Marco Sgarbanti

HIV-1, interferon and the interferon regulatory factor system: An interplay between induction, antiviral responses and viral evasion

Thirty years after the first isolation of the etiological agent of AIDS, the virus HIV-1 is still a major threat worldwide with millions of individuals currently infected. Although current combination therapies allow viral replication to be controlled, HIV-1 is not eradicated and persists in drug- and immune system-insensitive reservoirs and a cure is still lacking. Pathogens such as HIV-1 that cause chronic infections are able to adapt to the host in a manner that ensures long term residence and survival, via the evolution of numerous mechanisms that evade various aspects of the innate and adaptive immune response. One such mechanism is targeted to members of the interferon (IFN) regulatory factor (IRF) family of proteins. These transcription factors regulate a variety of biological processes including interferon induction, immune cell activation and downstream pattern recognition receptors (PRRs). HIV-1 renders IRFs harmless and hijacks them to its own advantage in order to facilitate its replication and evasion of immune responses. Type I interferon (IFN), the canonical antiviral innate response, can be induced in both acute and chronic HIV-1 infection in vivo, but in the majority of individuals this initial response is not protective and can contribute to disease progression. Type I IFN expression is largely inhibited in T cells and macrophages in order to successfully establish productive infection, whereas sustained IFN production by plasmacytoid dendritic cells is considered an important source of chronic immune activation, a hallmark to AIDS progression.

Therapeutics for HIV-1 reactivation from latency

Intensive combined antiretroviral therapy successfully suppresses HIV-1 replication and AIDS disease progression making infection manageable, but it is unable to eradicate the virus that persists in long-lived, drug-insensitive and immune system-insensitive reservoirs thus asking for life-long treatments with problems of compliance, resistance, toxicity and cost. These limitations and recent insights into latency mechanisms have fueled a renewed effort in finding a cure for HIV-1 infection. Proposed eradication strategies involve reactivation of the latent reservoir upon induction of viral transcription followed by the elimination of reactivated virus-producing cells by viral cytopathic effect or host immune response. Several molecules identified by mechanism-directed approaches or in large-scale screenings have been proposed as latency reversing agents. Some of them have already entered clinical testing in humans but with mixed or unsatisfactory results.

Role of Acetylases and Deacetylase Inhibitors in IRF-1-Mediated HIV-1 Long Terminal Repeat Transcription

Abstract: There is strong evidence that both transcriptional activation and silencing are mediated through the recruitment of enzymes that control reversible protein acetylation: histone acetylase (HAT) and histone deacetylase proteins. Acetylation is also a critical post‐translational modification of general and tissue‐specific transcription factors. In HIV‐1‐infected cells, the long terminal repeat (LTR) promoter, once organized into chromatin, is transcriptionally inactive in the absence of stimulation. LTR transcription is regulated by protein acetylation, since treatment with deacetylase inhibitors markedly induces transcriptional activity of the LTR. Besides cellular transcription factors involved in LTR activation, early in infection, and during reactivation from latency, we have previously shown that proteins of the IRF family play an important role. In particular, IRF‐1 is able per se to stimulate HIV‐1 LTR transcription even in the absence of Tat. IRF‐1 is also acetylated and associates with HATs such as p300/CBP and PCAF to form a multiprotein complex that assembles on the promoter of target genes. Here we show that CBP can be recruited by IRF‐1 to the HIV‐1 LTR promoter even in the absence of Tat and that treatment with deacetylase inhibitors, such as trichostatin A (TSA), increases LTR transactivation in response to both IRF‐1 and Tat. These results help to define the architecture of interactions between transcription factors binding HIV‐1 LTR and confirm the possibility that deacetylase inhibitors, such as TSA, combined with antiviral therapy may represent a valuable approach to control HIV‐1 infection.

Interferon regulatory factor‐1 acts as a powerful adjuvant in tat DNA based vaccination

Genetic vaccines are safe cost‐effective approaches to immunization but DNA immunization is an inefficient process. There is, therefore, a pressing need for adjuvants capable of enhancing the immunogenicity and effectiveness of these vaccines. This is particularly important for diseases for which successful vaccines are still lacking, such as cancer and infectious diseases including HIV‐1/AIDS. Here we report an approach to enhance the immunogenicity of DNA vaccines involving the use of transcription factors of the Interferon regulatory factor (IRF) family, specifically IRF‐1, IRF‐3, and IRF‐7 using the tat gene as model antigen. Balb/c mice were immunized by three intramuscular inoculations, using a DNA prime‐protein boost protocol, with a DNA encoding tat of HIV‐1 and the indicated IRFs and immune responses were compared to those induced by vaccination with tat DNA alone. In vivo administration of plasmid DNA encoding IRF‐1, or a mutated version of IRF‐1 deleted of the DNA‐binding domain, enhanced Tat‐specific immune responses and shifted them towards a predominant T helper 1‐type immune response with increased IFN‐γ production and cytotoxic T lymphocytes responses. Conversely, the use of IRF‐3 or IRF‐7 did not affect the tat‐induced responses. These findings define IRF‐1 and its mutated form as efficacious T helper 1‐inducing adjuvants in the context of tat‐based vaccination and also providing a new promising candidate for genetic vaccine development. J. Cell. Physiol. 224: 702–709, 2010.

Human Papillomavirus Type 16 E5 Protein Induces Expression of Beta Interferon through Interferon Regulatory Factor 1 in Human Keratinocytes

ABSTRACT Crucial steps in high-risk human papillomavirus (HR-HPV)-related carcinogenesis are the integration of HR-HPV into the host genome and loss of viral episomes. The mechanisms that promote cervical neoplastic progression are, however, not clearly understood. During HR-HPV infection, the HPV E5 protein is expressed in precancerous stages but not after viral integration. Given that it has been reported that loss of HPV16 episomes and cervical tumor progression are associated with increased expression of antiviral genes that are inducible by type I interferon (IFN), we asked whether E5, expressed in early phases of cervical carcinogenesis, affects IFN-β signaling. We show that the HPV type 16 (HPV16) E5 protein expression per se stimulates IFN-β expression. This stimulation is specifically mediated by the induction of interferon regulatory factor 1 (IRF-1) which, in turn, induces transcriptional activation of IRF-1-targeted interferon-stimulated genes (ISGs) as double-stranded RNA-dependent protein kinase R (PKR) and caspase 8. Our data show a new and unexpected role for HR-HPV E5 protein and indicate that HPV16 E5 may contribute to the mechanisms responsible for cervical carcinogenesis in part via stimulation of IFN-β and an IFN signature, with IRF-1 playing a pivotal role. HPV16 E5 and IRF-1 may thus serve as potential therapeutic targets in HPV-associated premalignant lesions.

IκB kinase ε targets interferon regulatory factor 1 in activated T lymphocytes.

ABSTRACT IκB kinase ε (IKK-ε) has an essential role as a regulator of innate immunity, functioning downstream of pattern recognition receptors to modulate NF-κB and interferon (IFN) signaling. In the present study, we investigated IKK-ε activation following T cell receptor (TCR)/CD28 stimulation of primary CD4+ T cells and its role in the stimulation of a type I IFN response. IKK-ε was activated following TCR/CD28 stimulation of primary CD4+ T cells; however, in T cells treated with poly(I·C), TCR/CD28 costimulation blocked induction of IFN-β transcription. We demonstrated that IKK-ε phosphorylated the transcription factor IFN regulatory factor 1 (IRF-1) at amino acid (aa) 215/219/221 in primary CD4+ T cells and blocked its transcriptional activity. At the mechanistic level, IRF-1 phosphorylation impaired the physical interaction between IRF-1 and the NF-κB RelA subunit and interfered with PCAF-mediated acetylation of NF-κB RelA. These results demonstrate that TCR/CD28 stimulation of primary T cells stimulates IKK-ε activation, which in turn contributes to suppression of IFN-β production.

On the Role of Interferon Regulatory Factors in HIV‐1 Replication

Abstract: Interferons (IFNs) are pleiotropic cytokines that possess several biological activities and play a central role in basic and applied research as mediators of antiviral and antigrowth responses, modulators of the immune system, and therapeutic agents against viral diseases and cancer. Interferon regulatory factors (IRFs) have been identified together with signal transducers and activators of transcription (STAT) from studies on the type I IFN as well as IFN‐stimulated (ISG) gene regulation and signaling. IRFs constitute a family of transcriptional activators and repressors implicated in multiple biological processes including regulation of immune responses and host defence, cytokine signaling, cell growth regulation, and hematopoietic development. All members share a well‐conserved DNA binding domain at the NH2‐terminal region that recognizes similar DNA sequences, termed IRF element (IRF‐E)/interferon‐stimulated response element (ISRE), present on the promoter of target genes. Recently, a sequence homologous to the ISRE has been identified downstream from the 5′ human immunodeficiency virus type 1 (HIV‐1) long terminal repeat (LTR). This sequence is a binding site for IRF‐1 and IRF‐2. Here we briefly summarize the role of IRFs in the regulation of HIV‐1 LTR transcriptional activity and virus replication. The overall effect of IRFs on HIV‐1 replication will also be discussed in the context of strategies carried out by the virus to counteract the IFN‐mediated host defences both in active replication and during the establishment of viral latency.

Type I IFN – A blunt spear in fighting HIV-1 infection

For more than 50 years, Type I Interferon (IFN) has been recognized as critical in controlling viral infections. IFN is produced downstream germ-line encoded pattern recognition receptors (PRRs) upon engagement by pathogen-associated molecular patterns (PAMPs). As a result, hundreds of different interferon-stimulated genes (ISGs) are rapidly induced, acting in both autocrine and paracrine manner to build a barrier against viral replication and spread. ISGs encode proteins with direct antiviral and immunomodulatory activities affecting both innate and adaptive immune responses. During infection with viruses, as HIV-1, that can establish a persistent infection, IFN although produced, is not able to block the initial infection and a chronic IFN-mediated immune activation/inflammation becomes a pathogenic mechanism of disease progression. This review will briefly summarize when and how IFN is produced during HIV-1 infection and the way this innate immune response is manipulated by the virus to its own advantage to drive chronic immune activation and progression to AIDS.

Analysis of the signal transduction pathway leading to human immunodeficiency virus-1-induced interferon regulatory factor-1 upregulation.

Abstract: Interferon (IFN) regulatory factors (IRFs) constitute a family of transcriptional activators and repressors involved in the regulation of immune system, host defense, and cell growth. All members share conserved DNA‐binding domains that recognize DNA sequences termed IRF‐binding elements/IFN‐stimulated response elements (IRF‐E/ISRE) present on the promoter of IFN‐α/β and IFN‐stimulated genes. An ISRE has been identified downstream of the transcription start site of the long terminal repeat (LTR) of human immunodeficiency virus‐1 (HIV‐1). Our previous results showed that among the IRF factors, IRF‐1 is able to stimulate HIV‐1 LTR transcription and its expression is induced by HIV‐1, early, upon infection and before the expression of Tat. In this study we investigated the signal transduction pathway leading to HIV‐1‐induced IRF‐1 expression. Key IRF‐1 promoter elements that mediate the activation of transcription upon induction by inflammatory cytokines are IFN‐γ‐activated sequences that bind members of the signal transducer and activator of transcription (STAT) family and binding sites for nuclear factor κB (NF‐κB). Both STAT‐1 and NF‐κB activation were examined to determine putative molecular targets whose inhibition resulted in the inhibition of HIV‐1 replication. The results show that at early time points after HIV‐1 infection, NF‐κB but not STAT‐1 is activated. Moreover, a significant decrease in HIV‐1 replication was observed upon de novo infection of Jurkat T cells expressing an NF‐κB super‐repressor (IκB‐α 2NΔ4). These results suggest that in early phases of HIV‐1 infection, before detectable cytokine production, NF‐κB seems responsible for HIV‐1‐induced IRF‐1 expression.

HIV-1 Tat Recruits HDM2 E3 Ligase To Target IRF-1 for Ubiquitination and Proteasomal Degradation

ABSTRACT In addition to its ability to regulate HIV-1 promoter activation, the viral transactivator Tat also functions as a determinant of pathogenesis and disease progression by directly and indirectly modulating the host anti-HIV response, largely through the capacity of Tat to interact with and modulate the activities of multiple host proteins. We previously demonstrated that Tat modulated both viral and host transcriptional machinery by interacting with the cellular transcription factor interferon regulatory factor 1 (IRF-1). In the present study, we investigated the mechanistic basis and functional significance of Tat−IRF-1 interaction and demonstrate that Tat dramatically decreased IRF-1 protein stability. To accomplish this, Tat exploited the cellular HDM2 (human double minute 2 protein) ubiquitin ligase to accelerate IRF-1 proteasome-mediated degradation, resulting in a quenching of IRF-1 transcriptional activity during HIV-1 infection. These data identify IRF-1 as a new target of Tat-induced modulation of the cellular protein machinery and reveal a new strategy developed by HIV-1 to evade host immune responses. IMPORTANCE Current therapies have dramatically reduced morbidity and mortality associated with HIV infection and have converted infection from a fatal pathology to a chronic disease that is manageable via antiretroviral therapy. Nevertheless, HIV-1 infection remains a challenge, and the identification of useful cellular targets for therapeutic intervention remains a major goal. The cellular transcription factor IRF-1 impacts various physiological functions, including the immune response to viral infection. In this study, we have identified a unique mechanism by which HIV-1 evades IRF-1-mediated host immune responses and show that the viral protein Tat accelerates IRF-1 proteasome-mediated degradation and inactivates IRF-1 function. Restoration of IRF-1 functionality may thus be regarded as a potential strategy to reinstate both a direct antiviral response and a more broadly acting immune regulatory circuit. Current therapies have dramatically reduced morbidity and mortality associated with HIV infection and have converted infection from a fatal pathology to a chronic disease that is manageable via antiretroviral therapy. Nevertheless, HIV-1 infection remains a challenge, and the identification of useful cellular targets for therapeutic intervention remains a major goal. The cellular transcription factor IRF-1 impacts various physiological functions, including the immune response to viral infection. In this study, we have identified a unique mechanism by which HIV-1 evades IRF-1-mediated host immune responses and show that the viral protein Tat accelerates IRF-1 proteasome-mediated degradation and inactivates IRF-1 function. Restoration of IRF-1 functionality may thus be regarded as a potential strategy to reinstate both a direct antiviral response and a more broadly acting immune regulatory circuit.