Fabio Romerio

The Human Factors YY1 and LSF Repress the Human Immunodeficiency Virus Type 1 Long Terminal Repeat via Recruitment of Histone Deacetylase 1

ABSTRACT Enigmatic mechanisms restore the resting state in activated lymphocytes following human immunodeficiency virus type 1 (HIV-1) infection, rarely allowing persistent nonproductive infection. We detail a mechanism whereby cellular factors could establish virological latency. The transcription factors YY1 and LSF cooperate in repression of transcription from the HIV-1 long terminal repeat (LTR). LSF recruits YY1 to the LTR via the zinc fingers of YY1. The first two zinc fingers were observed to be sufficient for this interaction in vitro. A mutant of LSF incapable of binding DNA blocked repression. Like other transcriptional repressors, YY1 can function via recruitment of histone deacetylase (HDAC). We find that HDAC1 copurifies with the LTR-binding YY1-LSF repressor complex, the domain of YY1 that interacts with HDAC1 is required to repress the HIV-1 promoter, expression of HDAC1 augments repression of the LTR by YY1, and the deacetylase inhibitor trichostatin A blocks repression mediated by YY1. This novel link between HDAC recruitment and inhibition of HIV-1 expression by YY1 and LSF, in the natural context of a viral promoter integrated into chromosomal DNA, is the first demonstration of a molecular mechanism of repression of HIV-1. YY1 and LSF may establish transcriptional and virological latency of HIV, a state that has recently been recognized in vivo and has significant implications for the long-term treatment of AIDS.

Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA

Background: Exosomes are extracellular vesicles that have been implicated in intercellular communication. Results: Exosomes that originate from human cells infected with HIV-1 contain virus-derived small noncoding RNA. Conclusion: Virus-derived small RNA present in exosomes exert functional consequences in naive recipient cells. Significance: Viral RNA molecules present in exosomes may be critical mediators of intercellular viral spread in infected hosts. Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 104–106 copies/ml TAR RNA in exosomes derived from infected culture supernatants and 103 copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.

An In Vitro System to Model the Establishment and Reactivation of HIV-1 Latency

HIV-1 establishes latency primarily by infecting activated CD4+ T cells that later return to quiescence as memory cells. Latency allows HIV-1 to evade immune responses and to persist during antiretroviral therapy, which represents an important problem in clinical practice. The lack of a valid cellular model to study HIV-1 latency has hindered advances in the understanding of its biology. In this study, we attempted to model HIV-1 latency using human primary CD4+ T cells infected in vitro with HIV-1 after activation with Ag-loaded dendritic cells and then brought back to quiescence through a resting phase in the presence of IL-7. During the resting phase, expression of cellular activation markers disappeared and cell proliferation and viral replication ceased, but resumed following restimulation of rested cells with Ag or mAbs directed to CD3/CD28. In addition, higher cell death rates were observed in HIV-1-infected than uninfected cultures during secondary but not primary stimulation. Thus, this system may allow us to study the biology of HIV-1 latency, as well as the mechanisms of CD4+ T cell death following HIV-1 reactivation.

Plasmacytoid dendritic cells accumulate and secrete interferon alpha in lymph nodes of HIV-1 patients.

Circulating plasmacytoid dendritic cells (pDC) decline during HIV-1 infection, but at the same time they express markedly higher levels of interferon alpha (IFNα), which is associated with HIV-1 disease progression. Here we show an accumulation of pDC in lymph nodes (LN) of treatment-naïve HIV-1 patients. This phenomenon was associated with elevated expression of the LN homing marker, CCR7, on pDC in peripheral blood of HIV-1 patients, which conferred increased migratory capacity in response to CCR7 ligands in ex vivo functional assays. LN-homed pDC of HIV-1 patients presented higher CD40 and lower BDCA2 levels, but unchanged CD83 and CD86 expression. In addition, these cells expressed markedly higher amounts of IFNα compared to uninfected individuals, and were undergoing faster rates of cell death. These results demonstrate for the first time that in asymptomatic, untreated HIV-1 patients circulating pDC up-regulate CCR7 expression, accumulate in lymph nodes, and express high amounts of IFNα before undergoing cell death. Since IFNα inhibits cell proliferation and modulates immune responses, chronically high levels of this cytokine in LN of HIV-1 patients may impair differentiation and immune function of bystander CD4+ T cells, thus playing into the mechanisms of AIDS immunopathogenesis.

MEK and ERK inhibitors enhance the anti-proliferative effect of interferon-α2b

Interferon (IFN)‐α, initially characterized as an antiviral cytokine, affects several cellular functions. It is used in clinical practice for the treatment of several tumors, including hematopoietic malignancies, due to its antiproliferative effects. To better characterize the molecular mechanism(s) underlying this property, we conducted our studies in purified primary CD4+ T cells stimulated with anti‐CD3 and interleukin (IL)‐2. Upon treatment with IFN‐α, the cells were blocked in the G0/G1 phase of the cell cycle and exhibited impaired entry into S phase and reduced proliferation. Moreover, we detected short‐ and long‐term inhibition of extracellular signal‐regulated kinase (ERK) and mitogen‐activated ERK‐regulating kinase (MEK) function, known to control cellular proliferation. The activity of the upstream regulators, Ras and Raf‐1, was not affected. Analysis of downstream events controlled by the MEK/ERK pathway showed reduced activity of cyclin‐dependent kinase (Cdk)‐2 and ‐4, high levels of the mitotic inhibitors, p21Waf1 and p27Kip1, and decreased cyclin D and E expression. When IFN‐α was used in combination with MEK and ERK inhibitors, we observed a dose‐dependent additive effect in reducing cellular proliferation. Our data demonstrate that IFN‐α may be associated with other molecules to inhibit cellular growth by targeting the MEK/ERK pathway. This may eventually lead to new clinical strategies to strengthen its anticancer effect.

Increased Interferon Alpha Expression in Circulating Plasmacytoid Dendritic Cells of HIV-1-Infected Patients

Background:The role of plasmacytoid dendritic cells (pDC) and interferon alpha (IFNα) in HIV-1 infection is still unclear. On one hand, HIV-1 disease is associated with a progressive decline of pDC, which displays reduced ability to produce IFNα after in vitro challenge. On the other hand, high IFNα serum levels in HIV-1-infected individuals have been proposed to promote immune hyper-activation and disease progression. Methods:We sought to determine whether disappearance of pDC in HIV-1 disease is due to homing in lymphoid tissues. We also studied IFNα and myxovirus resistance protein A (MxA) expression in unstimulated pDC and correlated these results with selected clinical and laboratory parameters. Results:We found that pDC decline markedly in peripheral blood of patients progressing to disease but at the same time express much higher levels of IFNα and MxA compared to control individuals. On the other hand, we observed steady pDC counts in lymph nodes of HIV-1 patients. The frequency of circulating pDC correlated directly with CD4 cell counts and inversely with viral load. However, we found no correlation between IFNα and MxA expression levels, CD4 counts, and viral load. Conclusions:Circulating pDC decline sharply in the course of HIV-1 disease, but express high levels of IFNα, which may represent a hallmark of systemic immune dysfunction.

Longitudinal Analysis of Distribution and Function of Plasmacytoid Dendritic Cells in Peripheral Blood and Gut Mucosa of HIV infected patients

Aberrant activation of plasmacytoid dendritic cells (pDCs) with excessive production of interferon alpha (IFNα) represents one of the hallmarks of immune activation during chronic phase of human immunodeficiency virus (HIV) infection. A number of studies have shown that disruption of mucosal integrity in the gut is a cause of persistent immune activation. However, little is known about the role that pDCs play in this process, and our current understanding comes from the simian immunodeficiency virus macaque model. Thus, in the present study we sought to investigate the frequency and function of pDCs in peripheral blood and gut samples from HIV-infected individuals before and 6 months after initiation of antiretroviral therapy (ART). We show that circulating pDCs were depleted in ART-naive HIV+ patients, and upregulated the gut-homing receptor CD103 compared with uninfected controls. By converse, pDCs accumulated in the terminal ileum of ART-naive HIV individuals compared with controls. Baseline levels of IFNα production and markers of immune activation in gut samples of ART-naive HIV subjects were elevated. All these parameters declined after 6 months of ART. Our results suggest that in chronic HIV infection, pDCs migrate from peripheral blood to the gut-associated lymphatic tissue, where they may contribute to immune activation.

Preferential Upregulation of Interferon-α Subtype 2 Expression in HIV-1 Patients

Humans tailor virus-specific immune responses through modulated expression of 12 different interferon (IFN)-alpha subtypes. However, exacerbated expression of certain IFN-alpha subtypes causes immunopathology in the context of autoimmune conditions and chronic viral infections. We showed that progression to AIDS is associated with elevated expression of IFN-alpha in unstimulated peripheral blood mononuclear cells. Here, we sought to determine whether distinct IFN-alpha subtypes are involved in this phenomenon. We used quantitative RT-PCR to assess expression levels of 12 IFN-alpha subtypes in peripheral blood mononuclear cells from normal donors and HIV-1 patients at CDC stage A and stage C of the disease. Three patterns of IFN-alpha subtype expression emerged. First, IFN-alpha2 and IFN-alpha6 mRNA levels were elevated in both patient groups. Second, IFN-alpha1/13, IFN-alpha8, IFN-alpha14, IFN-alpha16, IFN-alpha17, and IFN-alpha21 were upregulated in stage C but not stage A patients. Third, expression levels of IFN-alpha4, IFN-alpha5, IFN-alpha7, and IFN-alpha10 did not change among the three groups of volunteers. Among all other subtypes, IFN-alpha2 was preferentially upregulated, showing >60-fold higher levels in stage A and >400-fold in stage C patients compared with controls, which correlated with declining CD4 counts. Our results demonstrate that distinct IFN-alpha subtypes are sequentially activated during HIV-1 infection, which may be predictive of disease progression.

High levels of CD2 expression identify HIV-1 latently infected resting memory CD4+ T cells in virally suppressed subjects

ABSTRACT Resting memory CD4+ T cells are the largest reservoir of persistent infection in HIV-1-positive subjects. They harbor dormant, stably integrated virus despite suppressive antiretroviral therapy, posing an obstacle to a cure. Surface markers that identify latently infected cells remain unknown. Microarray analyses comparing resting latently infected and uninfected CD4+ T cells generated in vitro showed profound differences in the expression of gene programs related to transcriptional and posttranscriptional regulation, cell proliferation, survival, cycle progression, and basic metabolism, suggesting that multiple biochemical and metabolic blocks contribute to preventing viral production in latently infected cells. We identified 33 transcripts encoding cell surface markers that are differentially expressed between latently infected and uninfected cells. Quantitative reverse transcriptase PCR (RT-QPCR) and flow cytometry analyses confirmed that the surface marker CD2 was expressed at higher levels on latently infected cells. To validate this result in vivo, we sorted resting memory CD4+ T cells expressing high and low surface levels of CD2 from six HIV-1-infected subjects successfully treated with antiretroviral drugs for at least 3 years. Resting memory CD4+ CD2high T cells from all subjects harbored higher HIV-1 DNA copy numbers than all other CD4+ T cell subsets. Moreover, after ex vivo viral reactivation, robust viral RNA production was detected only from resting memory CD4+ CD2high T cells but not from other cell subsets. Altogether, these results show that a high CD2 expression level is a hallmark of latently infected resting memory CD4+ T cells in vivo.

Interferon- α 2b reduces phosphorylation and activity of MEK and ERK through a Ras / Raf -independent mechanism

Interferon (IFN)-α affects the growth, differentiation and function of various cell types by transducing regulatory signals through the Janus tyrosine kinase/signal transducers of activation and transcription (Jak/STAT) pathway. The signalling pathways employing the mitogen-activated ERK-activating kinase (MEK) and the extracellular-regulated kinase (ERK) are critical in growth factors signalling. Engagement of the receptors, and subsequent stimulation of Ras and Raf, initiates a phosphorylative cascade leading to activation of several proteins among which MEK and ERK play a central role in routing signals critical in controlling cell development, activation and proliferation. We demonstrate here that 24–48 h following treatment of transformed T- and monocytoid cell lines with recombinant human IFN-α2b both the phosphorylation and activity of MEK1 and its substrates ERK1/2 were reduced. In contrast, the activities of the upstream molecules Ras and Raf -1 were not affected. No effect on MEK/ERK activity was observed upon short-term exposure (1–30 min) to IFN. The anti-proliferative effect of IFN-α was increased by the addition in the culture medium of a specific inhibitor of MEK, namely PD98059. In conclusion, our results indicate that IFN-α regulates the activity of the MEK/ERK pathway and consequently modulates cellular proliferation through a Ras/Raf-independent mechanism. Targeting the MEK/ERK pathway may strengthen the IFN-mediated anti-cancer effect.