Department of Laboratory Medicine, University of Washington School of Medicine, Box 358070, 960 Republican Street, Seattle, WA 98109-8070, USA
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Evidence for HIV-1 persistence and replication in peripheral blood monocytes |
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HIV-1 infects and replicates primarily in CD4+ T cells and tissue macrophages, which accounts for many of the major aspects of HIV-1 pathogenesis in vivo. Previous studies showed that HIV-1 could be detected in blood monocytes.35 It was, however, not known whether the virus was produced or was maintained latently in monocytes in vivo.5 In vitro studies have shown that HIV-1 replication in freshly isolated blood monocytes and resting CD4+ T cells was blocked prior to the completion of reverse transcription and integration.6,7 However, treating, but not activating, resting CD4+ T cells with IL-2, IL-4, IL-7 and IL-15 was able to overcome this block, resulting in HIV-1 replication in resting CD4+ T cells.8 Thus, it is likely that replication of HIV-1 occurs in vivo in resting CD4+ T cells that are exposed to cytokines at sites of infection or in tissues.810 Whether cytokines similarly render monocytes susceptible to HIV-1 infection in vivo is possible, but still unknown. Sonza et al.11 showed recently that HIV-1 could be isolated when a patients monocytes had differentiated into MDM. These results indicate that monocytes harbour replication-competent HIV-1, and that HIV-1 could be produced upon the differentiation of monocytes into macrophages.6 Whether or not HIV could replicate in undifferentiated monocytes remained to be determined.
To examine the extent of HIV-1 replication in vivo in monocytes, and its role as a potential source of virus, we isolated highly purified CD14+ monocytes by a two-step procedure of immunoprecipitation depletion and cell sorting.10,12 The CD14+ monocytes purified by this assay represent the majority of heterogeneous blood monocytes circulating in vivo, because blood monocytes isolated by this method are not likely to have differentiated into macrophages as is the case during conventional adherence of PBMC in vitro.6 We measured the levels of cell-associated viral DNA, messenger RNA (mRNA) and genetic evolution of HIV-1 in patient samples obtained longitudinally during the course of HIV-1 infection.10,12 HIV-1 DNA was detected in CD14+ monocytes throughout the course of study in both patients without antiretroviral therapy and patients whose plasma viraemia had been <200 copies/mL for 3 years during highly active antiretroviral therapy (HAART). We analysed the decay of HIV-1 DNA in CD14+ monocytes in patients on effective HAART, and then compared the proviral decay in CD14+ monocytes with that in purified resting CD4+ T cells and activated CD4+ T cells. While significant variation in the decay slopes of HIV-1 DNA was seen between treated patients, viral decay in CD14+ monocytes was on average slower than that in activated or resting CD4+ T cells.10 We estimated the half-life of HIV-1 DNA in each cell compartment during the period when patients plasma virus was maintained at levels below the detection limit of conventional assays. Although there was a significant variation in all three cell compartments and between individual patients, the mean half-life of HIV-1 DNA in CD14+ monocytes [41.3 months, 95% confidence interval (CI): 17.9infinite] was longer than that in resting CD4+ T cells (23.5 months, CI: 12.5196.6) or activated CD4+ T cells (19.8 months, CI: 11.959.8).10 These apparent half-lives of HIV-1-infected cells from our patients were much longer than the estimated mean intermitotic lifespans of monocyte-macrophages (14 days), activated (2 days) and resting memory (6 months) CD4+ T cells, suggesting that these reservoirs may be renewed as a result of recent infection and/or ongoing virus replication in monocytes or in their precursor cells. To further assess these possibilities, we looked for the signs of viral replication by examining HIV-1 mRNA and sequence evolution in CD14+ monocytes. We determined HIV-1 transcription activity by assessing the levels of cell-associated multiply spliced (MS) (tat) and unspliced (US) (gag) viral mRNA in samples that were taken after plasma virus had been undetectable for more than 1 year, which would indicate continuous HIV-1 transcription in CD14+ monocytes.13,14 Both MS and US HIV-1 mRNA were detected consistently in CD14+ monocytes from each time point. The mean concentrations of gag and tat mRNA in CD14+ monocytes were significantly higher than those in resting CD4+ T cells. We also estimated HIV-1 transcriptional activity by measuring the ratio between viral DNA and RNA. The mean ratios of tat and gag mRNA/DNA for CD14+ monocytes were similar to those for activated CD4+ T cells, and were significantly higher than that for resting CD4+ T cells.10 These results indicate ongoing higher levels of viral transcription in CD14+ monocytes and activated CD4+ T cells compared with resting CD4+ T cells in patients with suppressive HAART. While the above data indicate ongoing viral transcriptional activity, the production of infectious virus could still be blocked at assembly by the protease inhibitor included in HAART. We therefore evaluated HIV-1 sequence evolution in CD14+ monocytes at time points pre-antiretroviral therapy and 3 years post-HAART, since mutational changes accumulate as a result of completed rounds of viral replication in vivo. We observed that sequence evolution varied by patient, as reported by others,15,16 and by cell population. When HIV-1 sequences from all treated patients were analysed together, we found significant genetic evolution in CD14+ monocytes. However, the sequence evolution in the two CD4+ T cell populations was not statistically significant,10 although HIV tends to evolve in activated and resting CD4+ T cells. Further comparison of HIV-1 sequences among CD14+ monocytes, activated and resting CD4+ T cells, and peripheral blood plasma by phylogenetic analyses showed that after prolonged HAART the viral populations related or identical to those found only in CD14+ monocytes were seen in plasma from three of the seven patients, suggesting that CD14+ monocytes appear to be a potential in vivo source of HIV-1.
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Models of HIV-1 persistence and replication in peripheral blood monocytes |
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In summary, given the fact that monocytes may circulate in peripheral blood for only 13 days before differentiating into macrophages in tissues, our finding of persistent HIV-1 in blood monocytes itself suggests ongoing renewal of infected monocytes by virus replication and/or recent infection in monocytes or their precursor cells. Our findings of more evident HIV-1 replication in CD14+ monocytes suggest that the HIV-1 pool in monocytes could be renewed, as a result of viral replication, more frequently in CD14+ monocytes than in resting CD4+ T cells. Therefore, although the long half-life of HIV-infected CD14+ monocytes appears to be similar to that of resting CD4+ T cells, HIV-1 could turn over at a higher rate in CD14+ monocytes than in resting CD4+ T cells in the presence of HAART. However, the viral pool in CD14+ monocytes could also be renewed by viruses produced from resting and activated CD4+ T cells, and tissue macrophages. The source of HIV-1 in blood monocytes and the contribution they make to the overall pool of HIV-1 replication require more extensive studies. The establishment of HIV-1 infection in CD14+ monocytes in the early stage of infection and ongoing viral replication in CD14+ monocytes present a major challenge to HIV-1 eradication with current regimens of HAART. New therapies with greater potency against viral production in blood monocytes may enhance the efficacy of antiretroviral therapy.
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Footnotes |
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References |
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