Host-virus interactions: HTLV antisense regulatory proteins play a role in the dysregulation of NF-\u3baB pathway

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-\u3baB 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-\u3baB pathway activation. We found that both HBZ and APH-2 antagonized the NF-\u3baB 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 I\u3baB-\u3b1 inhibitor was reduced. Moreover, we found that unlike HBZ, APH-2 formed complexes with an upstream inhibitor of the alternative NF-\u3baB 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 NF\u3baB promoter activation. Analyses are in progress to test the inhibitory effect of the antisense HBZ and APH-2 proteins on NF-\u3baB promoter activity in absence of TRAF3. The results of this study may contribute to clarify the effect of the alternative NF-\u3baB viral deregulation pathway in the expression of proinflammatory genes

2. CRISPR/Cas9 to study virus-host interactions

CRISPR relevance in virolog

5. CRISPR/Cas9 for the Study of the Interactions between Viruses and Host

CRISPR/Cas9 system as a powerful tool in virolog

NF-\u3baB activation mediated by HTLV-1 transactivator Tax protein is impaired in TRAF3 knock-out cells.

Investigate the role of Tax-1 and the viral antisense protein HBZ in NF-\u3baB deregulation in TRAF3 knock-out cell

TRAF3 involvement in the NF-\u3baB pathway deregulation mediated by HTLV-1 Tax protein

TRAF3 protein involvement in the NF-\u3baB pathway deregulatio

HLA-C and HIV-1: a matter of stability

HLA-C requires \u3b22-microglobulin (\u3b22m) for proper folding and cell surface expression. The HLA-C gene is highly polymorphic, encoding different allotypes classified in stable and unstable clusters, based on their binding stability to \u3b22m. Unstable HLA-C molecules release more \u3b22m than stable variants, generating more HLA-C free chains on the cell surface. HLA-C plays an important role in modulating HIV -1 infection. It has been reported that a higher HLA-C expression is associated with better HIV-1 infection control, while its lower expression leads to a rapid progression toward AIDS. Highly and lowly expressed HLA-C alleles have been defined as protective and non-protective, respectively, in relation to HIV-1 disease progression. Differently, in our previous studies, we reported that HIV-1 virions lacking HLA-C have reduced infectivity and increased susceptibility to neutralizing antibodies. The reasons behind this apparent contradiction were explained by our recent findings showing that HIV-1 Env interacts with HLA-C freechains, after their dissociation from \u3b22m, and that protective HLA-C alleles bind \u3b22m more efficiently (\u201cstable\u201d variants), compared to non-protective alleles presenting more free chains on the cell surface (\u201cunstable\u201d variants). It is known that patients affected by AIDS Dementia Complex (ADC), a very severe neurological condition of HIV-1 infection, present high level of \u3b22m in the cerebrospinal fluid. We propose that the outcome of HIV-1 infection might be driven both by HLA-C surface expression levels and by HLA-C/\u3b22m binding stability. According to this model, the expression of unstable variants leads to a higher \u3b22m shedding and to a reduction of immunocompetent HLA-C complexes expressed on the cell surface. Consequently, the increase of HLA-C free chains raises viral infectivity contributing to a rapid progression toward AIDS. This model was supported by our recent findings showing that unstable HLA-C variants are more frequently associated with the development of ADC in HIV-1 infected patients. To further investigate the contribution of HLA-C variants in HIV-1 infection, we used CRISPR/Cas9 to develop an HLA-C KO human cell line. The different HLA-C alleles have been then restored by transfection, generating cell lines expressing each a specific HLA-C allotype. To assign a binding stability score to each HLA-C variant, an acid wash treatment will be performed to study the kinetic of \u3b22m detachment. Furthermore, to evaluate the HIV-1 infectivity modulation, each HLA-C expressing cell line will be employed to produce HIV-1 pseudotyped viruses to be tested in infectivity assays. This study will allow us to stratify HLA-C alleles according to their binding stability to \u3b22m and to clarify the relationship between HLA-C allotypes binding stability, HIV-1 infection progression and HIV-1 related neurocognitive disorders onset