Preferential upregulation of interferon-alpha 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.

Human primary CD4 + T cells activated in the presence of IFN-alpha 2b express functional indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the catabolism of tryptophan. By creating a local microenvironment in which levels of tryptophan are low, IDO-expressing antigen-presenting cells (APC) could regulate T cell activation. This may be relevant to control both viral and bacterial replication as well as neoplastic cell growth. Interferon-alpha (IFN-alpha) is an antiviral cytokine affecting cellular differentiation. In addition, it reduces proliferation of CD4(+) T cells by several molecular mechanisms. To dissect the molecular steps responsible for the INF-mediated antiproliferative activity, we sought to determine whether activated primary CD4(+) T cells in the presence of IFN-alpha would produce IDO. We demonstrate here that IDO mRNA is not present in resting CD4(+) T cells. Stimulation with anti-CD3 plus interleukin-2 (IL-2) induces expression of IDO mRNA (about 2000 copies/150,000 cells), as determined by semiquantitative RT-PCR. When cells were stimulated in the presence of IFN-alpha, expression of IDO mRNA was significantly increased (more than 12,000 copies/150,000 cells). Functional analysis of IDO activity paralleled the results obtained with RT-PCR, demonstrating increased production of active enzyme in CD4(+) T cells stimulated in the presence of IFN-alpha. Our results indicate that IFN-alpha modulates levels of IDO produced by activated CD4(+) T cells. This would likely affect bystander cells by modifying levels of tryptophan in the local microenvironment.

Interferon-alpha2b reduces phosphorylation and activity of MEK and ERK through a Ras/Raf-independent mechanism.

Interferon (IFN)-alpha 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-alpha2b 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-alpha was increased by the addition in the culture medium of a specific inhibitor of MEK, namely PD98059. In conclusion, our results indicate that IFN-alpha 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.

The human factors YY1 and LSF repress the human immunodeficiency virus type 1 long terminal repeat via recruitment of histone deacetylase 1.

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.

IFN-alpha 2b reduces IL-2 production and IL-2 receptor function in primary CD4+ T cells.

Initially described as an antiviral cytokine, IFN-alpha has been subsequently shown to affect several cellular functions, including cellular differentiation and proliferation. For these reasons, IFN-alpha is currently used in clinical practice for the treatment of viral infections and malignancies. In this manuscript, we show two novel mechanisms concomitantly responsible for the antiproliferative effect of IFN-alpha. First, long-term treatment with IFN-alpha of primary CD4+ T cells reduced surface expression of CD3 and CD28. These events resulted in decreased phosphorylation of the mitogen-activated extracellular signal-regulated activating kinase and its substrate extracellular signal-regulated kinase, leading to diminished production of IL-2. Second, IFN-alpha treatment of primary CD4+ T cells reduced proliferative response to stimulation in the presence of exogenous IL-2 by markedly decreasing mRNA synthesis and surface expression of CD25 (alpha-chain), a critical component of the IL-2R complex. These results may be relevant for the antitumor effects of IFN-alpha and may help us to better understand its detrimental role in the inhibition of proliferation of the bulk of CD4+ T cells (uninfected cells) in HIV-infected persons, who are known to overproduce IFN-alpha.

Recombinant IFN-alpha (2b) increases the expression of apoptosis receptor CD95 and chemokine receptors CCR1 and CCR3 in monocytoid cells.

IFN-alpha-2b, known as potent immune modulator, can either inhibit or enhance immune cell activity within the tightly regulated microenvironment of inflammation, depending upon the concentration of the cytokine and the activation stage of the cell. Chemokine receptors, which not only mediate chemotaxis of immune cells to the site of inflammation but also affect cellular activation by transferring corresponding signals, represent yet another level of immune regulation. Here we demonstrate that IFN-alpha increases the expression of CCR1 and CCR3 in primary mononuclear phagocytes, as well as in the monocytoid cell line U937. Enhanced receptor mRNA expression correlated with functional readouts such as increased intracellular calcium mobilization and cell migration in response to ligands. Expression of CCR2b, CCR4, CCR5, and CXCR4 was unchanged or decreased after IFN-alpha treatment. These observations indicate a differentially regulated cellular signaling relationship of IFN-alpha pathways and chemokine receptor expression. We also provide evidence that, under these conditions, IFN-alpha treatment increased the expression of CD95 (Fas, Apo1), resulting in enhanced susceptibility to apoptosis. Taken together, these data add important information for the rational application of IFN-alpha (2b) in immune and cancer therapies.

In Vitro Techniques for Studies of HIV-1 Promoter Activity.

A rather unique feature of the human immunodeficiency virus type-1 (HIV-1) is the structural complexity of the regulatory sequences located in the long-terminal repeat (LTR) promoter region and the number of cellular and viral transcription factors known to interact with these sequences and modulate HIV gene expression see ref. 1). The HIV-1 LTR can be schematically divided into four functional regions: (1) the negative regulatory element (NRE) encompassing nuceotides -350 to -190 with respect to the transcription start site; (2) the enhancer (-140 to-81), containing two binding sites for the transcription factor NFκB; (3) the basal promoter (located between -80 and +1), including a typical TATAA box and three binding sites for the transcription factor Sp1; and (4) the trans-activation response (TAR) element, a bulged stem-and-loop structure present in the nascent RNA (+1 to +59) transcript that provides a binding site for Tat activation of HIV-1 transcription. In addition, a novel regulatory DNA element, named IST (Initiator of Short Transcripts), has been shown to be present in the HIV-1 LTR (position (-)5 to (+)26), encompassing the binding site for transcription factors YY1 and late SV40 transcription factor (LSF, or CP-2, or LBP-1) (see refs. 2 and 3). IST directs the RNA polymerase II to synthesize short (59-61 nt), correctly initiated, nonpolyadenylated transcripts that prematurely terminate at the TAR stem-loop structure. The function of these transcripts remains unclear (see ref. 4).

CD4 promoter transactivation by human herpesvirus 6.

The observation that human herpesvirus 6 (HHV-6) can induce CD4 gene transcription and expression in CD4(-) cells was reported several years ago (P. Lusso, A. De Maria, M. Malnati, F. Lori, S. E. DeRocco, M. Baseler, and R. C. Gallo, Nature 349:533-535, 1991) and subsequently confirmed (P. Lusso, M. S. Malnati, A. Garzino-Demo, R. W. Crowley, E. O. Long, and R. C. Gallo, Nature 362:458-462, 1993; G. Furlini, M. Vignoli, E. Ramazzotti, M. C. Re, G. Visani, and M. LaPlaca, Blood 87:4737-4745, 1996). Our objective was to identify the mechanisms underlying such phenomena. Using reporter gene constructs driven by the CD4 promoter, we report that HHV-6 can efficiently transactivate such genetic elements. Activation of the CD4 promoter occurs in the presence of the viral DNA polymerase inhibitor phosphonoformic acid, which limits expression to the immediate-early and early classes of viral genes. Using deletion mutants and specific CD4 promoter mutants, we identified an ATF/CRE binding site located at nucleotides -67 to -60 upstream of the CD4 gene transcription start site that is important for HHV-6 transactivation. The ATF/CRE site is also essential for CD4 promoter activation by forskolin, an activator of adenylate cyclase. Using electrophoretic mobility shift assays and specific antibodies, we showed that CREB-1 binds specifically to the -79 to -52 region of the CD4 promoter. Last, we have identified two open reading frames (ORFs) of HHV-6, U86 and U89 from the immediate-early locus A, that can transactivate the CD4 promoter in HeLa cells. However, transactivation of the CD4 promoter by ORFs U86 and U89 is independent of the CRE element, suggesting that additional HHV-6 ORFs are likely to contribute to CD4 gene activation. Taken together, our results will help to understand the complex interactions occurring between HHV-6 and the CD4 promoter and provide additional information regarding the class of transcription factors involved in the control of CD4 gene expression.

Interferon-gamma increases expression of chemokine receptors CCR1, CCR3, and CCR5, but not CXCR4 in monocytoid U937 cells.

Chemokine receptors (CR), which can mediate migration of immune cells to the site of inflammation, also function as coreceptors for human immunodeficiency virus (HIV) entry into CD4+ T lymphocytes and antigen-presenting cells. We demonstrate here that interferon-gamma (IFN-gamma) increases the expression of chemokine receptors CCR1, CCR3, and CCR5 in monocytoid U937 cells as detected by cell surface molecule labeling and mRNA expression, as well as by intracellular calcium mobilization and cell migration in response to specific ligands. The increased expression of these chemokine receptors also results in an enhanced HIV-1 entry into cells. Our data provide evidence for a relationship of cellular pathways that are induced by IFN-gamma with those that regulate chemokine receptor expression.

Repression of human immunodeficiency virus type 1 through the novel cooperation of human factors YY1 and LSF.

A subpopulation of stably infected CD4+ cells capable of producing virus upon stimulation has been identified in human immunodeficiency virus (HIV)-positive individuals (T.-W. Chun, D. Finzi, J. Margolick, K. Chadwick, D. Schwartz, and R. F. Siliciano, Nat. Med. 1:1284-1290, 1995). Few host factors that directly limit HIV-1 transcription and could support this state of nonproductive HIV-1 infection have been described. YY1, a widely distributed human transcription factor, is known to inhibit HIV-1 long terminal repeat (LTR) transcription and virus production. LSF (also known as LBP-1, UBP, and CP-2) has been shown to repress LTR transcription in vitro, but transient expression of LSF has no effect on LTR activity in vivo. We report that both YY1 and LSF participate in the formation of a complex that recognizes the initiation region of the HIV-1 LTR. Further, we have found that these factors cooperate in the repression of LTR expression and viral replication. This cooperative function may account for the divergent effects of LSF previously observed in vitro and in vivo. Thus, the cooperation of two general cellular transcription factors may allow for the selective downregulation of HIV transcription. Through this mechanism of gene regulation, YY1 and LSF could contribute to the establishment and maintenance of a population of cells stably but nonproductively infected with HIV-1.