Restriction Factors: From Intrinsic Viral Restriction to Shaping Cellular Immunity Against HIV-1

Antiviral restriction factors are host cellular proteins that constitute a first line of defense blocking viral replication and propagation. In addition to interfering at critical steps of the viral replication cycle, some restriction factors also act as innate sensors triggering innate responses against infections. Accumulating evidence suggests an additional role for restriction factors in promoting antiviral cellular immunity to combat viruses. Here, we review the recent progress in our understanding on how restriction factors, particularly APOBEC3G, SAMHD1, Tetherin, and TRIM5α have the cell-autonomous potential to induce cellular resistance against HIV-1 while promoting antiviral innate and adaptive immune responses. Also, we provide an overview of how these restriction factors may connect with protein degradation pathways to modulate anti-HIV-1 cellular immune responses, and we summarize the potential of restriction factors-based therapeutics. This review brings a global perspective on the influence of restrictions factors in intrinsic, innate, and also adaptive antiviral immunity opening up novel research avenues for therapeutic strategies in the fields of drug discovery, gene therapy, and vaccines to control viral infections

Viral and Cellular factors leading to the Loss of CD4 Homeostasis in HIV-1 Viremic Nonprogressors

Human immunodeficiency virus type 1 (HIV-1) viremic nonprogressors (VNPs) represent a very rare HIV-1 extreme phenotype. VNPs are characterized by persistent high plasma viremia and maintenance of CD41 T-cell counts in the absence of treatment. However, the causes of nonpathogenic HIV-1 infection in VNPs remain elusive. Here, we identified for the first time two VNPs who experienced the loss of CD41 homeostasis (LoH) after more than 13 years. We characterized in deep detail viral and host factors associated with the LoH and compared with standard VNPs and healthy controls. The viral factors determined included HIV-1 coreceptor usage and replicative capacity. Changes in CD41 and CD81 T-cell activation, maturational phenotype, and expression of CCR5 and CXCR6 in CD41 T-cells were also evaluated as host-related factors. Consistently, we determined a switch in HIV-1 coreceptor use to CXCR4 concomitant with an increase in replicative capacity at the LoH for the two VNPs. Moreover, we delineated an increase in the frequency of HLA-DR1CD381 CD41 and CD81 T cells and traced the augment of naive T-cells upon polyclonal activation with LoH. Remarkably, very low and stable levels of CCR5 and CXCR6 expression in CD41 T-cells were measured over time. Overall, our results demonstrated HIV-1 evolution toward highly pathogenic CXCR4 strains in the context of very limited and stable expression of CCR5 and CXCR6 in CD41 T cells as potential drivers of LoH in VNPs. These data bring novel insights into the correlates of nonpathogenic HIV1 infection. Importance: The mechanism behind nonpathogenic human immunodeficiency virus type 1 (HIV-1) infection remains poorly understood, mainly because of the very low frequency of viremic nonprogressors (VNPs). Here, we report two cases of VNPs who experienced the loss of CD41 T-cell homeostasis (LoH) after more than 13 years of HIV-1 infection. The deep characterization of viral and host factors supports the contribution of viral and host factors to the LoH in VNPs. Thus, HIV-1 evolution toward highly replicative CXCR4 strains together with changes in T-cell activation and maturational phenotypes were found. Moreover, we measured very low and stable levels of CCR5 and CXCR6 in CD41 T-cells over time. These findings support viral evolution toward X4 strains limited by coreceptor expression to control HIV-1 pathogenesis and demonstrate the potential of host-dependent factors, yet to be fully elucidated in VNPs, to control HIV-1 pathogenesis.This research was supported by a Gilead Fellowship (grant GLD15/0298) and La Caixa Foundation (grant LCF/PR/PR16/11110026). M.C.-L. is a Beatriu de Pinós postdoctoral fellow (grant BP 00075) supported by the Government of Catalonia’s Secretariat for Universities and Research of the Ministry of Economy and Knowledge. J.G.P. was supported by the ISCIII (grant CP15/00014). E.J.-M. was funded by Redes Temáticas de Investigación en SIDA (ISCIII RETIC RD16/0025/0041); Acción Estratégica en Salud; Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2008–2011; and Instituto de Salud Carlos III. E.J.-M. was cofunded by European Regional Development Fund/European Social Fund (FEDER) “Investing in your future.” J.M.-P. is supported by the Spanish Ministry of Science and Innovation (grant PID2019-109870RB-I00). J.G.P. and M.C.-L. designed the study, supervised experiments and data. J.G.P., M.C.-L., and A.K. contributed to data interpretation. M.C.-L., R.P., E.J.-M., M.P., and C.C. performed experiments, analyzed, and interpreted the data. J.D. carried out the clinical follow-up and patient identification. M.C.-L., D.O., M.P., and C.C. performed data analysis. M.C.-L., A.K., M.P., C.L.-G., B.C., J.M.-P., and J.G.P. performed manuscript writing, critical revision, and discussion. We declare no conflict of interest.S

Sex-specific innate immune selection of HIV-1 in utero is associated with increased female susceptibility to infection

Female children and adults typically generate more efficacious immune responses to vaccines and infections than age-matched males, but also suffer greater immunopathology and autoimmune disease. We here describe, in a cohort of>170 in utero HIV-infected infants from KwaZulu-Natal, South Africa, fetal immune sex differences resulting in a 1.5-2-fold increased female susceptibility to intrauterine HIV infection. Viruses transmitted to females have lower replicative capacity (p=0.0005) and are more type I interferon-resistant (p=0.007) than those transmitted to males. Cord blood cells from females of HIV-uninfected sex-discordant twins are more activated (p=0.01) and more susceptible to HIV infection in vitro (p=0.03). Sex differences in outcome include superior maintenance of aviraemia among males (p=0.007) that is not explained by differential antiretroviral therapy adherence. These data demonstrate sex-specific innate immune selection of HIV associated with increased female susceptibility to in utero infection and enhanced functional cure potential among infected males. Sex differences in the immune response to vaccines and infections have been well described in children and adults. Here the authors describe, in a cohort of 177 HIV-infected infants, innate immune sex differences in fetal life that increase female susceptibility to intrauterine HIV infection and increase the chances of subsequent HIV remission in infected males

Antibiotic Resistance Genes in the Bacteriophage DNA Fraction of Environmental Samples

Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, β-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to β-lactam antibiotics is conferred by β-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to β-lactam antibiotics, namely two β-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment

Antibiotic resistance genes in the viral DNA fraction of environmental samples = Gens de resistència a antibiòtics en el DNA de la fracció vírica de mostres ambientals

[spa] La tesi doctoral que es presenta a continuació té com a objectiu principal l’estudi de gens de resistència a antibiòtics de rellevància clínica en la fracció de DNA de partícules de bacteriòfags aïllades de diferents tipus de mostres ambientals per tal de determinar la importància dels bacteriòfags com a vehicles de mobilització de gens de resistència a antibiòtics entre bacteris. S’ha estudiat un ampli espectre de gens de resistència a antibiòtics com a representants dels grups principals descrits actualment en la nostra àrea geogràfica corresponent a tres β-lactamases (blaTEM, blaCTXM-1 i blaCTX-M-9), el gen mecA de resistència a meticil•lina en estafilococs, i els gens de resistència a quinolones qnrA i qnrS. Per això s’han analitzat diversos tipus de mostres procedents d’aigua residual municipal, d’aigua de riu i d’aigua residual amb contaminació fecal animal per tal de quantificar els gens de resistència a antibiòtics d’interès en DNA aïllat de bacteriòfags. Durant els diferents estudis s’ha intentat optimitzar la metodologia d’extracció de DNA de bacteriòfags així com els controls corresponents per garantir l’amplificació de DNA encapsidat i l’eliminació de qualsevol DNA lliure present a les mostres i de qualsevol possible vesícula amb DNA al seu interior. Per altra banda, també s’ha determinat la capacitat funcional dels gens de resistència detectats en DNA de fags i per això s’han realitzat experiments de transformació a partir de soques bacterianes sensibles a un determinat antibiòtic amb l’objectiu d’incorporar la resistència i per tant, esdevenir resistents a l’antibiòtic en qüestió. També, s’ha estudiat la influència de determinats compostos implicats en la inducció del cicle lític de bacteriòfags temperats, en l’augment en el nombre de còpies de gens de resistència a antibiòtics en DNA present en la fracció de fags de l’aigua residual. Finalment, degut a la importància de la transferència horitzontal de gens com a mecanisme de dispersió de la resistència a antibiòtics en el medi ambient i en clínica s’han dut a terme experiments de transducció per tal d’intentar reproduir in vitro el procés que tindria lloc de manera natural.[eng] The PhD thesis presented here has as a main objective the study of antibiotic resistance genes clinically relevant in the DNA fraction of bacteriophage particles isolated from environmental samples of different origin in order to determine the importance of bacteriophages as vehicles for the mobilization of antibiotic resistance genes between bacteria. Specifically, a broad range of antibiotic resistance genes were studied as representative of the main groups recently described in our geographical area belonging to three β-lactamases (blaTEM, blaCTXM-1 and blaCTX-M-9), the mecA gene conferring resistance to methicillin in staphylococci, and the quinolones resistance genes qnrA and qnrS. To achieve these goals samples of urban wastewater, river water and animal faecal wastes were analysed quantifying the antibiotic resistance genes of interest in bacteriophages DNA. During the development of this Thesis, it was attempted to optimize the available methodology for bacteriophage DNA extraction, as well as the necessary controls to guarantee the amplification of encapsidated DNA and to remove any free DNA in the samples and any possible vesicles containing DNA. In addition, the ability of phage-encoded genes to confer antibiotic resistance in bacterial strains was assessed by performing transformation experiments. It was also studied the influence of various compounds involved in the induction of the lytic cycle of temperate bacteriophages, on the abundance of antibiotic resistance genes in DNA from the phage fraction in wastewater samples. Finally, due to the importance of horizontal gene transfer as a mechanism for antibiotic resistance dissemination in clinical and environmental settings, transduction experiments were attempted to reproduce in vitro the process that would take place in nature. The research developed in this Thesis is divided into 5 studies included in 4 chapters: (1) Antibiotic resistance genes in the bacteriophage DNA fraction of water samples (wastewater, river water and animal wastewater); (2) Quinolone resistance genes (qnrA and qnrS) in bacteriophage particles from wastewater samples and the effect of inducing agents on packaged antibiotic resistance genes; (3) Evaluation of ARGs in the DNA of bacterial and bacteriophage fraction in wastewater samples from Tunisia and comparison with results obtained in Barcelona area; (4) Detection of quinolone-resistant Escherichia coli isolates belonging to clonal groups O25b:H4-B2-ST131 and O25b:H4-D-ST69 in water samples from Barcelona area. Each of the studies has given rise to a scientific article already published or submitted for scientific publication

Number of copies of <i>bla</i>_CTX-M genes (GC/ml) in urban sewage and river water samples in phage and bacterial DNA and box plot of averaged values.

<p>Number of copies of <i>bla</i>_CTX-M genes (GC/ml) in urban sewage and river water samples in phage and bacterial DNA and box plot of averaged values.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Electron micrographs of bacteriophages present in sewage and river water.

<p>A–B. Group of phages with <i>Myoviridae</i> and <i>Siphoviridae</i> morphology from sewage. C. <i>Myoviridae</i> phages from river water. D: group of <i>Siphoviridae</i> phages from sewage. E–F. <i>Myoviridae</i> phages from sewage. G: <i>Podoviridae</i> phage from sewage. H–I. <i>Siphoviridae</i> phages from sewage and river water respectively. Bar 200 nm.</p

Number of copies of <i>mec</i>A (GC/ml) in urban sewage and river water samples in phage and bacterial DNA and box plot of averaged values.

<p>Number of copies of <i>mec</i>A (GC/ml) in urban sewage and river water samples in phage and bacterial DNA and box plot of averaged values.</p