Skip Navigation

Blood Monocytes Harbor HIV Type 1 Strains with Diversified Phenotypes Including Macrophage-Specific CCR5 Virus

  1. Younong Xu1,
  2. Haiying Zhu1,
  3. Carrie K. Wilcox1,
  4. Angélique van't Wout2,a,
  5. Thomas Andrus1,
  6. Nicholas Llewellyn1,
  7. Leonidas Stamatatos4,
  8. James I. Mullins2,
  9. Lawrence Corey1,3 and
  10. Tuofu Zhu1,3
  1. 1Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, Washington
  2. 2Department of Microbiology, University of Washington School of Medicine, Seattle, Washington
  3. 3Programs in Infectious Diseases, Fred Hutchinson Cancer Research Center, Seattle, Washington
  4. 4Seattle Biomedical Research Institute, Seattle, Washington
  1. Reprints or correspondence: Dr. Tuofu Zhu, Dept. of Laboratory Medicine, Univ. of Washington School of Medicine, Box 358070, 1959 NE Pacific St., Seattle, WA 98195-8070 (tzhu{at}u.washington.edu).

  1. Presented in part: 12th Conference on Retroviruses and Opportunistic Infections, Boston, 22–25 February 2005 (abstract D118).

 
Next Section

Abstract

Background. Recent studies have shown that blood monocytes harbor human immunodeficiency virus type 1 (HIV-1) variants that are genotypically distinguishable from those in CD4+ T cells. However, the biological function of monocyte-derived HIV-1 remains unclear.

Methods. Using pseudovirus assay, we analyzed the phenotype conferred by monocyte-derived HIV-1 envelopes from 8 patients.

Results. All pseudoviruses carrying monocyte-derived HIV-1 envelopes used CCR5; however, their use of additional coreceptors delineated 4 phenotypes in which viruses used (1) CCR5 only, (2) CCR5 and CXCR4, (3) CCR3 and CCR5, or (4) multiple coreceptors, including CCR1, CCR3, GPR15, CCR5, and CXCR4. More importantly, we observed 2 distinct cell tropism phenotypes for pseudoviruses carrying monocyte-derived envelopes: (1) monocytederived, macrophage-specific R5 (MDMS-R5) virus that, using CCR5 only, could infect monocyte-derived macrophages (MDMs) but not CD4+ T cells and (2) dual tropic virus that infected both MDMs and primary CD4+ T cells. We found blood monocytes harboring viruses with multiple phenotypes as early as 25 days before seroconversion and as late as 9 years after seroconversion.

Conclusions. These data suggest that HIV-1 circulating in blood monocytes represents diverse HIV-1 with multiple phenotypes and that MDMS-R5 viruses may play an important role in infection with and persistence of HIV-1 within the monocyte/macrophage lineage.

Cells of the monocyte/macrophage lineage, including monocyte subsets within the blood, play an important role in HIV-1 persistence. Monocyte/macrophage tropism may also facilitate the transmission and establishment of HIV-1 infection in the new host [1–3]. Moreover, macrophage-tropic (i.e., M-tropic) HIV-1 variants are detectable during all stages of HIV-1 infection [4]. The chemokine receptor CCR5 serves as the principal coreceptor for entry of M-tropic HIV-1 into CD4-expressing T cells and monocytes/macrophages [5–7]. Simian immunodeficiency virus variants isolated from sooty mangabeys and chimpanzees inefficiently infect macrophages in vitro and are considerably impaired in their pathogenicity in vivo [8]. After infection with HIV-1, macrophages release several immunoregulatory and inflammatory factors, including tumor necrosis factor—α, interleukin (IL)—1, and IL-7, which, in turn, influence viral proliferation and disease associated with HIV-1 infection [9].

To understand the interaction between monocytes/macrophages and HIV-1 in vivo, as well as the association of this interaction with the pathogenesis of HIV-1 in patients, it is critical to study the HIV-1 strains that naturally exist in the monocytes and macrophages of patients. Several M-tropic HIV-1 variants have been previously isolated from unfractionated cellular and tissue sources. HIVBaL was derived from bronchopulmonary lavage cells with a 90% purity of lung macrophages [10]. HIVJRFL was recovered by coculturing normal peripheral blood lymphocytes (PBLs) activated by phytohemagglutinin with brain tissue from the frontal lobe that contained blood macrophages, brain microglial cells, and PBLs, among other cellular components, obtained from a patient who died with HIV dementia [11]. The wellcharacterized HIVADA was isolated by coculturing the peripheral blood mononuclear cells (PBMCs) of a patient with healthy, blood-derived monocytes [12]. Thus, none of these M-tropic HIV-1 strains were isolated from patients' purified monocytes/macrophages that were free of CD4+ T cell contamination. In addition, there are few published data on the coreceptor use and cell tropism of HIV-1 naturally existing in monocytes, most likely because monocytes have not been shown to support HIV-1 replication in vitro [13–15].

However, a recent study [16] demonstrated that HIV-1 replication occurs in monocytes in vivo, as was shown by the detection of HIV-1 transcripts (mRNA) and viral evolution in the blood monocytes of patients effectively treated with highly active antiretroviral therapy (HAART). Previous demonstrations of the recovery of replication-competent HIV-1 from the blood monocytes of patients receiving suppressive HAART strongly support this hypothesis [17, 18]. Furthermore, we have demonstrated that monocytes can harbor HIV-1 variants that are genetically distinguishable from those present in CD4+ T cells and that HIV-1 in blood monocytes is genetically identical to—or associated with—viral variants found in the blood plasma of patients after receipt of HAART for a prolonged period [16, 19, 20]. Other investigators have shown similar results for distinct HIV-1 populations in the blood monocytes and CD4+ T cells of patients who received suppressive HAART for a prolonged period [21, 22]. Taken together, these studies suggest that HIV-1 circulating in blood monocytes represents the residual replicating HIV-1 that might escape from antiretroviral therapy and might be produced from tissues macrophages [16, 19, 20, 23, 24].

In previous studies of HIV-1 circulating in blood monocytes, we used a 2-step strategy to isolate highly purified monocytes from peripheral blood [19] for polymerase chain reaction (PCR) analyses of DNA sequences of HIV-1 in blood monocytes [16, 19, 20]. In the present study, we determined the phenotypes of monocyte HIV-1 by use of the genotyped HIV-1 envelopes derived from highly purified monocytes in pseudovirus assays. Our results indicate that blood monocytes harbor diversified phenotypes of HIV-1, with multiple coreceptor use and cell tropisms throughout the course of HIV-1 infection. More importantly, we identified a group of monocyte-derived, macrophage-specific CCR5 (MDMS-R5) HIV-1 strains that use CCR5 as a coreceptor and infect primary MDMs but not primary CD4+ T cells.

Previous SectionNext Section

Materials and Methods

Study population. We examined 8 patients enrolled at the Primary Infection Clinic of the University of Washington (Seattle). The scientific and ethics review committees of the University of Washington approved the study, and written informed consent was obtained from all study participants. The human experimentation guidelines of the US Department of Health and Human Services, as well as those of the institutions of the investigators in the current study, were followed in obtaining clinical samples. Demographic and clinical data for patients are summarized in table 1. Of the 8 patients enrolled, 3 (patients 4 and 6) were receiving HAART from a point in time near seroconversion; 2 (patients 2 and 8) began receiving HAART 1520 and 3354 days after seroconversion, respectively; and 3 (patients 1, 3, and 7) had not received therapy. Longitudinal samples were collected over a period ranging from 25 days before seroconversion to 3409 days after seroconversion. The 3 therapy-naive patients (patients 1, 3, and 7) had relatively low viral loads, whereas all 5 patients receiving HAART maintained very low viral loads. Heterozygous CCR5 Δ32 genotypes were noted in 3 patients (patients 1, 3, and 8), whereas wild-type CCR5 was found in the remaining 5 patients.

Purification of monocytes and CD4+ T cells and amplification and cloning of HIV-1 envelopes from purified monocytes. A 2-step purification method (involving negative monocyte selection, followed by positive monocyte selection with the use of MicroBeads [Miltenyi Biotec]) [19, 20] was used to isolate highly purified CD14+ monocytes from the PBMCs of the patients. The purity of monocytes from all 8 patients, as determined using this method, was 99.11%–99.72%, with an average purity of 99.50% (figure 1) [20], as determined by fluorescence-activated cell sorting (FACS). The purified monocytes had no detectable cross-contamination of CD3+ T cells, including CD3+/CD4+ T cells, when analyzed using FACS (figure 1) or tested using semiquantitative reverse-transcriptase PCR analysis of T cell receptor (TcR) mRNA (detection limit, 10 TcR mRNA copies from 104 monocytes or 0.1% T cell contamination), as described elsewhere [17, 19, 20]. This method ensured our ability to amplify monocyte-derived HIV-1 fragments without detectable contamination from CD4+ T cells [19, 20]. Similarly, CD4+ T cells were purified first by use of the CD4+ T Cell Isolation Kit II (negative selection) and then by use of CD4+ Microbeads, each according to the manufacturer's protocols (Miltenyi Biotec) [19, 20]. Outer primers P17-2 and P18 and inner primers P15 and PE4 were used to amplify HIV-1 gp160 [16, 19, 25]. The PCR products of HIV-1 envelopes were directly cloned into the eukaryotic TA expression vector pCR3.1 (Invitrogen; Life Technologies). All sequences were aligned using Clustal W and were edited using MacClade software (version 4.06; Sinauer Associates), as described elsewhere [16, 19].

Figure 1
View larger version:
Figure 1

Estimation of T cell contamination of isolated CD14+ monocytes. A, Two-color fluorescence analysis of monocytes purified from patient 1. B, Purity of monocytes isolated from all 8 patients. *Percentage of CD14+/ CD3− monocytes. Purified cells were stained with a fluorescein isothiocyanate (FITC)— conjugated anti-CD14 or phycoerythrin (PE)— conjugated anti-CD3 monoclonal antibody (A, right). The cells were also stained with FITC- or PE-conjugated isotype IgG2a to control for nonspecific binding (A, left). The average purity of CD14+ monocytes (±SD) was 99.5% ± 0.21%. **Percentage of T cell receptor (TcR) mRNA within purified patient monocytes assessed by reverse-transcriptase polymerase chain reaction (RT-PCR). The detection limit of the RT-PCR is 10 copies TcR mRNA/104 monocytes or 0.1% T cell contamination. ND, not done; P1…P8, patient 1…patient 8.

Vectors. The Photinus pyralis luciferase expression vector pNL4-3-Luc-E−R− was provided by the National Institutes of Health AIDS Research and Reference Reagent Program [6]. The env expression vectors PCR3.1IIIB, PCR3.1ADA, PCR3.1JRCSF, PCR3.1SF2, and PCR3.1AMV were constructed by insertion of the whole envelope from IIIB, ADA, JRCSF, SF2, and AMV viruses into the TA expression vector PCR3.1. The full-length gp160 fragments amplified from 8 patients were inserted into pCR3.1 to construct env expression vectors.

Coreceptor use prediction by canonical site and position-specific scoring matrices (PSSMs). Coreceptor prediction by use of the PSSM method is described elsewhere [26]. In brief, a “training set” of third hypervariable (V3) amino acid sequences from viruses of known phenotype was used to generate a matrix of likelihood ratio scores for each site in the sequence. In general, the higher the score, the more similar the given V3 sequence is to an average actual X4 sequence, and the lower the score, the more similar the given V3 sequence is to an average actual R5 sequence. Coreceptor prediction by the canonical site method, in which a sequence is predicted to be X4 when it harbors arginine or lysine at V3 site 11 and/or site 25, is widely used for sequencebased prediction of the HIV-1 phenotype [27, 28].

Coreceptor determination with pseudoviruses. Luciferase reporter viruses were prepared in 293T cells by cotransfection of pNL4-3-Luc-E−R− vector and env expression plasmids with the use of lipofectamine, as described elsewhere [6]. Non—envelope-carrying pseudovirus was used as a background control. Pseudoviruses in supernatants of transfected 293T cells were quantified using a p24 ELISA Kit (NCI-Frederick; SAIC-Frederick). U87 cells expressing CD4 and coreceptors were used for infection with pseudoviruses as described elsewhere [6]. The luciferase activity of lysate was measured using a Fluoroskan AscentFL luminometer (MTX Labs) 2 days after infection. Coreceptor use was then determined by assessing the luciferase activity of viruses from each patient's HIV-1 envelope, compared with that of the pCR3.1 vector—based pseudoviruses in either U87.CD4.CXCR4 or U87.CD4.CCR5 cells [5–7]. Use of other chemokine receptors, such as CCR1, CCR2b, CCR4, CCR8, and BOB/GPR15, was determined using ghost cells expressing CD4 and corresponding coreceptors [29].

Infection of macrophages and CD4+ T cells with pseudoviruses. To determine the cell tropism of the HIV-1 variants, pseudoviruses carrying the HIV-1 envelopes isolated from the monocytes and CD4+ T cells of patients were used to infect purified MDMs and CD4+ T cells derived from the same healthy donor for each experiment. In brief, freshly isolated monocytes were seeded into 48-well plates at 200,000 cells/well and were cultured for 7 days with Iscove's modified Dulbecco's medium (Invitrogen) containing 10% human serum. Thereafter, the MDMs were infected with 20 ng/mL pseudovirus. After 2 days, infection of macrophages was determined by assessing luciferase activity, as described elsewhere [5–7]. Purified CD4+ T cells were stimulated with 2 μg/mL phytohemagglutinin (Sigma) for 72 h and infected with pseudoviruses in Iscove's modified Dulbecco's medium containing 10% human serum and 50 U/mL IL-2 (Gibco).

Previous SectionNext Section

Results

V3 loop sequence diversity and the predicted phenotype of HIV-1 clones isolated from monocytes. As expected, the V3 loop sequences of HIV-1 env from monocytes were relatively homogeneous at the early stages of infection and heterogeneous at later stages (table 2). The syncytium-inducing (SI) properties of T cell lines, as well as the CCR5 versus CXCR4 coreceptor specificities of HIV-1, are predicted by a canonical site change at amino acid position 11 or 25 in the V3 region [27, 28], as well as by computational tools that include the entire V3 loop sequence, including a PSSM [26]. Most sequences from all 8 patients had V3 sequences corresponding to the M-tropic, non—syncytium-inducing (NSI), and CCR5 phenotype (referred to as “NSI/R5 viruses”) by both the canonical site and PSSM methods. In addition, at 25 days before seroconversion, SI/X4 viruses were predicted by use of both methods in patient 4 (clone P4-1M-5); at a later stage of infection, SI/X4 viruses were predicted as a minor variant in patient 7 (clone P7-4M21) and patient 8 (clones P8-2M9 and P8-4M16). However, all 3 clones from patient 3 had canonical site changes that were predictive of X4 but were scored as R5 in the PSSM. In addition, P8-4M1, but not P8-4M16, was scored as X4 in the PSSM.

Table 1
View larger version:
Table 1

Demographic and clinical characteristics of study subjects.

Table 2
View larger version:
Table 2

V3 loop sequence alignment and coreceptor use of the monocyte-derived HIV-1 envelope.

We observed that there is a discrepancy between the predicted phenotype, as determined by canonical site or PSSM, and the observed phenotype in vitro, as determined using pseudoviruses (see below; all clones in patient 3 and clone P8-4M16 in patient 8). The computational predictions are based on the charges of V3 loop sequences or the canonical site at positions 11 and 25 in the V3 loop, without consideration of the role of other regions, such as V2, V4, and V5, which also influence coreceptor use. Coreceptor use determined by experimental observation is based on HIV-1 envelopes from patients that also contain diverse sequences in regions other than the V3 loop, such as V1, V2, V4, and V5. Our data suggest that, in addition to sequences of the V3 loop, other regions, such as V1, V2, V4, and V5, should be considered when coreceptor use is predicted.

Four phenotypes of coreceptor use of HIV-1 isolated from monocytes. To further assess coreceptor use of HIV-1 isolated from monocytes, pseudoviruses carrying HIV-1 envelope derived from monocytes were used to infect cell lines (U87 or ghost cells) expressing CD4 and various coreceptors. Infection was then evaluated by measuring luciferase activity. A total of 20 HIV-1 envelope clones isolated from monocytes at various points in time, from seroconversion to 9 years after seroconversion, were tested (tables 2 and 3). All pseudoviruses with monocyte-derived HIV-1 envelopes utilized CCR5. However, 4 distinct phenotypes of coreceptor use were observed with monocyte-envelope pseudoviruses. In the “exclusive R5 use” phenotype, all pseudoviruses with HIV-1 envelopes isolated from patients 5, 6, and 8, including those isolated at early stages of infection and at a time point of 9 years after seroconversion, used CCR5 exclusively. In the “dual R5/X4 use” phenotype, pseudoviruses with envelopes from 4 patients (patients 1, 2, 4, and 7) could use both CCR5 and CXCR4. Interestingly, the dual-R5/X4 viruses appeared 25 days before seroconversion in patient 4 and 7 days after seroconversion in patient 1, in the absence of HAART. In the “dual R5/R3 use” phenotype, pseudoviruses with envelopes from 4 patients, including all clones from patient 3, as well as one-third of the clones from patient 7, utilized CCR3 in addition to CCR5 for infection. In the multiple coreceptor use phenotype, some HIV-1 envelopes derived from monocytes showed broader use of coreceptors, including CCR1, CCR3, CCR5, and CXCR4 (at a time near seroconversion for patients 1 and 3 and at 8 years after seroconversion for patient 2), as well as GPR15, CCR5, and CXCR4 (before seroconversion, for patient 4).

Table 3
View larger version:
Table 3

Coreceptor use and cell phenotype of pseudoviruses carrying HIV-1 env from patients' monocytes.

Two phenotypes of cell tropism of HIV-1 isolated from monocytes. We tested the infectivity of these pseudoviruses in both purified MDMs and CD4+ T cells derived from the same HIV-seronegative donor for each experiment. All 20 clones isolated from monocytes were able to infect macrophages efficiently, and 12 could also infect CD4+ T cells (table 3). Pseudoviruses with 2 distinct phenotypes of cell tropism were observed. The first group included monocyte-derived, macrophagespecific, R5-using (i.e., MDMS-R5) viruses. Pseudoviruses carrying monocyte-derived envelopes from 3 patients (patients 5, 6, and 8), while using CCR5 only, could infect macrophages but not CD4+ T cells (table 3). The luciferase activities of these MDMS-R5 viruses infecting macrophages were consistent between the 2 donors. One clone (P7-4M23) from patient 7 was also macrophage specific while using both CCR5 and CCR3. The second group included dual M-tropic/T cell tropic(T-tropic) viruses. Pseudoviruses carrying monocyte-derived envelopes from 5 patients (patients 1–4 and patient 7) could infect both macrophages and CD4+ T cells while using multiple coreceptors (table 3). Although the luciferase activity of several pseudoviruses infecting macrophages was not as robust as that of the indicator cell lines, these data are consistent with a recent report using pseudoviruses carrying HIV-1 envelopes derived from brain tissue to infect macrophages [30].

Association of coreceptor use with cell tropism for HIV-1 clones isolated from monocytes. As shown in table 4, all 7 clones exclusively using CCR5 infected macrophages, although none of them could infect CD4+ T cells. In contrast, all clones with multiple coreceptor use (except clone P7-4M23, with coreceptor use of R5R3) could infect both CD4+ T cells and macrophages efficiently, suggesting an association between multiple coreceptor use and multiple cell tropisms for the HIV-1 clones isolated from monocytes.

Table 4
View larger version:
Table 4

Correlation of coreceptor use with cell tropism in monocyte-derived HIV-1 clones.

Previous SectionNext Section

Discussion

Previous studies from us and from other investigators showed that peripheral blood monocytes can harbor genetically diversified HIV-1 variants that are distinguishable from those present in blood CD4+ T cells [16, 19–22]. In the present study, we further demonstrated that HIV-1 strains in monocytes are also diverse in phenotype, with multiple coreceptor use and cell tropisms. In addition, we identified a group of HIV-1 strains that exclusively use CCR5 as a coreceptor and infect primary macrophages but not primary CD4+ T cells. Given that blood monocytes circulate in the peripheral blood for only a few days (half-life, 1–3 days), the diverse HIV-1 variants isolated from monocytes should be from sources of replicating virus that have recently infected blood monocytes.

One potential source of monocyte HIV-1 may be tissue macrophages that are productively infected and serve as a source of virus, especially in patients with end-stage disease [14, 31–37]. Recent studies indicated that, in patients receiving suppressive antiretroviral therapy, non-CD4+ T lymphocytes, including mainly monocytes/macrophages, are the major sources of plasma HIV-1 [16, 19, 23, 24, 38, 39]. In our study, 7 of 20 pseudoviruses with envelope clones isolated from blood monocytes at different time points, including a time point 9 years after seroconversion, used CCR5 and infected macrophages but not CD4+ T cells (tables 3 and 4). A previous study showed that recombinant HIV-1 viruses carrying C1 to the V3 region of envelope, amplified from the PBMCs of a long-term HIV-positive patient without clinical progression, demonstrated efficient growth in macrophages from several donors but attenuated growth in PBLs from different donors [40]. We cannot dismiss the possibility of an association between the HIV-1 gp160 of our patient carrying MDMS-R5 and the HIV-1 C1-V3 of the previously reported patient carrying the recombinant HIV-1. The exclusive infection of macrophages by these MDMS-R5 viruses in vitro may support the hypothesis that virus might be produced in tissue macrophages and released into the blood and then might preferentially infect blood monocytes that may differentiate into tissue macrophages [19, 20, 24]. Selective cell tropism via specific coreceptor(s) could enhance the infectivity of certain HIV-1 variants to specific target cells, such as monocytes/macrophages or CD4+ T cells, and would help to explain the compartmentalization observed between HIV-1 in blood monocytes and in CD4+ T cells [16, 19–23, 41].

The mechanism by which these MDMS-R5 viruses infect macrophages but not CD4+ T cells remains unclear. This MDMS-R5 HIV-1 phenotype may be related to the phenotype recently reported, which could use both CCR5 and CXCR4 to infect macrophages but could not utilize CCR5 to infect CD4+ T lymphocytes [42]. The restricted ability of these MDMS-R5 virus strains to use CCR5, depending on the cellular context, suggests that they likely differ from prototype R5 isolates in how they interact with CCR5 [42]. That the MDMS-R5 HIV-1 phenotype has not been identified previously may be a result of the fact that most HIV-1 strains studied in vitro are derived from isolates from PMBCs or lymphocyte-derived cloning.

Evidence indicates that a subset of monocytes (CD14low-CD16high) is increased in HIV-1—infected patients [43] and expresses higher levels of CCR5, which may potentially increase monocyte susceptibility to HIV infection [23]. Also, CD16+ monocytes have been shown to contribute to immune activation and HIV replication in resting T cells [44]. This CD16+ monocyte subset was further shown to be more permissive to infection and to preferentially harbor HIV-1 in vivo [45]. The CD14+ monocyte populations used to isolate HIV-1 envelopes in the present study should include diverse monocyte subsets (CD14lowCD16high, CD14highCD16−, and CD14highCD16high). The correlation of the HIV-1 phenotype including MDMS-R5 with the CD16+ monocyte subset warrants further investigation.

The unexpected finding from the present study is that some HIV-1 variants identified in blood monocytes use coreceptors such as CXCR4, CCR3, CCR1, and GPR15, in addition to CCR5, and infect CD4+ T cells (tables 3 and 4). It is possible that a portion of the HIV-1 population in blood monocytes may originate from CD4+ T cells or other cell or tissue compartments. The emergence of T-tropic HIV-1 in blood monocytes could indicate the ability of blood monocytes to support infection by HIV-1 of various phenotypes. However, the discovery of monocyte-derived HIV-1 provirus with T cell tropism in the present study does not necessarily indicate that T-tropic virus would productively replicate in monocytes/macrophages, because a previous study showed that replication of T-tropic virus in macrophages was blocked after entry [46]. Alternatively, such precursors as CD34+ progenitor cells in bone marrow could serve as target cells and become infected by HIV-1 variants with different phenotypes [47–50], and they could carry the virus through differentiation into blood monocytes (as reviewed in [24, 41]).

Interestingly, all monocyte viruses isolated from persons with early and late infection used CCR5 and infected macrophages (table 3), suggesting an important role of monocytes and related macrophages in the establishment and persistence of HIV-1 infection [1–4, 19, 27] (as reviewed in [23, 24, 41]). However, the dual M- and T-tropic viruses using dual-R5/X4 coreceptors appeared 7 days after seroconversion (in patient 1) and 25 days before seroconversion (in patient 4), in the absence of HAART. For patient 1, who is heterozygous for CCR5Δ32, the viruses infecting both macrophages and CD4+ T cells with multiple coreceptor use (R5/X4/R3) may have a selective advantage over the R5 virus. CCR3 use by these monocyte viruses may contribute to the dissemination of viral infection into other cell types expressing CCR3, such as microglia and T cells.

Taken together, these findings indicate that blood monocytes can harbor macrophage-specific MDMS-R5 viruses, as well as HIV-1 strains with multiple phenotypes at early and late stages of HIV-1 infection, with a majority of these viruses able to infect both macrophages and CD4+ T cells efficiently. These results suggest that blood monocytes, together with related tissue macrophages, may play an important role in the transmission, establishment, and persistence of HIV-1 infection.

Previous SectionNext Section

Acknowledgments

We thank Dr. Dan R. Littman for providing, through the AIDS Research and Reference Program of the National Institutes of Health, U87.CD4.CCR5 and U87.CD4.CXCR4 cells, pNL4-3-Luc-E−R−, and the human osteosarcoma cell line—derived GHOST (Hi 3) stably expressing CD4 together with 1 of the various coreceptors (CCR1, CCR2b, CCR3, CCR4,CXCR4, CCR5, CCR8, CXCR4/CCR5 or Bob/GOP15). We also thank M. Jenkins and C. Dai for technical assistance.

Previous SectionNext Section

Footnotes

  • ↵a Present affiliation: Department of Clinical Viro-Immunology, Sanquin Research, Plesmanlaan, Amsterdam, The Netherlands.

  • Potential conflicts of interest: none reported.

  • Financial support: Public Health Service (grants AI45402, AI49109, and AI55336 [to T.Z.]); National Institutes of Health (grants R01 HL072631 and P50 HG02360 [to A.v.W.]).

  • Received March 23, 2007.
  • Accepted July 6, 2007.
Previous Section
 

References

  1. ↵
    1. Roos M,
    2. Lange J,
    3. de Goede R,
    4. et al
    . Viral phenotype and immune response in primary human immunodeficiency virus type 1 infection. J Infect Dis 1992;165:427-32.
    Abstract/FREE Full Text
    1. Zhu T,
    2. Mo H,
    3. Wang N,
    4. et al
    . Genotypic and phenotypic characterization of HIV-1 patients with primary infection. Science 1993;261:1179-81.
    Abstract/FREE Full Text
  3. ↵
    1. van't Wout AB,
    2. Kootstra NA,
    3. Mulder-Kampinga GA,
    4. et al
    . Macrophage-tropic variants initiate HIV-1 infection after sexual, parenteral, and vertical transmission. J Clin Invest 1994;94:2060-7.
    MedlineWeb of Science
  4. ↵
    1. Schuitemaker H,
    2. Kootstra N,
    3. de Goede R,
    4. de Wolf F,
    5. Miedema F,
    6. Tersmette M
    . Monocytotropic human immunodeficiency virus type 1 (HIV-1) variants detectable in all stages of HIV-1 infection lack T-cell line tropism and syncytium-inducing ability in primary T-cell culture. J Virol 1991;65:356-63.
    Abstract/FREE Full Text
  5. ↵
    1. Alkhatib G,
    2. Combadiere C,
    3. Broder C,
    4. et al
    . CC CKR5: a RANTES, MIP- 1α, MIP-1β receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 1996;272:1955-8.
    Abstract/FREE Full Text
  6. ↵
    1. Deng H,
    2. Liu R,
    3. Ellmeier W,
    4. et al
    . Identification of a major co-receptor for primary isolates of HIV-1 [see comments]. Nature 1996;381:661-6.
    CrossRefMedline
  7. ↵
    1. Dragic T,
    2. Litwin V,
    3. Allaway GP,
    4. et al
    . HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 1996;381:667-73.
    CrossRefMedline
  8. ↵
    1. Hirsch VM,
    2. Sharkey ME,
    3. Brown CR,
    4. et al
    . Vpx is required for dissemination and pathogenesis of SIV(SM) PBj: evidence of macrophage-dependent viral amplification. Nat Med 1998;4:1401-8.
    CrossRefMedlineWeb of Science
  9. ↵
    1. Bendinelli M,
    2. Pistello M,
    3. Lombardi S,
    4. et al
    . Feline immunodeficiency virus: an interesting model for AIDS studies and an important cat pathogen. Clin Microbiol Rev 1995;8:87-112.
    Abstract/FREE Full Text
  10. ↵
    1. Gartner S,
    2. Markovits P,
    3. Markovitz D,
    4. Betts R,
    5. Popovic M
    . Virus isolation from and identification of HTLV-III/LAV-producing cells in brain tissue from a patient with AIDS. JAMA 1986;256:2365. [abstract].
    Abstract/FREE Full Text
  11. ↵
    1. Koyanagi Y,
    2. Miles S,
    3. Mitsuyasu RT,
    4. Merrill JE,
    5. Vinters HV,
    6. Chen ISY
    . Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms. Science 1987;236:819-22.
    Abstract/FREE Full Text
  12. ↵
    1. Gendelman HE,
    2. Orenstein JM,
    3. Martin MA,
    4. et al
    . Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes. J Exp Med 1988;167:1428-41.
    Abstract/FREE Full Text
  13. ↵
    1. Collman R,
    2. Hassan NF,
    3. Walker R,
    4. et al
    . Infection of monocyte-derived macrophages with human immunodeficiency virus type 1 (HIV-1): monocyte-tropic and lymphocyte-tropic strains of HIV-1 show distinctive patterns of replication in a panel of cell types. J Exp Med 1989;170:1149-63.
    Abstract/FREE Full Text
  14. ↵
    1. Sonza S,
    2. Maerz A,
    3. Deacon N,
    4. Meanger J,
    5. Mills J,
    6. Crowe S
    . Human immunodeficiency virus type 1 replication is blocked prior to reverse transcription and integration in freshly isolated peripheral blood monocytes. J Virol 1996;70:3863-9.
    Abstract/FREE Full Text
  15. ↵
    1. Naif HM,
    2. Li S,
    3. Alali M,
    4. et al
    . Definition of the stage of host cell genetic restriction of replication of human immunodeficiency virus type 1 in monocytes and monocyte-derived macrophages by using twins. J Virol 1999;73:4866-81.
    Abstract/FREE Full Text
  16. ↵
    1. Zhu T,
    2. Muthui D,
    3. Holte S,
    4. et al
    . Evidence for human immunodeficiency virus type 1 replication in vivo in CD14+ monocytes and its potential role as a source of virus in patients on highly active antiretroviral therapy. J Virol 2002;76:707-16.
    Abstract/FREE Full Text
  17. ↵
    1. Sonza S,
    2. Mutimer HP,
    3. Oelrichs R,
    4. et al
    . Monocytes harbour replicationcompetent, non-latent HIV-1 in patients on highly active antiretroviral therapy. AIDS 2001;15:17-22.
    CrossRefMedlineWeb of Science
  18. ↵
    1. Lambotte O,
    2. Taoufik Y,
    3. de Goër MG,
    4. Wallon C,
    5. Goujard C,
    6. Delfraissy JF
    . Detection of infectious HIV in circulating monocytes from patients on prolonged highly active antiretroviral therapy. J Acquir Immune Defic Syndr 2000;23:114-9.
    MedlineWeb of Science
  19. ↵
    1. Fulcher JA,
    2. Hwangbo Y,
    3. Zioni R,
    4. et al
    . Compartmentalization of human immunodeficiency virus type 1 between blood monocytes and CD4+ T cells during infection. J Virol 2004;78:7883-93.
    Abstract/FREE Full Text
  20. ↵
    1. Llewellyn N,
    2. Zioni R,
    3. Zhu H,
    4. et al
    . Continued evolution of HIV-1 circulating in blood monocytes with antiretroviral therapy: genetic analysis of HIV-1 in monocytes and CD4+ T cells of patients with discontinued therapy. J Leukoc Biol 2006;80:1118-26.
    Abstract/FREE Full Text
  21. ↵
    1. Delobel P,
    2. Sandres-Sauné K,
    3. Cazabat M,
    4. et al
    . Persistence of distinct HIV-1 populations in blood monocytes and naive and memory CD4 T cells during prolonged suppressive HAART. AIDS 2005;19:1739-50.
    MedlineWeb of Science
  22. ↵
    1. Potter SJ,
    2. Lemey P,
    3. Achaz G,
    4. et al
    . HIV-1 compartmentalization in diverse leukocyte populations during antiretroviral therapy. J Leukoc Biol 2004;76:562-70.
    Abstract/FREE Full Text
  23. ↵
    1. Crowe S,
    2. Zhu T,
    3. Muller WA
    . The contribution of monocyte infection and trafficking to viral persistence, and maintenance of the viral reservoir in HIV infection. J Leukoc Biol 2003;74:635-41.
    Abstract/FREE Full Text
  24. ↵
    1. Zhu T
    . HIV-1 in peripheral blood monocytes: an underrated viral source. J Antimicrob Chemother 2002;50:309-11.
    FREE Full Text
  25. ↵
    1. Zhu T,
    2. Wang N,
    3. Carr A,
    4. et al
    . Genetic characterization of human immunodeficiency virus type 1 in blood and genital secretions: evidence for viral compartmentalization and selection during sexual transmission. J Virol 1996;70:3098-107.
    Abstract/FREE Full Text
  26. ↵
    1. Jensen MA,
    2. Li FS,
    3. van't Wout AB,
    4. et al
    . Improved coreceptor usage prediction and genotypic monitoring of R5-to-X4 transition by motif analysis of human immunodeficiency virus type 1 env V3 loop sequences. J Virol 2003;77:13376-88.
    Abstract/FREE Full Text
  27. ↵
    1. Fouchier RAM,
    2. Groenink M,
    3. Kootstra NA,
    4. et al
    . Phenotype-associated sequence variation in the third variable domain (V3) of the human immunodeficiency virus type 1 gp120 molecule. J Virol 1992;66:3183-7.
    Abstract/FREE Full Text
  28. ↵
    1. Chesebro B,
    2. Nishio J,
    3. Perryman S,
    4. et al
    . Identification of human immunodeficiency virus envelope gene sequences influencing viral entry into CD4-positive Hela cells, T-leukemic cells and macrophages. J Virol 1991;65:5782.
    Abstract/FREE Full Text
  29. ↵
    1. Morner A,
    2. Bjorndal A,
    3. Albert J,
    4. et al
    . Primary human immunodeficiency virus type 2 (HIV-2) isolates, like HIV-1 isolates, frequently use CCR5 but show promiscuity in coreceptor usage. J Virol 1999;73:2343-9.
    Abstract/FREE Full Text
  30. ↵
    1. Peters PJ,
    2. Bhattacharya J,
    3. Hibbitts S,
    4. et al
    . Biological analysis of human immunodeficiency virus type 1 R5 envelopes amplified from brain and lymph node tissues of AIDS patients with neuropathology reveals two distinct tropism phenotypes and identifies envelopes in the brain that confer an enhanced tropism and fusigenicity for macrophages. J Virol 2004;78:6915-26.
    Abstract/FREE Full Text
  31. ↵
    1. Gartner S,
    2. Markovits P,
    3. Markovitz DM,
    4. Kaplan MH,
    5. Gallo RC,
    6. Popovic M
    . The role of mononuclear phagocytes in HTLV-III/LAV infection. Science 1986;233:215-9.
    Abstract/FREE Full Text
    1. Wiley CA,
    2. Schrier RD,
    3. Nelson JA,
    4. Lampert PW,
    5. Oldstone MBA
    . Cellular localization of human immunodeficiency virus infection within the brain of acquired inmmune deficiency syndrome patients. Proc Natl Acad Sci USA 1986;83:7089-93.
    Abstract/FREE Full Text
    1. Ho DD,
    2. Rota TR,
    3. Hirsch MS
    . Infection of monocyte/macrophages by human T lymphotropic virus type III. J Clin Invest 1986;77:1712-5.
    MedlineWeb of Science
    1. Orenstein JM,
    2. Fox C,
    3. Wahl SM
    . Macrophages as a source of HIV during opportunistic infections. Science 1997;276:1857-61.
    Abstract/FREE Full Text
    1. Embretson J,
    2. Zupancic M,
    3. Ribas JL,
    4. et al
    . Massive covert infection of helper T lymphocytes and macrophages by HIV during the incubation period of AIDS. Nature 1993;362:359-62.
    CrossRefMedline
    1. Cavert W,
    2. Haase AT
    . A national tissue bank to track HIV eradication and immune reconstruction. Science 1998;280:1865-6.
    Abstract/FREE Full Text
  37. ↵
    1. Veazey RS,
    2. DeMaria M,
    3. Chalifoux LV,
    4. et al
    . Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection. Science 1998;280:427-31.
    Abstract/FREE Full Text
  38. ↵
    1. Chun TW,
    2. Davey RT Jr.,
    3. Ostrowski M,
    4. et al
    . Relationship between preexisting viral reservoirs and the re-emergence of plasma viremia after discontinuation of highly active anti-retroviral therapy [see comments]. Nat Med 2000;6:757-61.
    CrossRefMedlineWeb of Science
  39. ↵
    1. Bailey JR,
    2. Sedaghat AR,
    3. Kieffer T,
    4. et al
    . Residual human immunodeficiency virus type 1 viremia in some patients on antiretroviral therapy is dominated by a small number of invariant clones rarely found in circulating CD4+ T cells. J Virol 2006;80:6441-57.
    Abstract/FREE Full Text
  40. ↵
    1. Grovit-Ferbas K,
    2. Ferbas J,
    3. Gudeman V,
    4. et al
    . Potential contributions of viral envelope and host genetic factors in a human immunodeficiency virus type 1-infected long-term survivor. J Virol 1998;72:8650-8.
    Abstract/FREE Full Text
  41. ↵
    1. Zhu T
    . HIV-1 genotypes in peripheral blood monocytes. J Leukoc Biol 2000;68:338-44.
    Abstract/FREE Full Text
  42. ↵
    1. Yi Y,
    2. Shaheen F,
    3. Collman RG
    . Preferential use of CXCR4 by R5X4 human immunodeficiency virus type 1 isolates for infection of primary lymphocytes. J Virol 2005;79:1480-6.
    Abstract/FREE Full Text
  43. ↵
    1. Thieblemont N,
    2. Weiss L,
    3. Sadeghi HM,
    4. Estcourt C,
    5. Haeffner-Cavaillon N
    . CD14lowCD16high: a cytokine-producing monocyte subset which expands during human immunodeficiency virus infection. Eur J Immunol 1995;25:3418-24.
    MedlineWeb of Science
  44. ↵
    1. Ancuta P,
    2. Weiss L,
    3. Haeffner-Cavaillon N
    . CD14+CD16++ cells derived in vitro from peripheral blood monocytes exhibit phenotypic and functional dendritic cell-like characteristics. Eur J Immunol 2000;30:1872-83.
    CrossRefMedlineWeb of Science
  45. ↵
    1. Gray L,
    2. Sterjovski J,
    3. Churchill M,
    4. et al
    . Uncoupling coreceptor usage of human immunodeficiency virus type 1 (HIV-1) from macrophage tropism reveals biological properties of CCR5-restricted HIV-1 isolates from patients with acquired immunodeficiency syndrome. Virology 2005;337:384-98.
    CrossRefMedline
  46. ↵
    1. Schmidtmayerova H,
    2. Alfano M,
    3. Nuovo G,
    4. Bukrinsky M
    . Human immunodeficiency virus type 1 T-lymphotropic strains enter macrophages via a CD4- and CXCR4-mediated pathway: replication is restricted at a postentry level. J Virol 1998;72:4633-42.
    Abstract/FREE Full Text
  47. ↵
    1. Folks TM,
    2. Kessler SW,
    3. Orenstein JM,
    4. Justement JS,
    5. Jaffe ES,
    6. Fauci AS
    . Infection and replication of HIV-1 in purified progenitor cells of normal human bone marrow. Science 1988;242:919-22.
    Abstract/FREE Full Text
    1. McElrath MJ,
    2. Pruett JE,
    3. Cohn ZA
    . Mononuclear phagocytes of blood and bone marrow: comparative roles as viral reservoirs in human immunodeficiency virus type 1 infections. Proc Natl Acad Sci USA 1989;86:675-9.
    Abstract/FREE Full Text
    1. Scadden DT,
    2. Zeira M,
    3. Woon A,
    4. et al
    . Human immunodeficiency virus infection of human bone marrow stromal fibroblasts. Blood 1990;76:317-22.
    Abstract/FREE Full Text
  50. ↵
    1. Voulgaropoulou F,
    2. Tan B,
    3. Soares M,
    4. Hahn B,
    5. Ratner L
    . Distinct human immunodeficiency virus strains in the bone marrow are associated with the development of thrombocytopenia. J Virol 1999;73:3497-504.
    Abstract/FREE Full Text
| Table of Contents

This Article

  1. J Infect Dis. (2008) 197 (2): 309-318. doi: 10.1086/524847
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most