of CXCR4-utilizing isolates in MDMs by addition of CC chemokines. We were unable to induce the replication of the tissue culture-adapted virus NL4-3; however, we were able to enhance the replication of the primary CXCR4-utilizing isolate 2005. This observation indicates that although signaling through CC chemokine receptors may promote the replication of some primary CXCR4-utilizing isolates, multiple defects may exist in tissue culture-adapted isolates which impede their ability to replicate in MDMs.

The mechanism by which envelope or CC chemokine signaling promotes replication in MDMs may involve changes in the cytoskeleton. Bukrinskaya et al. have demonstrated that after viral entry, reverse transcription complexes rapidly localize to the cytoskeletal compartment (5). In addition, they demonstrated that actin polymerization is a prerequisite for efficient reverse transcription (5). CCR5 stimulation by CC chemokines promotes actin polymerization (35). Because M-tropic envelopes share with CC chemokines the ability to transduce signals through CCR5, it will be interesting to determine whether CCR5-utilizing envelopes also induce actin polymerization. Thus, our data are consistent with a model of viral replication in macrophages in which envelope-CCR5 signaling promotes postentry events by mediating necessary changes in the cytoskeletal architecture.

The failure of most primary CXCR4-utilizing viruses to replicate in macrophages suggests that engagement of CXCR4 by the envelope does not provide a sufficient stimulus to overcome the observed block to reverse transcription. However, since some primary CXCR4-utilizing isolates can replicate in macrophages at low levels, engagement of and signaling through CCR5 cannot be an absolute requirement for replication in MDMs. It is possible that some primary CXCR4-utilizing envelopes, unlike NL4-3, provide a sufficient signal through CXCR4. Alternatively, Chackerian et al. (7) have suggested that a viral factor in addition to envelope must play a role in overcoming the block to reverse transcription in macrophages. We can speculate that a virion-associated factor, if sufficiently active, can augment or replace the stimulation provided by envelope-mediated signaling. In this regard, both Nef and Vpr are incorporated into virions (4, 14, 34, 47, 50) and have been implicated in the replication of HIV in macrophages (22, 27). Of note, the Nef protein enhances the infectivity of macrophages at an early step after viral entry (25).

In conclusion, we have demonstrated that HIV and SIV M-tropic envelopes signal in macrophages through CCR5. In contrast, two T-tropic envelopes were unable to stimulate MDMs in a similar manner. We have further shown that CCR5-utilizing HIV envelopes differ in their capacity to transduce signals through CCR5. Finally, we present data indicating that envelope-mediated CCR5 signal transduction promotes the replication of M-tropic HIV isolates in macrophages. These findings have potential implications for our understanding of the role of viral envelope-mediated signal transduction in the pathogenesis of HIV disease.


    ACKNOWLEDGMENT

James Arthos and Andrea Rubbert contributed equally to this work.


    FOOTNOTES

* Corresponding author. Mailing address: NIH, Bldg. 10, Rm. 6A-08, 10 Center Dr., Bethesda, MD 20892. Phone: (301) 402-3547. Fax: (301) 480-5244. E-mail: jarthos{at}nih.gov.

dagger Present address: Department of Clinical Medicine, University of Cologne, Cologne, Germany.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Journal of Virology, July 2000, p. 6418-6424, Vol. 74, No. 14
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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