- Open Access
MicroRNA profile changes in human immunodeficiency virus type 1 (HIV-1) seropositive individuals
- Laurent Houzet†1,
- Man Lung Yeung†1,
- Valery de Lame2,
- Dhara Desai2,
- Stephen M Smith2 and
- Kuan-Teh Jeang1Email author
© Houzet et al; licensee BioMed Central Ltd. 2008
Received: 10 December 2008
Accepted: 29 December 2008
Published: 29 December 2008
MicroRNAs (miRNAs) play diverse roles in regulating cellular and developmental functions. We have profiled the miRNA expression in peripheral blood mononuclear cells from 36 HIV-1 seropositive individuals and 12 normal controls. The HIV-1-positive individuals were categorized operationally into four classes based on their CD4+ T-cell counts and their viral loads. We report that specific miRNA signatures can be observed for each of the four classes.
MiRNAs are single-stranded small oligoribonucleotides of 19–25 nt in size that originate from larger RNA polymerase II (RNAP II) transcripts [1–3]. They have been described in plants, invertebrates, and vertebrates. There is evidence that miRNAs function in cellular development, differentiation, proliferation, apoptosis, and metabolism [1, 4, 5]. Perturbed expression of miRNAs is also implicated in cancers and viral infections [6–11].
The course of human immunodeficiency virus (HIV-1) infection in cells is impacted by the action of several hundred host proteins [12–16]. Viral replication appears to be modulated also by the expression of human microRNAs [17–20]. In turn, the expression of HIV-1 proteins in cells  or the in vivo infection by virus  (as monitored by cells harvested from infected individuals) can change human miRNA profiles. To date, a systematic investigation of how human miRNA patterns vary at various stages of HIV-1 infection has not been performed. Here, using patient peripheral blood mononuclear cells (PBMCs), we present miRNA profiling of four classes of HIV-1 seropositive individuals. We report that HIV-1 infection generally resulted in the down regulation of most human miRNAs in vivo.
MicroRNA expression is deregulated in HIV infected patients
Primary PBMC samples are expected to show some degree of individual-to-individual variability. To analyze the raw miRNA readouts, we applied two levels of filtering. First, we considered only those miRNAs that were at least two fold or more changed (either up or down) when compared to normal controls. Second, we discarded miRNA changes that were not replicated in more than 50% of the patients in any of the four different classes. When these two filters were applied to the 327 miRNA readouts, 62 miRNAs satisfied both criteria (Figure 1B). The frequencies of these 62 miRNA changes were then compared between class I, II, III, and IV patients using JMP software and BRB array tools (see Materials and Methods). The resulting in silico clustering patterns indicated a closer relatedness in the frequencies of miRNA changes between class II and class III patients; and between class I and class IV patients (Figure 1B). It is unclear at this juncture what these relationships mean biologically.
Class-specific signatures in HIV-1 patient PBMCs
miRNA profiles are changed in PBMCs treated ex vivo with T-cell activating or inactivating stimuli
Several highly abundant T-cell specific miRNAs were down regulated
We describe here miRNA changes in PBMCs from 36 HIV-1 seropositive individuals categorized into four descriptive classes (Figure 1A). Our findings revealed miRNA signature profiles which are sufficiently distinctive that different classes of HIV-1 infected persons could be distinguished using these biomarkers (Figure 3). Because only a small fraction of PBMCs are infected by HIV-1 in vivo, our "Patients" miRNA changes are more likely results of bystander effects [26, 27] than outcomes of direct cellular infection by HIV-1. Indeed, the "Patients"-specific miRNA profile did not match well the miRNA changes in virus infected PBMCs (Figure 4).
While the description of signature profiles is interesting, a question remains why do the miRNAs change during HIV-1 infection? The answer is unknown; however, one view is that the virus may benefit from altering the host cell's normal miRNA milieu. This view emerges from the idea that certain host cell miRNAs may serve innate antiviral defenses. Two types of extant findings support the above notion. First, miRNA-processing enzymes such as Drosha and Dicer have been knocked down to reduce the cell's processing of mature miRNAs [22, 28, 29]. When mammalian miRNAs were thusly reduced, virus replication in cells became more robust. Second, when putative anti-viral miRNAs have been knocked down directly using chemically modified antisense-oligoribonucleotides, or antagomirs [17, 19, 30], these knock downs also enhanced viral replication. Collectively, these findings are compatible with some cellular miRNAs acting physiologically to suppress viral infection. Indeed, miR-150 and miR-223 have been shown to target the HIV-1 genome, restricting virus expression . Our observed down modulation of these two miRNAs in T-cells (Figure 6) would suggest an in vivo setting which favors HIV-1 replication. A second view is that cellular miRNAs could be co-opted by viruses to enhance propagation. Thus, it has been reported that human miR-122 interacts with the 5' UTR of hepatitis C virus (HCV) RNA. MiR-122, rather than antagonizing HCV replication, appears to augment intracellular viral production [31, 32]. These two views when taken together argue that down regulation of anti-viral miRNAs and up regulation of virus-augmenting miRNAs may be beneficial objectives for the virus to achieve in vivo.
MiRNAs target cellular mRNAs and proteins, and miRNAs are also involved in the differentiation of hematopoietic cells and the regulation of immune cell function and activity . Since one miRNA could potentially target one hundred discrete mRNAs through imperfect complementarity, another outcome of miRNA profile changes may be to alter the landscape of host cell proteins . We note that most "Patients" miRNAs are down regulated (Figure 2), suggesting that the mRNA/protein targets of these miRNAs might be commensurately up regulated in vivo. Because many host cell proteins act to modulate HIV-1 replication , a careful and detailed analyses of how some of these host factors match as targets of our "Patients" miRNAs would be highly informative.
The above discussions suggest miRNA changes as causative of pathogenic manifestations. On the other hand, it cannot be excluded that the miRNA alterations may simply be consequences of viral pathogenesis. In this respect, HIV/SIV disease progression has been correlated with systemic immune activation [34–37]. We note that our "Patients" miRNA profiles are more consistent with T-cell immune activation (Figure 4) than immune inactivation (Figure 5). Time will tell whether it is miRNA changes that result in immune activation/inactivation or vice versa. We caution that because our PBMC samples have not been fractionated into cellular subsets, some of the differences in miRNA signatures could be explained by in-/out- fluxes of different cell types. Nevertheless, the current picture paints an interplay between cellular miRNAs and viruses which is complex; and one which has evolved into an apparent equilibrium between the host and the pathogen, creating a milieu for moderate and persistent in vivo viral infection . Finally, this miRNA analysis, although still in its early stages, might be adapted usefully in the future to staging patients for antiretroviral therapy.
Materials and methods
Patients and cells
Normal and human immunodeficiency virus-infected patient PBMCs were obtained from the NIH blood bank and Saint Michael's Medical Center. The study protocol was approved by the St. Michael's Medical Center's Institutional Review Board. Written informed consent was obtained from each subject. The IRB approval letter and the signed, informed consents are available for review. Plasma viral loads were quantified by the Bayer SIV bDNA assay (Bayer Reference Testing Laboratory, Emeryville, CA) . Peripheral blood CD4+ T-cell concentrations were quantified using standard techniques, as previously described . PBMCs were isolated using standard Ficoll separation procedure. Ficoll-purified PBMCs were directly lysed for RNA isolation or stored in liquid nitrogen.
RNA-primed array-based Klenow extension analysis
RNAs with a cutoff size < 200 nts were hybridized on a microarray printed with 327 probes complementary to mature miRNAs. The probe design and the experimental procedures are the same as previously described . After hybridization, excessive RNA was removed by washing in 0.1 × SSC. Unhybridized probes were removed using exonuclease I (New England Biolabs) for 3 hours. Since the probe design contains a stretch of thymidine, polyadenylation from the 3'end of the hybridized miRNAs was achieved by addition of biotin-label dATP (Enzo Life Sciences). Detection of the labeled miRNA under the 532 nm wavelength was facilitated by addition of streptavidin-conjugated Alexa-flur-555. Data points collected from GenePix 4000B (Molecular Devices) were exported into BRBarray tools (developed by Richard Simon and Amy Peng Lam; http://linus.nci.nih.gov/BRB-ArrayTools.html) and JMP software (SAS) for further analysis. Microarray signal normalization was performed using the "median-normalization" procedure. This method is applicable for normalizing arrays in which the majority of data points do not change significantly in values. Essentially, the log-intensities of an array and the reference array are normalized to a median value such that the unchanged gene-by-gene difference between the normalized array and the reference array is 0. The linearity of the microarray readouts has been previously validated using quantitative RT-PCR assays.
This study was supported in part by the NIH Bench-to-Bedside Program, the Intramural AIDS Targeted Anti-viral Program (IATAP), and intramural funding from NIAID, NIH. We thank the NIAID microarray core facility for advice and assistance.
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