JOURNAL OF VIROLOGY,0022-538X/02/$04.00�0 DOI: 10.1128/JVI.76.2.707–716.2002

Jan. 2002, p. 707–716 Vol. 76, No. 2

Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Evidence for Human Immunodeficiency Virus Type 1 Replication InVivo in CD14� Monocytes and Its Potential Role as a Source of

Virus in Patients on Highly Active Antiretroviral TherapyTuofu Zhu,1,2* David Muthui,1 Sarah Holte,3 David Nickle,2 Feng Feng,1 Scott Brodie,1

Yon Hwangbo,1 James I. Mullins,1,2 and Lawrence Corey1,2,4

Departments of Laboratory Medicine1 and Microbiology,2 University of Washington School of Medicine,Seattle, Washington 98195, and Programs in Biostatistics3 and Infectious Diseases,4

Fred Hutchinson Cancer Research Center, Seattle, Washington 98104

Received 23 May 2001/Accepted 28 September 2001

In vitro studies show that human immunodeficiency virus type 1 (HIV-1) does not replicate in freshlyisolated monocytes unless monocytes differentiate to monocyte-derived macrophages. Similarly, HIV-1 mayreplicate in macrophages in vivo, whereas it is unclear whether blood monocytes are permissive to productiveinfection with HIV-1. We investigated HIV-1 replication in CD14� monocytes and resting and activated CD4�

T cells by measuring the levels of cell-associated viral DNA and mRNA and the genetic evolution of HIV-1 inseven acutely infected patients whose plasma viremia had been <100 copies/ml for 803 to 1,544 days duringhighly active antiretroviral therapy (HAART). HIV-1 DNA was detected in CD14� monocytes as well as inactivated and resting CD4� T cells throughout the course of study. While significant variation in the decayslopes of HIV-1 DNA was seen among individual patients, viral decay in CD14� monocytes was on averageslower than that in activated and resting CD4� T cells. Measurements of HIV-1 sequence evolution and theconcentrations of unspliced and multiply spliced mRNA provided evidence of ongoing HIV-1 replication, morepronounced in CD14� monocytes than in resting CD4� T cells. Phylogenetic analyses of HIV-1 sequencesindicated that after prolonged HAART, viral populations related or identical to those found only in CD14�

monocytes were seen in plasma from three of the seven patients. In the other four patients, HIV-1 sequencesin plasma and the three cell populations were identical. CD14� monocytes appear to be one of the potentialin vivo sources of HIV-1 in patients receiving HAART.

Highly active antiretroviral therapy (HAART) has generallybeen successful in reducing human immunodeficiency virustype 1 (HIV-1) RNA in plasma to “undetectable” levels (�50copies/ml), with dramatic improvements in the clinical courseof HIV-1 infection (3, 19, 20, 34, 44). However, low levels ofviral replication persist in persons on prolonged HAART (6,13, 17, 19a, 26a, 32, 38a, 47, 58, 61). While resting CD4� T cellsare a potential reservoir for virus persistence (9, 10, 15, 16, 57),recent studies suggest the existence of other sources of emer-gent HIV-1 upon discontinuing HAART (8, 60).

Blood monocytes, derived from mononuclear phagocyteprecursor cells in bone marrow, may circulate in peripheralblood for 1 to 3 days before entering tissues and differentiatingto tissue-specific macrophages (29). CD14 is expressed exclu-sively on the mononuclear phagocyte lineage, at high levels onthe surfaces of most blood monocytes (48) and at lower butdetectable levels in macrophages in tissues such as lung (49).However, CD14 is absent in macrophages from small intestine,T cells, B cells, and natural killer (NK) cells (48). Tissue mac-rophages may be productively infected with HIV-1 and simianimmunodeficiency virus–HIV-1 chimeras and act as viral res-ervoirs (18, 24, 25, 27, 33). In vitro studies suggest that HIV-1does not replicate in freshly isolated peripheral blood mono-

cytes unless monocytes differentiate to monocyte-derived mac-rophages (11, 30, 31, 39, 50). Although HIV-1 can be detectedin blood monocytes (18, 23, 26, 27), it is unknown whether thevirus is produced or is maintained latently in monocytes in vivo(28, 45). Most recently, infectious HIV-1 has been isolatedfrom monocyte-derived macrophages of patients on prolongedHAART (51), indicating that monocytes harbor replication-competent HIV-1 and confirming that HIV-1 can be producedafter monocytes differentiate to macrophages (11, 26, 30, 31,50). Whether HIV-1 replicates in undifferentiated bloodmonocytes remains unclear. In the present study, we investi-gated HIV-1 replication in purified CD14� monocytes withoutthe aid of in vitro adherence, activation, and differentiation(11, 26, 30, 31, 50). Moreover, we compared the replicationcharacteristics of HIV-1 in CD14� monocytes with those inactivated and resting CD4� T cells. Finally, we determined thecontribution of these three cell populations to HIV-1 persis-tence in patients on prolonged HAART.

(This study was presented in part at the 4th InternationalWorkshop on HIV, Cells of Macrophage Lineage, and otherReservoirs, Donnini, Florence, Italy, 1 to 4 December 1999,and the 1st International Workshop on Acute HIV-1 Infection2000, Arlington, Va., 16 to 17 October 2000.)

MATERIALS AND METHODS

Study patients. We studied seven homosexual men who initiated HAARTcontaining indinavir (2,400 mg/day), zidovudine (600 mg/day), and lamivudine(300 mg/day) between 18 and 121 days after the onset of symptoms of acute HIV

* Corresponding author. Mailing address: Department of Labora-tory Medicine, University of Washington, P.O. Box 358070, Room 362,960 Republican St., Seattle, WA 98195-8070. Phone: (206) 732-6079.Fax: (206) 732-6055. Email: tzhu@u.washington.edu.

707

infection (Table 1) (3, 44). All patients were highly compliant with therapy. Sixof them had maintained undetectable levels of plasma viremia (�50 HIV-1 RNAcopies/ml of plasma, determined by a Roche ultrasensitive reverse transcriptasePCR [RT-PCR] assay) for 803 to 1,544 days with 1 or 2 transient episodes ofplasma viremia (�100 copies/ml), while patient 7 had consistently maintainedplasma HIV-1 RNA levels of fewer than 50 copies/ml. Leukapheresis from eachpatient included a time point prior to or on the day of the initiation of therapy(sample I) and two or three time points at 346 to 1,630 days into therapy(samples II, III, and IV).

Isolation of CD14� monocytes and resting and activated CD4� T lympho-cytes. CD14� monocytes, resting CD4� T lymphocytes, and activated CD4� Tlymphocytes were purified from peripheral blood mononuclear cells (PBMC) bynegative selection using magnetic bead depletion followed by fluorescent-acti-vated cell sorting (FACS) (7, 10, 15, 16). Specifically, monocytes were selectedusing monoclonal antibodies (MAbs) against CD3, CD8, CD19, and CD16 ex-pressed on the surfaces of T cells, CD8� T cells, B cells, and NK cells, respec-tively. The resulting cells were further positively sorted by FACS of monocyteswith antibodies to CD14. Resting CD4� T cells (HLA-DR�, CD25�, CD38�,and CD69�) were purified from PBMC by negative selection and an additional

6-day incubation to remove residual activated cells as described previously (7,10). To purify activated CD4� T cells, negative selection was used to depleteCD8� T cells, B cells, NK cells, and monocytes/macrophages. The resulting cellswere then selected by FACS using antibodies to activated cell surface markersHLA-DR, CD25, CD38, and CD69. The purity of isolated cells was analyzed byflow cytometry.

Quantification of HIV-1 DNA. Genomic DNA was isolated from purified cellsusing the QIAmp tissue kit (Qiagen) according to the manufacture’s protocol.HIV-1 DNA copies were quantified by real-time PCR (TagMan) (3, 5). Theresults shown in Fig. 2 and Table 2 are the mean values of three independentmeasurements for each sample. The detection limits for this assay were 5 copiesper 1 to 5 �g of total DNA per PCR, as described previously (3, 5).

Quantification of cell-associated unspliced (US) and multiply spliced (MS)viral mRNA and virion RNA in plasma. Cell-associated RNA was isolated withthe QIAmp RNA kit (Qiagen), digested with DNase I, and reverse transcribedas described previously (5) with antisense primer DM104 (residues 1674 to 1645of HIV-1 HXB2 sequence in GenBank, 5�-AGTCTCTAAAGGGTTCCTTTGGTCCTTGTC-3�) for US gag or primer DT1R (8556 to 8525, 5�-GCAATCAAGAGTAAGTCGATCAAGCGGTGGTA-3�) for MS tat. cDNA diluted in 10-fold series in triplicate was used for nested PCR with the following primers:DM102 (HIV-1 gag, residues 1395 to 1427, 5�-GAGACCATCAATGAGGAAGCTGCAGAATGGGAT-3�) and DM104 for a first round of PCR and SK38 andSK39 (41) for a second round of PCR. For tat, DT1F (residues 5780 to 7807,5�-TGGGTGTCGACATAGCAGAATAGGCATT-3�) and DT1R were used forthe first round of amplification, and DT2F (residues 5831 to 5859, 5�-GGAGGCCAGTAGATCATAGACTAGAGCCCT-3�) and DT2R (residues 8451 to8432, 5�-TCTCTGTCTCTCTCTCCACCTTCTTCTTC-3�) were used for sec-ond-round reactions. PCR products were separated on polyacrylamide gels afterliquid hybridization with 32P-labeled probes (SK39 for gag; DMTP1, residues6025 to 6059, 5�-GGGCTGGAGGTGGGTTGCTTTGATAGAGAATCTTG-3�, for tat), blotted, and autoradiographed. Conditions of PCR and liquid hy-bridization procedure have been described previously (5, 63). Controls withoutRT were negative, which confirmed the absence of viral DNA. Each PCRamplification contained primers for GAPDH (glyceraldehyde-3-phosphate de-hydrogenase) (14) as an internal control for the amount of amplifiable cDNA.The amounts of gag and tat HIV-1 mRNA were calculated following limitingdilution with the computer program QUALITY (40). Viral RNA was isolatedfrom plasma (63) containing fewer than 50 HIV-1 RNA copies/ml and thenHIV-1 gag was quantified as described above. Utilization of nested PCR plus 32Pliquid hybridization was sensitive enough to detect one copy per �g of cDNA perreaction and specific for the detection of HIV-1 (data not shown). The averagesof two independent measurements for HIV-1 mRNA and virion RNA for eachsample are shown in Table 1 and Fig. 3.

PCR, sequencing, and sequence analyses. Cellular DNA and cDNA which hadbeen reverse transcribed from plasma viral RNA with primer PE2 were used toamplify HIV-1 env gp120 sequences using a nested PCR with the outer primersPE0 and PE2 and the inner primers PE1 and P2 (63). Multiple independent PCRproducts generated from target sources containing 20 to 200 copies of HIV-1DNA or cDNA of each sample (purified cells or plasma) were cloned andsequenced (63). Twelve to seventeen clone sequences were aligned by usingClustal W (52). Likelihood ratio tests were implemented through MODELTEST(38) and used to derive a maximum-likelihood model (PAUP 4.0; Sinauer As-sociates, Inc., Sunderland, Mass.) of evolution that statistically fit the data whilemaking the fewest assumptions about the evolution of the sequences themselves.Parameters derived from the best-fit model were applied to the data sets toobtain maximum-likelihood distances in PAUP*. The distances were used toconstruct neighbor-joining (42) trees from which we calculated the most recentcommon ancestor (MRCA) for the entire ingroup of each patient. We thencalculated the distances to the MRCA for each sequence and divided thesedistances into two groups, those from MRCA to sample I and those from MRCAto sample II. A t test was performed to compare the mean distances between theMRCA and sequences from samples I and II.

Statistical analyses. Estimates of decay slopes of proviral DNA were obtainedwith linear random effects regression models (12) of log-transformed data be-ginning at the time the patient started HAART. Cell-associated HIV-1 DNAlevels prior to the initiation of treatment were used as the DNA levels attreatment start for individuals who did not have these measurements on the dayof treatment initiation. The associated coefficient of time covariant provided anestimate of the mean decay slope, while individual decay slopes were estimatedusing empirical Bayes methods (12). All comparisons of means (DNA, mRNA,and ratios) and decay slopes (DNA) were made with generalized estimatingequations (12) to account for correlations which could arise for different cell

TABLE 1. Characteristics of HIV-1-infected patientsreceiving HAART

Patientand

sample

Time (days)a of: No. ofCD4�

cells/mm3

No. ofHIV-1 RNAcopies/ml of

plasmabSampling HAARTinitiation

Serocon-version

1 90 62I 64 452 38,900II 729 860 �50 (20)III 954 986 �50 (12)IV 1,458 1,002 �50 (31)

2 121 41I 41 702 492,000II 1,051 842 �50 (20)III 1,235 815 �50 (8)IV 1,751 788 �50 (7)

3 79 58I 78 777 22,200II 425 1,423 �50 (25)III 739 1,518 �50 (14)IV 1,153 1,265 �50 (6)

4 20 30I 10 381 3,500,000II 423 928 �50 (30)III 1,251 841 �50 (12)

5 18 18I 18 755 6,000,000II 578 1,061 �50 (14)III 941 1,001 �50 (17)

6 80 60I 35 435 67,862II 820 718 �50 (18)III 1,017 848 �50 (10)IV 1,202 803 �50 (11)

7 21 20I 20 680 12,500II 485 729 �50 (4)III 725 801 �50 (4)IV 1,205 832 �50 (5)

a Days after the onset of acute syndrome of HIV infection (3, 44).b Levels of plasma viral RNA were measured by a commercial RT-PCR assay

(Amplicor ultrasensitive HIV-1 monitor assay; Roche) (3, 44). Numbers in pa-rentheses are levels of virion RNA determined by a sensitive limiting-dilutionRT-PCR (see Materials and Methods).

708 ZHU ET AL. J. VIROL.

types sampled from the same individual and repeated sampling of individualsover time.

Nucleotide sequence accession numbers. The nucleotide sequences reportedin this paper have been submitted to GenBank and were given accession num-bers AF405731 to AF406313.

RESULTS

Decay rate of HIV-1 DNA in CD14� monocytes and acti-vated and resting CD4� T cells. We used negative selectionand FACS to isolate monocytes (CD14�), activated CD4� Tcells (CD69�, CD25�, CD38�, and HLA-DR�), and restingCD4� T cells (CD69�, CD25�, CD38�, and HLA-DR�). Asshown in Fig. 1, these cells were highly pure and free fromcontamination with the other two cell populations. HIV-1DNA was detected in all three cell types throughout the study(Fig. 2 and Table 2). The mean numbers of copies of HIV-1DNA before the initiation of treatment were 272 � 26.2 per106 CD14� monocytes, 1,247 � 115.1 per 106 activated CD4�

T cells, and 1,286 � 102.7 per 106 resting CD4� T cells (P �0.01 for the comparison of monocytes versus activated or rest-ing CD4� T cells). HIV-1 DNA levels decreased over thecourse of therapy in all three cell compartments. Previousstudies have shown an initial fast decay of HIV-1 in a variety ofsample types (CD4� T cells, resting CD4� T cells, bulk PBMC,and/or blood plasma) that was followed by a much slower andvarying decay after the initiation of HAART (4, 17, 22, 35, 36,55). We were not able to define a similar decay in the three cell

populations during early treatment because of the inability toaccess large volumes of PBMC via leukapheresis at such fre-quent time points. We compared the decay of HIV-1 DNA inthese three cell compartments between samples I and II andfound that the viral decay was significantly slower in CD14�

monocytes (mean decay rate of �0.0195 [range, �0.0260 to�0.0130] log10 per month) and resting CD4� T cells (�0.0191[�0.0290 to �0.00092]) than in activated CD4� T cells(�0.0380 [�0.0490 to �0.0271]) during the first 2 years oftreatment. We then focused on the decay of HIV-1 DNA inCD14� monocytes and activated and resting CD4� T cellsafter patients had attained less than 50 RNA copies per ml ofplasma during HAART. We estimated the decay rate in eachcell compartment by using only HIV-1 DNA copies of samplesII to IV (Table 1) from each patient. As shown in Fig. 2 andTable 2, there was a significant variation in the viral decay ratein all three cell compartments and among individual patients,ranging from �0.0193 log10 HIV-1 DNA copies per month(half-life [t1/2], 15.6 months) to �0.0058 (t1/2, infinite) forCD14� monocytes (Fig. 2A), �0.0281/month (t1/2, 10.7months) to 0.0001 (t1/2, infinity) for resting CD4� T cells (Fig.2B), and �0.0239 (t1/2, 12.6 months) to �0.0045/month (t1/2, 67months) for activated CD4� T cells (Fig. 2C). The mean decayrate of HIV-1 DNA in CD14� monocytes (�0.0073; 95%confidence interval [CI], �0.0168 to �0.0022) was significantlylower than that estimated in the activated CD4� T cells(�0.0152; CI, �0.0253 to �0.0050) (P � 0.0002). No signifi-

FIG. 1. Representative two-color fluorescence analysis of enriched CD14� monocytes (A and B) and activated (C and D) and resting (E andF) CD4� T cells. Purified cells were stained either with unconjugated MAb (negative control) or with fluorescein isothiocyanate (FITC)- orphycoerythrin (PE)-conjugated MAb. The purity of CD14� monocytes obtained by using a combination of negative selection and FACS (seeMaterials and Methods) was 98.31 to 99.99% (mean � standard deviation, 99.37% � 0.58%). There was no contamination of T cells (A) or B cells(B). Activated CD4� T cells, also purified by both negative selection and FACS for the activation markers HLA-DR, CD25, CD38, and CD69,showed a purity of 98.02 to 99.95% (99.21% � 0.63%) without CD14� monocytes (C) and resting CD4� T cells (D). Resting CD4� T cells wereisolated by negative selection with a 6-day incubation as described previously (7, 10, 15). These cells had undetectable (below the negative control)contamination with CD14� monocytes (E) and activated CD4� T cells (F). The unstained cells in the left bottom corner of panels E and F arered blood cells.

VOL. 76, 2002 HIV-1 REPLICATION IN CD14� MONOCYTES 709

cant differences were seen in the rate of viral decay betweenCD14� monocytes and resting CD4� T cells (�0.0128; CI,�0.0240 to �0.0015) (P � 0.668) (Table 2). The correspondingestimated t1/2 of HIV-1 DNA were 41.3 months in CD14�

monocytes, 23.6 months in resting CD4� T cells, and 19.8months in activated CD4� T cells (Table 2). We could notdetermine the impact of intermittent episodes of plasma vire-mia on viral decay (38a), because all patients except one (pa-tient 7) had at least one documented episode of low-levelviremia (�100 HIV RNA copies/ml).

HIV-1 transcription activity in CD14� monocytes and acti-vated and resting CD4� T cells. The apparent half-lives ofHIV-1-infected cells from our patients (Table 2) were muchlonger than the estimated mean intermitotic life spans (21, 22,35, 36, 54–56) of monocytes/macrophages (41.3 months versus14 days) and activated (19.8 months versus 2 days) and restingmemory (23.6 versus 6 months) CD4� T cells, suggesting thatthese reservoirs may be renewed as a result of continued viralreplication. We then examined HIV-1 transcription activity byassessing the levels of cell-associated MS (tat) and US (gag)viral mRNA in samples II and/or III. HIV-1 mRNA in samplesII and III, taken after plasma virus had been undetectable for301 to 1,028 days (Table 1), would indicate ongoing HIV-1transcription in vivo (17, 32, 43). We detected both MS and USHIV-1 mRNA in all three cell populations (Fig. 3). The meanconcentrations of gag and tat mRNA showed significant differ-ences between the three cell populations (Fig. 3A and B). We

also estimated HIV-1 transcriptional activity by measuring theratio between viral DNA and RNA in these three cell popu-lations. The mean mRNA/DNA ratios of tat and gag forCD14� monocytes were similar to that for activated CD4� Tcells and were significantly higher than that for resting CD4�

T cells (Fig. 3C and D), indicating higher levels of viral tran-scription in CD14� monocytes and activated CD4� T cellsthan in resting CD4� T cells.

HIV-1 sequence evolution in CD14� monocytes and acti-vated and resting CD4� T cells. While the above data indicateongoing viral transcriptional activity, the production of infec-tious virus could still be blocked at assembly by the proteaseinhibitor included in HAART. We therefore evaluated HIV-1sequence evolution in all three cell populations, since muta-tional changes accumulate as a result of completed rounds ofviral replication in vivo. As shown in Table 3, the geneticdistances from the deduced MRCA to sequences in sample IIwere longer than those for sample I in most patients (Table 3),suggesting sequence evolution that varied by patient (8, 60, 61)and by cell population. Four of seven patients had minor se-quence evolution over the course of follow-up; three others(patients 1, 6, and 7) exhibited significant sequence evolution.When HIV-1 sequences in CD14� monocytes from all sevenpatients were analyzed together, we found a significant differ-ence between the mean genetic distances from MRCA to sam-ple I (mean, 0.43) and from MRCA to sample II (0.68) (P �0.02). For activated and resting CD4� T cells, the mean dis-

FIG. 2. Decay rates of HIV-1 DNA in peripheral blood CD14� monocytes (A), resting CD4� T cells (B), and activated CD4� T cells (C) inseven acutely infected patients taking HAART who had undetectable levels of plasma virus. Patients 1 to 6 had maintained undetectable levelsof plasma viremia with one or two episodes of low-level plasma viremia (�100 copies/ml), while patient 7 had consistently maintained plasmaHIV-1 RNA at levels lower than 50 copies/ml. Average decay slopes were estimated using linear random-effects regression (12) for one phasedecay. The bold line in each panel represents the average slope and intercept of the decay in that cell compartment, and the dashed lines withdifferent symbols represent predicted individual intercepts and decay slopes. The data are summarized in Table 2.

TABLE 2. Decay of HIV-1 infected CD14� monocytes and resting and activated CD4� T cells

Cell typeDecay slope (per mo) t1/2 (mo)

Mean 95% CI Mean 95% CI

CD14� monocytes �0.0073 �0.0168 to 0.0022 41.3 17.9 to �Resting CD4� T cells �0.0128 �0.0240 to �0.0015 23.6 12.5 to 196.6Activated CD4� T cells �0.0152 �0.0253 to �0.0050 19.8 11.9 to 59.8

710 ZHU ET AL. J. VIROL.

tances from MRCA to sample II (0.70 for activated CD4� Tcells and 0.54 for resting CD4� T cells) tended to be longerthan those from MRCA to sample I (0.51 for activated and0.50 for resting CD4� T cells). However, the sequence evolu-tion in the two CD4�-T-cell populations was not statisticallysignificant (P � 0.08 for activated CD4� T cells; P � 0.45 forresting CD4� T cells).

Comparison of HIV-1 sequences in plasma and purifiedcells: evidence for production of HIV-1 virions from provirusin CD14� monocytes. We were able to detect low levels ofplasma viremia (4 to 30 HIV-1 RNA copies/ml of plasma) atthe time corresponding to sample II or III (Table 1), indicatingongoing production of HIV-1 in vivo after prolonged HAART(13). We then compared HIV-1 env sequences from plasma

with proviral sequences obtained from purified cell popula-tions. In patients 2, 3, 4, and 6, HIV-1 sequences in plasma andin all three cell types were homogenous and indistinguishable(data not shown) within each individual. However, there weretwo distinct but associated groups of sequences in patient 1(Fig. 4). Group 1 encompassed the major variant populationsfound in all cell compartments. Group 2 sequences werelargely observed in sample II (639 days posttherapy) and wereclosely related to a variant that was derived from CD14�

monocytes of sample I (26 days before therapy) (Fig. 4). Se-quences similar to this monocyte-associated lineage were notdetected in activated or resting CD4� T cells of sample I bysequencing of additional clones or by screening of PCR prod-ucts with a quantitative homoduplex tracking assay capable of

FIG. 3. Comparison of levels of HIV-1 mRNA in CD14� monocytes and activated CD4� T cells and resting CD4� T cells in seven patientsfollowing 1 to 3 years of HAART. (A) Levels of MS tat mRNA in the three cell types. Pairwise comparisons of the mean level of the log of tatmRNA using general estimating equations (12) detected significant differences between all three cell types (P � 0.001 for resting compared toactivated cells, P � 0.001 for resting cells compared to monocytes, P � 0.003 for activated cells compared to monocytes. (B) Corresponding levelsof MS gag mRNA. Again, significant differences were found between all three cell types (P � 0.001 for resting compared to activated cells, P �0.001 for resting cells compared to monocytes, P � 0.020 for activated cells compared to monocytes). (C) Ratio of HIV-1 tat mRNA to DNA. (D)Ratio of HIV-1 gag mRNA to DNA. Pairwise comparisons of the log of means ratios of both tat and gag mRNA and DNA suggest significantdifferences between resting CD4� T cells and both monocytes and activated CD4� T cells (for both gag and tat, P � 0.001 for resting cells comparedto activated cells and monocytes; for tat, P � 0.984 for activated cells compared to monocytes; for gag, P � 0.828 for activated cells compared tomonocytes).

VOL. 76, 2002 HIV-1 REPLICATION IN CD14� MONOCYTES 711

detecting minor variants (63). However, both groups of se-quences were observed in blood plasma at a time point corre-sponding to sample II, suggesting that both groups of provi-ruses were able to produce virus. In patient 7, HIV-1sequences were homogenous before therapy (sample I) andremained so in resting and activated CD4� T cells after 464days of therapy (sample II), whereas new variants with signif-icant evolution (Table 3) were seen in CD14� monocytes ofsample II as well as in plasma 2 months after sample II (group2 of patient 7 in Fig. 4). In patient 5, two groups of variantswith and without a 54-bp deletion in HIV-1 region C3 (clones5 M2-2 and 5 M2-1, respectively, in Fig. 5) were identified inCD14� monocytes of sample II (560 days posttherapy). Wethen performed additional PCR studies to assess the represen-tation of virus with the 54-bp deletion in all three cell popu-lations and plasma. The sequence populations without the54-bp deletion (5 M2-1-like, 358 bp) (Fig. 5B) were detected inall cells and plasma, whereas the sequences with the 54-bpdeletion (5 M2-2-like, 304 bp) (Fig. 5B) were detected only inCD14� monocytes and plasma at day 560 posttherapy (sampleII). These results are consistent with the production of HIV-1from CD14� monocytes under suppressive HAART.

DISCUSSION

Previous in vitro studies showed that HIV-1 replication infreshly isolated blood monocytes and resting CD4� T cells wasblocked prior to the completion of reverse transcription and

integration (50, 59). However, a recent study showed thattreating, but not activating, resting CD4� T cells with thecytokines interleukin 2 (IL-2), IL-4, IL-7, and IL-15 was able toovercome this block, resulting in HIV-1 replication in restingCD4� T cells (53). Thus, it is likely that replication of HIV-1in vivo occurs in resting T cells that are exposed to cytokines atsites of infection or in tissues (53, 62). Whether cytokinessimilarly render monocytes susceptible to HIV-1 infection invivo is unknown. Since the original presentation of these re-sults, Sonza et al. have shown that HIV-1 can be isolated whenpatient’s monocytes are differentiated into monocyte-derivedmacrophages (51). Our studies were conducted on freshly iso-lated patient CD14� monocytes without adherence-induceddifferentiation, which is known to alter the susceptibility ofmononuclear phagocytes to HIV-1 replication (11, 26, 30, 31,39, 50, 51). Our findings indicate that HIV-1 replicates inCD14� monocytes in vivo, even in patients receiving HAART.Our studies also confirm that HIV-1 replication can occur inactivated and to lesser extent in resting CD4� T cells in pa-tients undergoing suppressive HAART (62).

A recent study concluded that in five of eight patients whodiscontinued HAART, a rebound HIV-1 in plasma could haveresulted from the activation of virus in resting CD4� T cells(60). Another study showed that HIV-1 reservoirs other thanresting CD4� T cells could prompt the emergence of plasmavirus (8). In three of our seven patients, the viral populationsthat were closely related or identical to those detected eitherinitially (patients 1 and 7) (Fig. 4) or only (patient 5) (Fig. 5)in CD14� monocytes were seen in plasma after prolongedHAART, indicating that CD14� monocytes may serve as apotentially important source of HIV-1 in patients takingHAART. This hypothesis is supported by our findings ofhigher levels of HIV-1 transcripts and sequence evolution inCD14� monocytes than in resting CD4� T cells, which sug-gests a higher level of HIV-1 replication in CD14� monocytesthan in resting CD4� T cells. The differences in both HIV-1DNA and RNA between the three cell populations, the con-sistency between the DNA and RNA data, and the indepen-dently performed sequence analyses confirm our findings andverify the lack of “contamination” within the laboratory as anexplanation of our results. While we demonstrate CD14�

monocytes as a potential reservoir of HIV-1 replication inpatients on HAART, it is possible that other reservoirs, espe-cially in tissues (18, 24, 25, 27, 33, 59), may contribute to thepersistence of HIV-1. In four other patients, HIV-1 sequencepopulations in plasma were identical to those isolated fromresting CD4� T cells (8, 59) which were also identical to viralsequences from CD14� monocytes and activated CD4� Tcells, suggesting that not only resting CD4� T cells (60) butalso CD14� monocytes and activated CD4� T cells are poten-tial viral sources in these patients.

Although the apparent long half-life of HIV-1-infectedCD14� monocytes appears to be similar to that in restingCD4� T cells, HIV-1 could turn over at a higher rate in CD14�

monocytes than in resting CD4� T cells in the presence ofHAART. Given the fact that monocytes may circulate in pe-ripheral blood for only a few days before differentiating tomacrophages in tissues (29), the persistence of HIV-1 in bloodmonocytes itself suggests ongoing virus replication and/or re-cent infection in monocytes. Our findings of more evident

TABLE 3. Analysis of HIV-1 genetic distances inpatients receiving HAARTa

Patient Cell type

Mean geneticdistance (%) from

MRCA to: P value Continuedevolution

Sample I Sample II

1 Monocytes 0.51 1.24 0.0309 YesActivated T cells 0.91 2.01 0.0074 YesResting T cells 0.44 0.63 0.4465 No

2 Monocytes 0.75 0.84 0.4841 NoActivated T cells 0.75 0.75 0.5035 NoResting T cells 0.78 0.82 0.2138 No

3 Monocytes 0.21 0.25 0.3547 NoActivated T cells 0.33 0.26 0.3465 NoResting T cells 0.24 0.25 0.8285 No

4 Monocytes ND ND ND NDActivated T cells ND ND ND NDResting T cells 0.60 0.57 0.5089 No

5 Monocytes 0.28 0.28 0.9897 NoActivated T cells 0.20 0.25 0.4048 NoResting T cells 0.20 0.21 0.6969 No

6 Monocytes 0.64 0.68 0.3782 NoActivated T cells 0.64 0.66 0.6914 NoResting T cells 0.63 0.71 0.0059 Yes

7 Monocytes 0.20 0.77 �0.0001 YesActivated T cells 0.25 0.28 0.4215 NoResting T cells 0.44 0.63 0.4465 No

a ND, not done.

712 ZHU ET AL. J. VIROL.

FIG. 4. Phylogenetic tree analysis of HIV-1 envelope sequences in CD14� monocytes (red circles), activated (blue circles) and resting CD4�

T cells (green circles), and plasma (black circles) of patients receiving HAART. The number within each circle is the sample number (i.e., 1 and2 indicate samples I and II). Numbers in parentheses are the numbers of sequences that were identical in the sample to the left. The outgroup isa patient (46) who was not on therapy but whose samples were obtained over a time frame similar to that of the ingroup. Numbers on thesesequences are relative sampling times. The bar indicates genetic distance.

VOL. 76, 2002 HIV-1 REPLICATION IN CD14� MONOCYTES 713

HIV-1 replication in CD14� monocytes suggest that the HIV-1pool in monocytes could be renewed, as a result of viral rep-lication, more frequently in CD14� monocytes than in restingCD4� T cells. However, the viral pool in CD14� monocytes, aswell as in resting CD4� T cells, could also be renewed by virusproduced from activated CD4� T cells. The source of HIV-1 inblood monocytes and the role they play in the overall pool ofHIV-1 replication remain to be defined. It is possible thatinfected monocytes produce relatively small amounts of virusbut are a major carrier of virus into tissue sites where tissuemacrophages may produce virus. Hence, blood monocytes mayserve as an indirect source of HIV-1. One potential explana-tion for pronounced viral replication in CD14� monocytes isthat antiretroviral drugs may not block viral replication inmonocytes/macrophages as efficiently as in CD4� T cells (1, 2,24, 37). As reported elsewhere (26), we have not found evi-dence for the evolution of drug resistance in patients takingHAART (T. Zhu et al., unpublished data). The establishmentof HIV-1 infection in CD14� monocytes during primary infec-tion and the ongoing viral replication in CD14� monocytes aswell as in CD4� T cells and other tissues constitute the majorproblem of HIV-1 eradication. Therapies with greater potencyagainst viral production in monocytes may provide more com-plete suppression of HIV-1.

ACKNOWLEDGMENTS

We are grateful to K. Diem, M. Berrey, T. Shea, L. Stensland, H.Liu, and E. Peterson for assistance.

We acknowledge financial support from NIH grants (AI41535,AI45206, AI35605, AI45402, and AI 49109).

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