Transcriptome Sequencing of Peripheral Blood Mononuclear Cells from Elite Controller-Long Term Non Progressors

The elite controller (EC)-long term non-progressor (LTNP) phenotype represent a spontaneous and advantageous model of HIV-1 control in the absence of therapy. The transcriptome of peripheral blood mononuclear cells (PBMCs) collected from EC-LTNPs was sequenced by RNA-Seq and compared with the transcriptomes from other phenotypes of disease progression. The transcript abundance estimation combined with the use of supervised classification algorithms allowed the selection of 20 genes and pseudogenes, mainly involved in interferon-regulated antiviral mechanisms and cell machineries of transcription and translation, as the best predictive genes of disease progression. Differential expression analyses between phenotypes showed an altered calcium homeostasis in EC-LTNPs evidenced by the upregulation of several membrane receptors implicated in calcium-signaling cascades and intracellular calcium-mobilization and by the overrepresentation of NFAT1/Elk-1-binding sites in the promoters of the genes differentially expressed in these individuals. A coordinated upregulation of host genes associated with HIV-1 reverse transcription and viral transcription was also observed in EC-LTNPs -i.e. p21/CDKN1A, TNF, IER3 and GADD45B. We also found an upregulation of ANKRD54 in EC-LTNPs and viremic LTNPs in comparison with typical progressors and a clear alteration of type-I interferon signaling as a consequence of viremia in typical progressors before and after receiving antiretroviral therapy.We want to particularly acknowledge the patients in this study for their participation and to the HIV BioBank integrated in the Spanish AIDS Research Network and collaborating Centres (http://hivhgmbiobank.com/donor-area/hospitals-and-centres-transferring-samples/?lang = en) for the generous gifts of clinical samples used in this work. The HIV BioBank and the AIDS Immunopathogenesis Unit are integrated in the Spanish AIDS Research Network. The HIV BioBank is partially funded by the RD16/0025/0019 project as part of the Plan Nacional R + D + I and cofinanced by ISCIII- Subdirección General de Evaluación and el Fondo Europeo de Desarrollo Regional (FEDER)”. This work was partially supported by Instituto de Salud Carlos III and co-funded by European Regional Development Fund (ERDF) “A way to build Europe” (projects RD12/0017/0015 and RD16CIII/0002/0001, Plan Estatal de I + D + I 2013–2016), the French Government’s Investissement d’Avenir program, Laboratoire d’Excellence Integrative Biology of Emerging Infectious Diseases (n°ANR-10-LABX-62-IBEID) and the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 681137. FDF was supported by the Spanish Government’s Sara Borrell postdoctoral ProgramS

Transcriptomic Evidence of the Immune Response Activation in Individuals With Limb Girdle Muscular Dystrophy Dominant 2 (LGMDD2) Contributes to Resistance to HIV-1 Infection

LGMDD2 is a rare form of muscular dystrophy characterized by one of the three heterozygous deletions described within the TNPO3 gene that result in the addition of a 15-amino acid tail in the C-terminus.TNPO3 is involved in the nuclear import of splicing factors and acts as a host cofactor for HIV-1 infection by mechanisms not yet deciphered. Further characterization of the crosstalk between HIV-1 infection and LGMDD2 disease may contribute to a better understanding of both the cellular alterations occurring in LGMDD2 patients and the role of TNPO3 in the HIV-1 cycle. To this regard, transcriptome profiling of PBMCs from LGMDD2 patients carrying the deletion c.2771delA in the TNPO3 gene was compared to healthy controls. A total of 545 differentially expressed genes were detected between LGMDD2 patients and healthy controls, with a high representation of G protein-coupled receptor binding chemokines and metallopeptidases among the most upregulated genes in LGMDD2 patients. Plasma levels of IFN-β and IFN-γ were 4.7- and 2.7-fold higher in LGMDD2 patients, respectively. An increase of 2.3-fold in the expression of the interferon-stimulated gene MxA was observed in activated PBMCs from LGMDD2 patients after ex vivo HIV-1 pseudovirus infection. Thus, the analysis suggests a pro-inflammatory state in LGMDD2 patients also described for other muscular dystrophies, that is characterized by the alteration of IL-17 signaling pathway and the consequent increase of metallopeptidases activity and TNF response. In summary, the increase in interferons and inflammatory mediators suggests an antiviral environment and resistance to HIV-1 infection but that could also impair muscular function in LGMDD2 patients, worsening disease evolution. Biomarkers of disease progression and therapeutic strategies based on these genes and mechanisms should be further investigated for this type of muscular dystrophy.This study was funded by Asociación Conquistando Escalones, French Agency for Research on AIDS and Viral Hepatitis (ANRS grant ECTZ107263), Instituto de Salud Carlos III (PI19CIII/00004), NIH grant R01AI143567, the Spanish Ministry of Science and Innovation (PID2019-110275RB I00) and Fundación Isabel Gemio. It has been conducted within the Spanish AIDS Research Network (RIS) and Centro de Investigación Biomédica en Red de Enfermedades Infecciosas, funded by Instituto de Salud Carlos 640 III (Plan Estatal de I+D+I 2013-2016) and co-funded by European Regional Development Fund (ERDF) “A way to build Europe” (RD16CIII/0002/0001).S

Characterization of LEDGF/p75 genetic variants and association with HIV-1 disease progression

BACKGROUND: As Lens epithelium-derived growth factor (LEDGF/p75) is an important co-factor involved in HIV-1 integration, the LEDGF/p75-IN interaction is a promising target for the new class of allosteric HIV integrase inhibitors (LEDGINs). Few data are available on the genetic variability of LEDGF/p75 and the influence on HIV disease in vivo. This study evaluated the relation between LEDGF/p75 genetic variation, mRNA expression and HIV-1 disease progression in order to guide future clinical use of LEDGINs. METHODS: Samples were derived from a therapy-naïve cohort at Ghent University Hospital and a Spanish long-term-non-progressor cohort. High-resolution melting curve analysis and Sanger sequencing were used to identify all single nucleotide polymorphisms (SNPs) in the coding region, flanking intronic regions and full 3'UTR of LEDGF/p75. In addition, two intronic tagSNPs were screened based on previous indication of influencing HIV disease. LEDGF/p75 mRNA was quantified in patient peripheral blood mononuclear cells (PBMC) using RT-qPCR. RESULTS: 325 samples were investigated from patients of Caucasian (n = 291) and African (n = 34) origin, including Elite (n = 49) and Viremic controllers (n = 62). 21 SNPs were identified, comprising five in the coding region and 16 in the non-coding regions and 3'UTR. The variants in the coding region were infrequent and had no major impact on protein structure according to SIFT and PolyPhen score. One intronic SNP (rs2737828) was significantly under-represented in Caucasian patients (P<0.0001) compared to healthy controls (HapMap). Two SNPs showed a non-significant trend towards association with slower disease progression but not with LEDGF/p75 expression. The observed variation in LEDGF/p75 expression was not correlated with disease progression. CONCLUSIONS: LEDGF/p75 is a highly conserved protein. Two non-coding polymorphisms were identified indicating a correlation with disease outcome, but further research is needed to clarify phenotypic impact. The conserved coding region and the observed variation in LEDGF/p75 expression are important characteristics for clinical use of LEDGINs.This work was partly supported by the Flemish Agency for Innovation by Science and Technology (CellCoVir - IWT file nr 60813). Linos Vandekerckhove is supported by the National Fund for Scientific Research – Belgium as Principal Investigator. The Spanish RIS cohort and HIV BioBank are integrated in the Spanish AIDS Research Network supported by Instituto de Salud Carlos III (Grant RD06/0006/0035) and Fundación para la Investigación y Prevención del SIDA en España (FIPSE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Insight in miRNome of Long-Term Non-Progressors and Elite Controllers Exposes Potential RNAi Role in Restraining HIV-1 Infection

Long-term non-progressors (LTNP) and elite controllers (EC) represent spontaneous natural models of e cient HIV-1 response in the absence of treatment. The main purposes of this work are to describe the miRNome of HIV-1 infected patients with di↵erent extreme phenotypes and identify potentially altered pathways regulated by di↵erentially expressed (DE) miRNAs. The miRNomes from peripheral blood mononuclear cells (PBMCs) of dual phenotype EC-LTNP or LTNP with detectable viremia and HIV-infected patients with typical progression before and after treatment, were obtained through miRNA-Seq and compared among them. The administration of treatment produces 18 DE miRNAs in typical progressors. LTNP condition shows 14 DE miRNA when compared to typical progressors, allowing LTNP phenotype di↵erentiation. A set of four miRNAs: miR-144-3p, miR-18a-5p, miR-451a, and miR-324 is strongly downregulated in LTNP and related to protein regulation as AKT, mTOR, ERK or IKK, involved in immune response pathways. Deregulation of 28 miRNA is observed between EC-LTNP and viremic-LTNP, including previously described anti-HIV miRNAs: miR-29a, associated with LTNP phenotype, and miR-155, targeting different pre-integration complexes such as ADAM10 and TNPO3. A holistic perspective of the changes observed in the miRNome of patients with different phenotypes of HIV-control and non-progression is provided.This study has been conducted within the Spanish AIDS Research Network (RIS), funded by Instituto de Salud Carlos III (Plan Estatal de I+D+I 2013-2016) and co-funded by European Regional Development Fund (ERDF) “A way to build Europe” (RD12/0017/0015 and RD16CIII/0002/0001 projects), and the European Union’s Horizon 2020 Research and Innovation Programme under grant number 681137. The HIV BioBank, integrated in the Spanish AIDS Research Network, is partially funded by the RD16/0025/0019 project as part of the Plan Nacional R + D + I and cofinanced by ISCIII- Subdirección General de Evaluación and el Fondo Europeo de Desarrollo Regional (FEDER). R.A.-S. was supported by the Ministry of Innovation, Science and Universities predoctoral funding (FPU18/05527) and H.E.T.-T. by the ISCIII-PFIS predoctoral program (FI14CIII/00014).S

Different Expression of Interferon-Stimulated Genes in Response to HIV-1 Infection in Dendritic Cells Based on Their Maturation State

Dendritic cells (DCs) are professional antigen-presenting cells whose functions are dependent on their degree of differentiation. In their immature state, DCs capture pathogens and migrate to the lymph nodes. During this process, DCs become resident mature cells specialized in antigen presentation. DCs are characterized by a highly limiting environment for human immunodeficiency virus type 1 (HIV-1) replication due to the expression of restriction factors such as SAMHD1 and APOBEC3G. However, uninfected DCs capture and transfer viral particles to CD4 lymphocytes through a trans-enhancement mechanism in which chemokines are involved. We analyzed changes in gene expression with whole-genome microarrays when immature DCs (IDCs) or mature DCs (MDCs) were productively infected using Vpx-loaded HIV-1 particles. Whereas productive HIV infection of IDCs induced expression of interferon-stimulated genes (ISGs), such induction was not produced in MDCs, in which a sharp decrease in ISG- and CXCR3-binding chemokines was observed, lessening trans-infection of CD4 lymphocytes. Similar patterns of gene expression were found when DCs were infected with HIV-2 that naturally expresses Vpx. Differences were also observed under conditions of restrictive HIV-1 infection, in the absence of Vpx. ISG expression was not modified in IDCs, whereas an increase of ISG- and CXCR3-binding chemokines was observed in MDCs. Overall these results suggest that sensing and restriction of HIV-1 infection are different in IDCs and MDCs. We propose that restrictive infection results in increased virulence through different mechanisms. In IDCs avoidance of sensing and induction of ISGs, whereas in MDCs increased production of CXCR3-binding chemokines, would result in lymphocyte attraction and enhanced infection at the immune synapse.IMPORTANCE In this work we describe for the first time the activation of a different genetic program during HIV-1 infection depending on the state of maturation of DCs. This represents a breakthrough in the understanding of the restriction to HIV-1 infection of DCs. The results show that infection of DCs by HIV-1 reprograms their gene expression pattern. In immature cells, productive HIV-1 infection activates interferon-related genes involved in the control of viral replication, thus inducing an antiviral state in surrounding cells. Paradoxically, restriction of HIV-1 by SAMHD1 would result in lack of sensing and IFN activation, thus favoring initial HIV-1 escape from the innate immune response. In mature DCs, restrictive infection results in HIV-1 sensing and induction of ISGs, in particular CXCR3-binding chemokines, which could favor the transmission of HIV to lymphocytes. Our data support the hypothesis that genetic DC reprograming by HIV-1 infection favors viral escape and dissemination, thus increasing HIV-1 virulence.We thank Olga Palao (AIDS Immunopathogenesis Unit) and A. Zaballos (Genomics Unit, Instituto de Salud Carlos III) for their secretarial and technical assistance, respectively. We also greatly appreciate our patients for their willingness to participate.Funding for this research was provided by: Ministerio de Economía y Competitividad (SAF2013-44677-R, PI16CIII/00034, ISCIII-FIS Nº) Instituto de Salud Carlos III (Spanish AIDS Research Network RD16CIII/0002/0001)S