HTLV ANTISENSE PROTEINS ROLE IN THE NF-\u3baB MODULATION.

The retrovirus HTLV-1 is the causative agent of adult T-cell leukemia, whereas the genetically related sierotype HTLV-2 is sporadically associated with neurological diseases. The HTLV-1 genome encodes regulatory proteins, such as the oncoprotein Tax and the antisense proteins HBZ, involved into T-cells proliferation and transformation. Tax-1, HBZ, and the HTLV-2 homologs, Tax-2 and APH-2 interact with many host cell factors imparing cell signaling pathways involved in the mechanisms of survival, and proliferation, including the NF-\u3baB pathway. The aim of this study is to investigate the involvement of the regulatory proteins HBZ and APH-2 in the constitutively Tax-mediated NF-\u3baB activation. We demonstrated that HBZ and APH-2 differ in the NF-\u3baB promoter suppression. The APH-2 protein, differently from HBZ, localizes into the cytoplasm in presence of Tax, where it prevents the degradation of the inhibitor I\u3baB, hindering the nuclear translocation of p65. Unlike HBZ, we found that APH-2 interacts with the E3 ubiquitin ligase TRAF3, an upstream inhibitor of the alternative NF-\u3baB pathway. By generating a TRAF3-KO cell line applying the CRISPR/Cas9 technique, we are investigating the HBZ and APH-2 activity on the alternative NF-\u3baB cell signaling. This study may provide insight into the effect of host-viral interactions in human viral oncogenesis

HTLV-1 BASIC LEUCIN ZIPPER FACTOR AND ITS HOMOLOGOUS APH-2 IMPAIR NF-\u3baB ACTIVATION MEDIATED BY THE VIRAL ONCOPROTEIN TAX.

HTLV-1 and HTLV-2 are complex retroviruses which share a similar genomic organization but differ in their pathobiology. HTLV-1, the first human retrovirus discovered, is the causal agent of an aggressive adult T-cell leukemia, whereas HTLV-2 is associated with a few cases of neurological disease. Both virus genomes encode an oncogenic protein, Tax, required for viral replication and capable to induce cell transformation. In addition, HTLV-1 and -2 generate an antisense transcript, named HBZ and APH-2, respectively, crucial for viral infection. Comparative studies between HTLV-1 regulatory proteins, Tax-1 and HBZ, and the HTLV-2 homologs, Tax-2 and APH-2, may highlight the contribution of viral proteins to oncogenesis. The purpose of this study is to investigate the functional role of the viral regulatory proteins HBZ and APH-2 in the NF-\u3baB cell signaling, which is constitutively activated by Tax in infected cells. We demonstrated that APH-2 and HBZ differ in their suppression of the NF-\u3baB promoter activity. Unlike HBZ, the APH-2 protein is recruited by Tax in cytoplasmic structures and prevents the degradation of the inhibitor I\u3baB, impairing p65 nuclear translocation. Furthermore, we found that APH-2, but not HBZ, forms complexes with the adaptor protein TRAF3, an upstream inhibitor of the alternative NF-\u3baB pathway. We generated a TRAF3-KO cell line applying the CRISPR/Cas9 technique, which will allow us to investigate the HBZ and APH-2 role in modulating the alternative NF-\u3baB cell signaling. This study may provide insight into the effect of host-viral interactions in human viral oncogenesis

A CRISPR/Cas9 based approach to study the implication of HTLV regulatory proteins in the NF-κB modulation.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), an aggressive form of T-cell malignancy with no cure. The HTLV-1 oncoprotein Tax plays a key role in CD4+ T-cell transformation, mainly through constitutive activation of both the canonical and the alternative NF-κB pathways. The HTLV-1 basic zipper protein (HBZ), encoded by the antisense viral genome strand, plays an essential role in the oncogenic process in concert with Tax, mediating T-cell proliferation. Unlike HTLV-1, the genetically related retrovirus HTLV-2 is not associated with ATL diseases. Functional comparisons between HTLV-1 regulatory proteins, Tax-1 and HBZ, and the HTLV-2 homologs, Tax-2 and APH-2, may highlight different mechanisms of their oncogenic potential. The aim of this study is to investigate how the antisense proteins HBZ and APH-2 impaired the NF-κB pathway activation. We found that both HBZ and APH-2 antagonized the NF-κB promoter activity mediated by Tax, but not in the same extent. Analyzing the intracellular distribution of the antisense proteins, we found that APH-2 is retained in cytoplasm complexes, whereas HBZ is mainly distributed into the nucleus. We observed that in presence of APH-2 and Tax-2, the degradation of the IκB-α inhibitor was reduced. Moreover, we found that unlike HBZ, APH-2 formed complexes with an upstream inhibitor of the alternative NF-κB pathway, the TNF receptor-associated factor 3, TRAF3. We generated a TRAF3 knock-out cell line applying the CRISPR/Cas9-mediated genome editing. By luciferase assays, we showed that TRAF3 is not required for Tax mediated NFκB promoter activation. Analyses are in progress to test the inhibitory effect of the antisense HBZ and APH-2 proteins on NF-κB promoter activity in absence of TRAF3. The results of this study may contribute to clarify the effect of the alternative NF-κB viral deregulation pathway in the expression of proinflammatory genes

TRAF3 Is Required for NF-\u3baB Pathway Activation Mediated by HTLV Tax Proteins

Human T-cell leukemia viruses type 1 (HTLV-1) and type 2 (HTLV-2) share a common genome organization and expression strategy but have distinct pathological properties. HTLV-1 is the etiological agent of Adult T-cell Leukemia (ATL) and of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), whereas HTLV-2 does not cause hematological disorders and is only sporadically associated with cases of subacute myelopathy. Both HTLV genomes encode two regulatory proteins that play a pivotal role in pathogenesis: the transactivating Tax-1 and Tax-2 proteins and the antisense proteins HBZ and APH-2, respectively. We recently reported that Tax-1 and Tax-2 form complexes with the TNF-receptor associated factor 3, TRAF3, a negative regulator of the non-canonical NF-kappa B pathway. The NF-kappa B pathway is constitutively activated by the Tax proteins, whereas it is inhibited by HBZ and APH-2. The antagonistic effects of Tax and antisense proteins on NF-kappa B activation have not yet been fully clarified. Here, we investigated the effect of TRAF3 interaction with HTLV regulatory proteins and in particular its consequence on the subcellular distribution of the effector p65/RelA protein. We demonstrated that Tax-1 and Tax-2 efficiency on NF-kappa B activation is impaired in TRAF3 deficient cells obtained by CRISPR/Cas9 editing. We also found that APH-2 is more effective than HBZ in preventing Tax-dependent NF-kappa B activation. We further observed that TRAF3 co-localizes with Tax-2 and APH-2 in cytoplasmic complexes together with NF-kappa B essential modulator NEMO and TAB2, differently from HBZ and TRAF3. These results contribute to untangle the mechanism of NF-kappa B inhibition by HBZ and APH-2, highlighting the different role of the HTLV-1 and HTLV-2 regulatory proteins in the NF-kappa B activation

A potential role of RUNX2- RUNT domain in modulating the expression of genes involved in bone metastases: an in vitro study with melanoma cells

Ectopic expression of RUNX2 has been reported in several tumors. In melanoma cells, the RUNT domain of RUNX2 increases cell proliferation and migration. Due to the strong link between RUNX2 and skeletal development, we hypothesized that the RUNT domain may be involved in the modulation of mechanisms associated with melanoma bone metastasis. Therefore, we evaluated the expression of metastatic targets in wild type (WT) and RUNT KO melanoma cells by array and real-time PCR analyses. Western blot, ELISA, immunofluorescence, migration and invasion ability assays were also performed. Our findings showed that the expression levels of bone sialoprotein (BSP) and osteopontin (SPP1) genes, which are involved in malignancy-induced hypercalcemia, were reduced in RUNT KO cells. In addition, released PTHrP levels were lower in RUNT KO cells than in WT cells. The RUNT domain also contributes to increased osteotropism and bone invasion in melanoma cells. Importantly, we found that the ERK/p-ERK and AKT/p-AKT pathways are involved in RUNT-promoted bone metastases. On the basis of our findings, we concluded that the RUNX2 RUNT domain is involved in the mechanisms promoting bone metastasis of melanoma cells via complex interactions between multiple players involved in bone remodeling

HIV-1-Associated Neurocognitive Disorders: Is HLA-C Binding Stability to β2-Microglobulin a Missing Piece of the Pathogenetic Puzzle?

AIDS dementia complex (ADC) and HIV-associated neurocognitive disorders (HAND) are complications of HIV-1 infection. Viral infections are risk factors for the development of neurodegenerative disorders. Aging is associated with low-grade inflammation in the brain, i.e., the inflammaging. The molecular mechanisms linking immunosenescence, inflammaging and the pathogenesis of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease, are largely unknown. ADC and HAND share some pathological features with AD and may offer some hints on the relationship between viral infections, neuroinflammation, and neurodegeneration. β2-microglobulin (β2m) is an important pro-aging factor that interferes with neurogenesis and worsens cognitive functions. Several studies published in the 80–90s reported high levels of β2m in the cerebrospinal fluid of patients with ADC. High levels of β2m have also been detected in AD. Inflammatory diseases in elderly people are associated with polymorphisms of the MHC-I locus encoding HLA molecules that, by associating with β2m, contribute to cellular immunity. We recently reported that HLA-C, no longer associated with β2m, is incorporated into HIV-1 virions, determining an increase in viral infectivity. We also documented the presence of HLA-C variants more or less stably linked to β2m. These observations led us to hypothesize that some variants of HLA-C, in the presence of viral infections, could determine a greater release and accumulation of β2m, which in turn, may be involved in triggering and/or sustaining neuroinflammation. ADC is the most severe form of HAND. To explore the role of HLA-C in ADC pathogenesis, we analyzed the frequency of HLA-C variants with unstable binding to β2m in a group of patients with ADC. We found a higher frequency of unstable HLA-C alleles in ADC patients, and none of them was harboring stable HLA-C alleles in homozygosis. Our data suggest that the role of HLA-C variants in ADC/HAND pathogenesis deserves further studies. If confirmed in a larger number of samples, this finding may have practical implication for a personalized medicine approach and for developing new therapies to prevent HAND. The exploration of HLA-C variants as risk factors for AD and other neurodegenerative disorders may be a promising field of study

Development of new edited cell models to analyze pros and cons of the CRISPR/Cas9 technique

The CRISPR/Cas9 system is a powerful genome-editing tool and its great potential is thoroughly recognized. CRISPR/Cas9 has been applied in several fields, such as plant engineering, development of new cell lines, and animal models. CRISPR/Cas9 exploits a small single guide RNA to direct the Cas9 endonuclease activity against a specific genome sequence, where it introduces a double strand break. The DNA damage is then repaired by the non-homologous and joining or the homology direct repair processes. While the first one allows a gene knock out, the homology direct repair bases its activity on a repair template, thus allowing the introduction of specific modification within a gene target. In the present work, we aimed to analyze the pros and cons of the CRISPR/Cas9 system by developing new edited cell models, that will be useful to study the involvement of specific factors in different human diseases. Particularly, viral infections, cancer diseases, and neurodegenerative disorders represent a major issue for public health. The CRISPR/Cas9 system was fundamental to develop new cell lines useful to study host-retrovirus interaction, the involvement of specific proteins in tumorigenesis and cancer progression, and the importance of particular proteins in the onset of neurodegenerative disorders. Retroviral infection: host-virus interaction HIV-1 and HTLV-1 represent the two major human retroviruses. To assess how specific host proteins modulate HIV-1 infection, different 293T packaging cell lines were developed. Specifically, CRISPR/Cas9 was used to produce 293T ACOT8, HDAC6 \u394BUZ, and HLA-C KO cell lines. To investigate the involvement of TRAF3 in the deregulation of the NF-kB pathway mediated by the HTLV protein Tax1, a TRAF3 KO cell model was developed. Cancer diseases Malignant melanoma and pancreatic cancer are two of the most frequent morbidities. To analyze the oncogenic potential of RUNX2 in the development and progression of malignant melanoma, the Mel-HO RUNX2 KO melanoma cell line was developed through CRISPR/Cas9. To clarify the role of GNA15 in the molecular mechanisms leading to pancreatic adenocarcinoma, CRISPR/Cas9 was employed to generate PT45 GNA15 KO adenocarcinoma cells. Neurodegenerative diseases Neurodegenerative disorders are becoming even more frequent especially due to the increase in the average age of the population. Nowadays no effective cures are available. To understand the molecular mechanism by which GPR3 induces amyloid \u3b2 deposition in Alzheimer\u2019s disease, CRISPR/Cas9 was used to develop H4 Swe GPR3 KO neuroglioma cells. To fully investigate the involvement of the palmitoyl-protein thioesterase 1 PPT1, in the neuronal ceroid lipofuscinoses neurological disorder, SH-SY5Y PPT1 KO neuroblastoma cell line was developed. The CRISPR/Cas9 pros and cons are analyzed for a full technique understanding, with particular regard to its potential limits, such as off-targets, in vitro genome manipulation, delivery systems, and editing screening. As any technology revolution, CRISPR/Cas9 represents the linchpin of fierce debates involving many scientists and researchers to regulate its employment for gene therapy, germline, and human embryos manipulation

HTLV Deregulation of the NF-κB Pathway: An Update on Tax and Antisense Proteins Role

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), an aggressive CD4+/CD25+ T-cell malignancy and of a severe neurodegenerative disease, HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The chronic activation or deregulation of the canonical and non-canonical nuclear factor kappa B (NF-κB) pathways play a crucial role in tumorigenesis. The HTLV-1 Tax-1 oncoprotein is a potent activator of the NF-κB transcription factors and the NF-κB response is required for promoting the development of HTLV-1 transformed cell lines. The homologous retrovirus HTLV-2, which also expresses a Tax-2 transforming protein, is not associated with ATL. In this review, we provide an updated synopsis of the role of Tax-1 in the deregulation of the NF-κB pathway, highlighting the differences with the homologous Tax-2. Special emphasis is directed toward the understanding of the molecular mechanisms involved in NF-κB activation resulting from Tax interaction with host factors affecting several cellular processes, such as cell cycle, apoptosis, senescence, cell proliferation, autophagy, and post-translational modifications. We also discuss the current knowledge on the role of the antisense viral protein HBZ in down-regulating the NF-κB activation induced by Tax, and its implication in cellular senescence. In addition, we review the recent studies on the mechanism of HBZ-mediated inhibition of NF-κB activity as compared to that exerted by the HTLV-2 antisense protein, APH-2. Finally, we discuss recent advances aimed at understanding the role exerted in the development of ATL by the perturbation of NF-κB pathway by viral regulatory proteins

CRISPR/Cas9 as a tool for the study of the interactions between viruses and host

CRISPR/Cas9 in virolog

Involvement of HLA-C binding stability to \u3b22-microglobulin in HIV-1 infection

MHC-I is a heterotrimeric complex composed by HLA/\u3b22-microglobulin/peptide. HLA-C binding to \u3b22-microglobulin is weaker compared to HLA-A and -B. The HLA-C gene encodes different allotypes classified in stable and unstable clusters based on their binding stability to \u3b22-microglobulin. Unstable HLA-C molecules release more \u3b22-microglobulin than stable variants. The ratio of complete heterotrimers and HLA-C free chains at the cell surface is involved in controlling HIV-1 infection. We recently reported that HIV-1 Env associates with HLA-C during viral budding, increasing viral infectivity and spreading. We aimed to address how the binding stability of different HLA-C allotypes to \u3b22-microglobulin influences HIV-1 infectivity. We showed that HIV-1 virions produced in PBMCs with unstable HLA-C variants are more infectious then those produced in PBMCs with stable ones. To deeply investigate the role of each HLA-C variant in HIV-1 infection modulation, 293T HLA-C-/- packaging cells were developed using CRISPR/Cas9. The different HLA-C alleles were then restored, developing cell lines expressing specific HLA-C allotypes. To analyse the binding stability to \u3b22-microglobulin of each HLA-C variant, an acid wash treatment will be performed to assign a binding stability score ranking. Moreover, to investigate how HLA-C variants modulate HIV-1 infectivity, the homozygous HLA-C cells will be employed to produce HIV-1 pseudotyped viruses to evaluate their infectivity through a luciferase infectivity assay. This study will allow us to stratify the HLA-C alleles according to their binding stability to \u3b22-microglobulin. Furthermore, these analyses will be fundamental to clarify the relationship among HLA-C allotypes binding stability and HIV-1 infection progression