The mechanism by which the uninfected CD4+ T cell population is inexorably depleted during the asymptomatic phase of HIV-1 infection remains elusive. From
in vitro functional studies using synthetic peptides, we have concluded that the crown of the gp120 V3 loop participates in the process of antigen presentation between an HIV-infected macrophage and a responding CD4+ T cell, driving the T cell to an accelerated and enhanced AICD [
17]. In the present study, we monitored changes in the transcriptional profile generated by specific signaling that is associated with the V3 epitope, in a primary CD4+ enriched population using oligonucleotide microarrays. Our results provided evidence that the V3 epitope on the surface of macrophages exerts differential and profound effects on cell cycle, gene expression, energy and metabolic demands, orchestrating the proliferation and immune response of the responding CD4+ T cells during antigen presentation.
Microarray technology and the synchronous development of bioinformatics tools that enable a more integrative and function-orientated analysis have shed some light on the immunological imprint of HIV-1 and HIV-1 accessory proteins on a variety of immune cell types [
24]. Most of the studies on the putative HIV-1-induced modulation of host gene expression have been mostly performed in established human cell lines that harbor defects in crucial cellular functions, such as cell cycle, proliferation and apoptosis. This is the first study, to our knowledge, that explored the impact of a defined 15-amino acid V3 epitope of the gp120 on the transcriptional processes of physiological primary human CD4+ enriched cells. A negative selection procedure was preferred to avoid any putative antibody-mediated signaling events. Moreover, the Affymetrix chip used included genes from the whole genome and not only genes related to immune response, thus providing an integral icon of V3 impact on responding T cells. The validity of microarray results was confirmed on a set of genes from three independent donors by real-time reverse transcription polymerase chain reaction quantitative real-time PCR (qRT-PCR).
To decipher the cellular processes most affected by the V3 epitope, we classified genes with modulated transcriptional expression into (a) functional categories through IPA, and (b) into enriched gene modules descriptive of biological processes, molecular functions and cellular compartments by DAVID. Moreover, the data set was mined for networks of interconnected modulated genes, and for overrepresented pathways within the IPA library of canonical pathways.
We focused our analysis on the identification of biological themes and functional gene clusters, as a coordinated change among many gene products may produce potent biological effects, while the effect of each individual gene could be less obvious. Nevertheless, we did not underrate the role of each individual gene exhibiting an extensive modulation, as interpreting the pathways and functions of a set of differentially-expressed genes can only assess genes of known functions operating by known mechanisms. Often these well-annotated genes represent a minority of genes identified. As half of the genome is poorly annotated [
25], the truly new knowledge arising from microarray experiments resides in the functions of the poorly annotated genes.
Cell cycle control was the most overrepresented biological function among classifications of genes affected by V3
The nucleolus, which is mainly associated with ribosome biogenesis, was the most highly overrepresented gene module that emerged from DAVID analysis. An emerging body of evidence indicates that the nucleolus is a dynamic structure involved in the response to cellular stress [
26,
27], in regulation of cell cycle and cell growth [
28] and can also be a target for virus infection [
29]. Intriguingly,
NOC2L, the most significantly mRNA up-regulated gene due to V3 treatment, is located in the nucleolus. NOC2L transcribes an inhibitor of histone acetyltransferase with a poorly defined role. Evidence suggests that NOC2L directly interacts with p53 tumor suppressor inhibiting p53-activated gene expression and p53- dependent apoptosis [
22].
NOC2L also regulates transcription of
CDKN1A[
30],
PMAIP1[
31] and of
TP53I3[
32], affecting cell cycle progression and cell survival.
Prominent within the classifications were groups of genes associated with the cell cycle. Cell cycle was not only the most significantly enriched functional category, but also was the theme of the second highest network. Gene annotation clustering by DAVID highlighted the M phase of the mitotic cell cycle and specifically sister chromatide segregation and ATP binding phosphoproteins as to play crucial role on cell cycle perturbation by the V3 epitope. Canonical pathway analysis highlighted the intracellular cascades that impair cell cycle progression focusing on the regulatory role of stathmin1 (
STMN1) in microtubule dynamics, on the mitotic role of Polo-like kinases, and on G2/M DNA damage checkpoint regulation, the second checkpoint enforced during the cell cycle.
STMN1 is required for orderly progression through mitosis and is overexpressed across a broad range of human malignancies. Polo-like kinases are required at several key points through mitosis, starting from control of the G2/M transition through phosphorylation of CDC25C and mitotic cyclins and in the DNA damage checkpoint adaptation. Noteworthy, aberrant activation patterns of the Polo-like kinase pathway were observed in the CD4+ T cells from SIV-infected Rhesus Macaques (RM) following T-cell receptor stimulation [
33]. These results imply that the V3 epitope infers cell cycle progression propelling cell division in responding CD4 T cells, a process that requires profound changes in the microtubule network that culminates in the formation of the spindle apparatus and the vigilance of DNA integrity/content by regulators of G2/M DNA damage checkpoint. The transcription of genes encoding cyclins, cyclin-dependent kinases and cell cycle regulators (
cyclin I, cyclin D2, MKI67, CDC2, CCNB1) and the E2F cycle-specific transcription factors (
EIF2S1, EIF3S9, EIF4A1) was enhanced presumably in order to control tightly the above-mentioned processes. This is consistent with earlier studies demonstrating that HIV-1 envelope glycoproteins expressed on T cells provoke aberrancies in cell cycle regulatory proteins, specifically accumulation of CCNB1 and hyperphosphorylation of p34cdc2 (CDK1) kinase elicited by cell-cell contact with T cells expressing CD4 coreceptors [
34].
In chronically infected HIV-patients with active viral replication, cell cycle dysregulation (CCD) is associated with increased T-cell susceptibility to apoptosis and involves increased activation of the G2/M phase-associated CCNB/p34-cdc2 complex and abnormal nucleolar structure with dysregulation of nucleolin turnover [
35,
36]. Unscheduled p34-cdc2 activation may induce cell death by a mechanism called mitotic catastrophe [
37] and CCD is consistently associated with increased levels of activation-induced apoptosis [
38]. Moreover, a significantly larger fraction of CD4+ CD25+ T cells from untreated HIV+ individuals expressed cyclin A and/or cyclin B1 than was the case with those from HIV- individuals [
39].
In the most significantly enriched functional categories and networks in our study, were genes associated with gene expression, DNA replication recombination and repair, and cellular development. They indicate that the V3 epitope-CCR5 interaction alters the gene activity facilitating T cell differentiation towards an immune response state. Summarizing, our data suggest that V3 signaling incites CCD in responding CD4+ T cells. This is consistent with our previous findings that the V3 epitope condemns unifected CD4+ T cells to AICD, as CCD has been associated with increased levels of AICD, and there have been observations that this V3 epitope increases the intracellular Ca
++ levels in responding CD4+ T cells during antigen presentation [
17].
The enhanced and accelerated proliferation induced by V3 presumably demands of the T cell increased energy to cope with the required amino acid and lipid metabolism. The AMP-activated protein kinase (AMPK), the most significant enriched pathway in our analysis, is a key regulator of cellular energy homeostasis and was recently shown to be activated by TCR-dependent Ca
++ signals [
40]. Of special interest was also the up-regulation of genes involved in the synthesis of cholesterol, the basic component of lipid rafts. These highly ordered plasma membrane microdomains are reported to play a key organizing role in the formation of immunological synapses, in TCR activation and HIV infection [
41]. The ATP-binding cassette transporter G1 (ABCG), a lipid transporter that promotes cholesterol efflux and regulates its subcellular distribution, was the gene that exhibited the most significant down-regulation in response to V3. Armstrong et al., in a recent study, showed that the absence of ABCG1 in CD4
+ T cells resulted in hyperproliferation
in vitro, when cells are stimulated via the TCR [
42].
Genes associated with immune responses and antigen presentation were significantly affected by the V3 epitope
The presence of V3 induced increased transcription of genes that orchestrate immune response in responding CD4+ T cells, with type I interferons and NFkB being the key genes according to the IPA network. IFN-α is produced by plasmacytoid dendritic cells (pDCs) and elicits an antiviral program protecting CD4+ T cell population against HIV-1. IFN-α inhibits HIV-1 replication, formation of infectious viral particles and induces apoptosis of infected CD4
+ T cells. Thus, it may also account for the depletory effect of HIV on uninfected CD4+ T cells. It has been shown that when pDC and CD4+ T cells bind non-infectious HIV-1, uninfected pDC will produce IFN-α and CD4+ T cells will express TRAIL and DR5, resulting in the preferential apoptosis of uninfected T helper cells [
43]. The protective/immunopathogenic balance is statistically tipped in favor of immunopathogenesis, due to the fact that non-infectious HIV-1 particles have been estimated to outnumber infectious particles.
It is worth noting that a panel of interferon-stimulated genes (ISGs) were up-regulated by the V3 epitope, namely
IFI6,
IRF7,
SP100 and
IFRD1. The integral role of these ISGs in HIV pathogenesis is underpinned by studies that corroborate the observed transcriptional up-regulation in activated CD4+ T cells of untreated HIV-positive individuals (IFI6, IRF7) [
39], in an HIV-infected CD4+ T cell population derived from human tonsils (IFI6, IRF7, SP100) [
44] and in lymphatic tissue of HIV-1 positive individuals throughout all stages of infection (IFI6) [
45]. In addition, IFNα levels and ISG expression are increased in progressor as compared with non-progressor HIV-infected individuals [
46,
47]. The highly up-regulated transcriptional expression of ISGs in both AGMs, SMs and RMs during acute SIV infection persists only in pathogenic RMs during the chronic phase of the infection while it is down-regulated to baseline in AGMs and SMs [
48,
49]
Although NFκB did not display any significant transcriptional change, IPA network analysis attached importance to the role of NF-κΒ in regulating immune response. Transcriptional regulation by NF-κB is crucial in determining T cell fate and it has been implicated in the onset of AICD [
50]. Interestingly, it has been shown that NF-κB activity is considerably enhanced after binding of NF-κB -NFAT5 complexes to κB elements of NF-κB -responsive genes [
51].
In our proposed model [
52], the V3 loop subverts the process of antigen presentation in order to facilitate HIV-1 entry in CD4+ T cells. "CD28 signaling in T cells", the major driver of positive immune response that elicits sustained IL-2 secretion, T cell division, clonal expansion and differentiation was conspicuous among the enriched canonical pathways. The pleiotropic impact of V3 on the CD28 signaling pathway involved the remarkable up-regulation of the transcription factor, NFAT5, the amplification of key genes acting in the PI3K/
AKT cascade, as well as the up-regulation of three molecules that participate in CD3/CD28-triggered actin polymerization. NFAT5 is a member of the Rel family of transcription factors, which comprises of the NF-kB and NFAT proteins, which are major regulators of immune response [
53]. It has been postulated that expression of NFAT5 is correlated with the duration of antigen presentation. Prolonged TCR-dependent stimulation leads to sustained activation of calcineurin and subsequent expression of NFAT5. It has also been suggested that NFAT5 participates in specific aspects of host defense by up-regulating TNF family genes that are culpable of AICD and other target genes in T cells [
54]. Such mechanisms, along with the observed increased expression of NFAT5 by the V3, support our hypothesis that the interaction of V3 with the CCR5 receptor, during antigen presentation, results in persistent activation of the responding CD4 T cells leading subsequently to AICD [
17].
V3 treatment also triggered the significant transcriptional up-regulation of two key genes in the
PI3K/AKT cascade,
PI3KR1 and
PDPK1, that have been shown to play a role in IL-2 induced T cell proliferation. A variety of HIV proteins (gp120, gp160, Tat, Nef) impinge on pleiotropic PI3 pathway in order to facilitate HIV infection. The observed up-regulation of mRNA levels of
PI3KR1 is reconciled with reports that associate gp120 of HIV-1 with elevated PI3-kinase activity and calcium mobilization through the chemokine receptors, CCR5 and CXCR4, in primary CD4 lymphocytes and PI-3K- mediated TNF-α production in macrophages. These studies showed that inhibition of the PI3-kinase signaling pathway suppresses virus infection post-viral entry and post-reverse transcription but prior to HIV gene expression [
55,
56].
The V3 epitope appeared to interfere with the CD28 signaling pathway by up-regulating
N-WASP,
PAK1 and
ARP2/3, the central genes of the host cell actin remodeling machinery. Profound actin rearrangements at the immunological synapse triggered by TCR/CR28 stimulation lead to segregation of surface receptors and receptor-proximal signaling machineries to modulate, amplify and stabilize output signals to ensure ample T cell activation. It has been also proposed that gp120 modulates the synapse triggering a rapid, actin-dependent recruitment of CD4, CXCR4, and LFA-1 on the target cell during cell-cell transmission between X4 HIV-1-infected effector T cell and CD4+/CXCR4+ target T cell [
57]. It is interesting to note that that Nef impairs the ability of infected lymphocytes to form immunological synapses with antigen-presenting cells lowering TCR-mediated stimulation [
58]. Nef reduces actin polymerization inhibiting N-WASP that activates Arp2/3-mediated actin nucleation in lymphocytes [
59]. It is tempting to speculate that Nef manipulating the formation of immunological synapses might counteract the activity of V3. To ensure its transmission, the virus needs to establish a balance between the apoptosis-prone activation state of the recipient T cell and the replication unfavorable environment of a resting T cell [
60]. Thus, V3-CCR5 signaling acts as a double-edged sword; V3 renders cells hyper-responsive to stimulation favoring HIV infection and vigorous T cell proliferation, but in the case of an abortive entry of HIV into a T cell, the cell undergoes sustained proliferation but is condemned to AICD due to the absence of Nef to balance the homeostatic mechanisms of apoptosis.
Studies on the transcriptional imprint of gp120 have been previously performed in astrocytes [
61], keratinocytes [
62], natural killers [
63], pDCs [
58] MMDCs [
64] and PBMCs [
16]. In these studies, gp120 generated subtle changes in the transcriptional profile of astrocytes and induced apoptotic genes of the Fas/FasL pathway in keratinocytes. In NK cells, that lack CD4 receptor, gp120 signaling through CCR5 elicited up-regulation of genes involved in apoptosis and down-regulation of genes involved in proliferation and survival. In pDCs, gp120 interfered with the transcription of genes associated with TLR9 activation and decreased the ability of pDCs to secrete antiviral and inflammatory factors, while in monocyte-macrophage dendritic cells, no significant effect of gp120 on the transcriptional state was found. The transcriptional changes by gp120 in these type of cells were less profound than the modifications in T cells, which is consistent with the fact that these type of cells are less conducive to HIV-1 infection. In an elegant study by Cicala et al [
16], in which the effect of R5 and X4 gp120 on PBMCs was discriminated, it was shown that R5 gp120s exerted a greater impact on the transcriptional profile of responding cells than X4 gp120, and the cell cycle and proliferation were among the gene categories strongly modulated by V3. Interestingly, treatment of cells from CCR5Δ32 homozygous donors with gp120 derived from an R5 virus, demonstrated that the majority of responses required the engagement of the CCR5 coreceptor. Although in the present study we investigated V3-specific signals in a CD4+ enriched population, our findings are consistent with those reported by Cicala et al.