Not so long ago I wrote about a study from Vicente Planelles’s research group that found HIV-infected T cells in vivo show evidence of Vpr-induced cell cycle arrest. The authors of that paper were surprised by the proportion of infected cells that had lost CD4 expression and were thus double negative CD4-/CD8- T cells. Further confirmation of this finding now comes from The Swiss HIV Cohort Study in a new paper published in J. Virology.
The researchers took a careful and detailed approach to investigate the extent of HIV infection in multiple different cell types using samples from 12 infected individuals (six on long-term combination ART, six untreated or off treatment for at least 8 months). Included in the evaluation were unfractionated PBMC (peripheral blood mononuclear cells), total CD4+ T-cells, resting or activated CD4+ T-cells, CD8-/CD4- double negative T-cells and blood monocytes. The researchers ascertained the extent of infection by comparing the results of different assays: HIV DNA, HIV-RNA mapping to the gag gene (unspliced RNA), multiply spliced HIV-RNA coding for tat, rev or nef (multiply spliced RNA) and extracellular, virion-enclosed genomic HIV-RNA. This approach allowed the identification of three categories of HIV-infected cells expressing viral RNA which could be discerned by virtue of their dissimilar frequencies; cells expressing unspliced RNA were most frequent, followed by those expressing multiply spliced RNA, with the least frequent population being those expressing virion-enclosed genomic HIV-RNA.
Frequencies of all types of infected cells were substantially lower in treated participants. Also, in treated participants, essentially all infected cells were contained in the CD4+ T cell fraction. But in individuals not on ART, generally less than 50% of the infected cells identified in PBMC were CD4+ T cells. The researchers also found that while virion-enclosed genomic HIV-RNA was detected only at low levels in CD4+ T-cells from both groups of participants, it was present at substantial levels in PBMC from untreated individuals. These findings prompted an effort to identify these infected cells; after ruling out monocyte/macrophages as the source, the researchers homed in on double negative CD4-/CD8- T cells. This analysis revealed that in untreated participants, 50% of unspliced RNA, 41% of multiple spliced RNA and 64% of PBMC associated virion-enclosed genomic HIV-RNA was attributable to double negative T cells. Further analysis showed that cells expressing virion-enclosed genomic HIV-RNA were approximately 10 times more frequent among CD4-/CD8- T-cells than in CD4+ T cells. In a comparison of treated vs. untreated participants, HIV-infected CD4-/CD8- T-cells expressing viral RNA were at least 500 times less frequent in individuals on ART.
To confirm that these infected double negative T cells were previously CD4+ T cells that had downregulated the CD4 receptor, the researchers looked for CD4 messenger RNA and also compared viral sequences between the different infected cell populations. Results of both analyses were consistent with CD4 downregulation in this cell population.
The study authors conclude by noting that “our data suggest that contrary to current belief, productive HIV infection in peripheral blood may be restricted to a major extent to T-helper lymphocytes of a CD4-/CD8- double negative phenotype.” They also note that CD4 downregulation may significantly impact previous estimates of the lifespan of infected cells, because “activated CD4+T-cells with transcriptionally active proviral integrants may have an extremely short half-life, simply because they may rapidly switch to a CD4- phenotype and would not be identifiable as T-helper cells in cell sorting experiments using the CD4 marker.” The researchers recommend that the pan-T cell marker CD3 should be used in future studies of cellular HIV replication in order to ensure that double negative cells are not missed; they also stress the need to look for the presence of this population in other anatomical sites (such as the lymphoid tissue and gut).
In terms of the mechanism of CD4 downregulation, the authors note that the HIV proteins Vpu and Nef have been shown to exert this effect in vitro. It is also known that there is a transient downregulation of CD4 that occurs when CD4+ T cells are activated normally via their T cell receptor, so the finding is consistent with a preponderance of viral replication occurring in activated cells. Perhaps another possibility suggested by Vicente Planelles’s prior work is that cell cycle arrest is preventing or delaying the re-expression of the CD4 molecule by HIV-infected cells. This study is likely to generate many new lines of investigation regarding the role of CD4 downregulation in HIV infection.
JVI Accepts, published online ahead of print on 3 July 2007
J. Virol. doi:10.1128/JVI.00492-07
Philipp Kaiser, Beda Joos, Barbara Niederöst, Rainer Weber, Huldrych F Günthard, Marek Fischer*, and The Swiss HIV Cohort Study
Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zürich Rämistrasse 100 8091 Zürich, Switzerland; http://www.shcs.ch
HIV-1 transcription is subject to substantial fluctuation during the viral life cycle. Due to the low frequencies of HIV-1 infected cells and because latently and productively infected cells collocate in vivo, little quantitative knowledge has been attained about the range of in vivo HIV-1 transcription in peripheral blood cells (PBMC). By combining cell sorting, terminal dilution of intact cells and highly sensitive, patient-specific PCR assays, we dissected PBMC obtained from HIV-1 infected patients according to their degree of viral transcriptional activity and their cellular phenotype. Regardless of treatment status, the bulk of infected cells exhibited a CD4+ phenotype but transcribed HIV-1 provirus at low levels presumably insufficient for virion production. Furthermore, expression of activation markers on the surface of these CD4+ T-lymphocytes showed little or no association with viral transcriptional enhancement.
In contrast, HIV-infected T-lymphocytes of a CD4-/CD8- phenotype occurring exclusively in untreated patients, exhibited elevated viral transcription rates. This cell type harbored a substantial proportion of all HIV-RNA+ cells, of intracellular viral RNAs and the majority of cell-associated virus particles. In conjunction with the observation that HIV-quasispecies in CD4+ and CD4-/CD8- T-cells were phylogenetically closely related, these findings provide evidence that CD4 expression is downmodulated during transition to productive infection in vivo. The abundance of viral RNA in CD4-/CD8- T-cells from viremic patients and the almost complete absence of viral DNA and RNA in this cell type during antiretroviral treatment identifies HIV+ CD4-/CD8 T-cells as the major cell-type harboring productive infection in peripheral blood.
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