Despite advances in the treatment of HIV-1 a cure remains elusive. A significant barrier to the eradication of the virus from an infected individual is a pool of cells infected with transcriptionally silent proviruses. A key pillar of the strategy to eradicate latent viruses has been called 'kick and kill', whereby the latent virus is stimulated to transcribe rendering the host cell vulnerable to eradication by cytotoxic T cells. Optimising the reactivation signal is therefore critical to this approach. Here the established model system of latency 'J-lat' is used to probe optimum reactivation signals. Single clones are observed to respond to maximal stimulation with a single agent with a fixed proportion of cells. Here it is shown that this proportion can be overcome by dosing with two agents in combination and critically that maximum synergies between agents occur at concentrations of agents close to those achieved in vivo. The role of SETDB1 recruitment by the recently described HUSH complex is examined using shRNA knockdowns of these proteins. Knockdown does not increase expression from the majority of J-lat clones tested. Viral factors which influence silencing and reactivation from latency have not been explored to the same extent. Here mutations affecting the binding of splicing factors to HIV-1 mRNA were cloned into laboratory viruses. A reduction in splice factor binding is seen to change the use of splice junctions required for the production of Tat mRNA; in turn this alters the rate at which proviruses are silenced. In addition the threshold for transcription in response to stimulation is increased in mutants with reduced splice factor binding.

Freie Universiẗat, Diss., 2006--Berlin.

Eradication of latent human immunodeficiency virus (HIV) infection is a global health challenge. Reactivation of HIV latency and killing of virus-infected cells, the so-called “kick and kill” or “shock and kill” approaches, are a popular strategy for HIV cure. While antiretroviral therapy (ART) halts HIV replication by targeting multiple steps in the HIV life cycle, including viral entry, integration, replication, and production, it cannot get rid of the occult provirus incorporated into the host-cell genome. These latent proviruses are replication-competent and can rebound in cases of ART interruption or cessation. In general, a very small population of cells harbor provirus, serve as reservoirs in ART-controlled HIV subjects, and are capable of expressing little to no HIV RNA or proteins. Beyond the canonical resting memory CD4+ T cells, HIV reservoirs also exist within tissue macrophages, myeloid cells, brain microglial cells, gut epithelial cells, and hematopoi-etic stem cells (HSCs). Despite a lack of active viral production, latently HIV-infected subjects con-tinue to exhibit aberrant cellular signaling and metabolic dysfunction, leading to minor to major cellular and systemic complications or comorbidities. These include genomic DNA damage; telo-mere attrition; mitochondrial dysfunction; premature aging; and lymphocytic, cardiac, renal, he-patic, or pulmonary dysfunctions. Therefore, the arcane machineries involved in HIV latency and its reversal warrant further studies to identify the cryptic mechanisms of HIV reservoir formation and clearance. In this review, we discuss several molecules and signaling pathways, some of which have dual roles in maintaining or reversing HIV latency and reservoirs, and describe some evolving strategies and possible approaches to eliminate viral reservoirs and, ultimately, cure/eradicate HIV infection.

Human immunodeficiency virus-1 (HIV-1) establishes a persistent proviral reservoir by integrating into the genome of infected host cells. Current antiretroviral treatments (ART) do not target this persistent population of proviruses which include latently infected cells that upon treatment interruption can be reactivated to contribute to HIV-1 rebound. Deep sequencing of persistent HIV-1 proviruses has revealed that greater than 90% of integrated HIV-1 genomes are defective and unable to produce infectious virions. We hypothesized that intragenic elements in the HIV genome support transcription of aberrant HIV-1 RNAs from defective proviruses that lack long terminal repeats (LTRs). Using an intact provirus detection assay, I observed that resting CD4+ T cells and monocyte-derived macrophages (MDMs) are biased towards generating defective HIV-1 proviruses. Multiplex reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) identified env and nef transcripts which lacked 5’ untranslated regions (UTR) in acutely infected CD4+ T cells and MDMs indicating transcripts are generated that do not utilize the promoter within the LTR. 5’UTR-deficient env transcripts were also identified in a cohort of people living with HIV-1 (PLWH) on ART, suggesting that these aberrant RNAs are produced in vivo. Using 5’ rapid amplification of cDNA ends (RACE), I mapped the start site of these transcripts within the Env gene. This region bound several cellular transcription factors and functioned as a transcriptional regulatory element that could support transcription and translation of downstream HIV-1 RNAs. Transient expression of an HIV-1 5’UTR deletion construct in HEK293T cells demonstrated that HIV-1 transcripts and proteins are still produced when the 5’UTR is absent. These studies provide mechanistic insights into how defective HIV-1 proviruses are persistently expressed to potentially drive inflammation in PLWH.

Convolutional neural networks are commonly used when training deep networks with time-independent data and have demonstrated positive results in predicting DNA binding sites for DNA-binding proteins. Based upon the success of convolutional neural networks in predicting DNA binding sites of proteins, this project intends to determine if a convolutional neural network could predict possible HIV-B provirus integration sites. When exploring existing research, little information was found regarding DNA sequences targeted by HIV for integration, few, if any, have attempted to use artificial neural networks to identify these sequences and the integration sites themselves. Using data from the Retrovirus Integration Database, we train a convolutional artificial neural network to determine if it can detect potential target sites for HIV integration. The analysis and results reveal that the created convolutional neural network is able to predict HIV integration sites in human DNA with an accuracy that exceeds that of a potential random binary classifier. When analyzing the datasets separated by the neural network, the relative distribution of the different nucleotides in the immediate vicinity of HIV integration site reveals that some nucleotides are disproportionately occurring less often at these sites compared to nucleotides in randomly sampled human DNA. / Konvolutionella artificiella nätverk används vanligen vid tidsoberoende datamängder. Konvolutionella artificiella nätverk har varit framgångsrika med att förutse bindningssiter för DNA-bindande proteiner. Med de framsteg som gjorts med konvolutionella artificiella nätverk vill detta projekt bestämma huruvida det går att med ett konvolutionellt artificiella nätverk förutsäga möjliga siter för HIV-B integration i mänskligt DNA. Våran eftersökning visar att det finns lite kunskap om huruvida det finns nukleotidsekvenser i mänskligt DNA som främjar HIV integration. Samtidigt har få eller inga studier gjorts med konvolutionella artificiella nätverk i försök att förutsäga integrationssiter för HIV i mänskligt DNA. Genom att använda data från Retrovirus Integration Database tänker vi träna ett konvolutionellt artificiellt nätverk med syftet att försöka bestämma huruvida det tränade konvolutionella artificiella nätverket kan förutspå potentiella integrationssiter för HIV. Våra resultat visar att det skapade konvolutionella artificiella nätverket kan förutsäga HIV integration i mänskligt DNA med en träffsäkerhet som överträffar en potentiell slumpmässig binär klassificerare. Vid analys av datamängderna separerade av det neurala nätverket framträder en bild där vissa nukleotider förekommer oproportionerligt mindre frekvent i närheten av integrationssiterna i jämförelse med nukleotider i slumpmässigt genererad mänsklig DNA.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

No description available.

The shortage of human tissues and organs for allotransplantation can be overcome by xenotransplantation. As a source of organs, the miniature pig is convenient. However, the presence of pathogens transmissible to the recipients, especially porcine endogenous retrovirus (PERV), represents a threat for successfull xenotransplantation. Infectious PERVs contain three classes of envelope glycoprotein. Two classes, PERV-A and PERV-B are polytropic, they can infect human, pig and mink cells in vitro. PERV-C is evolutionary young, ecotropic isolate that can infect pig only. We previously detected a new full-lenght, but replication-defective PERV-A isolate dubbed (MAMBA) with high transcriptional activity in Large-White pig from a Czech breed. To support our results with PERV-MAMBA epigenetic regulation in pig tissues, in vitro DNA methylation essay was accomplished. Methylated or non-methylated reporter plasmids containing provirus 5' LTR were transfected into 293T cells and luciferase activity was measured. In both cases, methylated LTR decreased significantly expression of luciferase. Thus, PERV LTR-driven transcription is sensitive to DNA methylation. We also used PERV-A MAMBA provirus to study recombination between two pig endogenous retroviruses. We prepared 293T and BeWo cell clones harboring PERV-A...

A via sexual é a principal forma de transmissão inter-humana da infecção pelo vírus da imunodeficiência humana (HIV). Com o incremento do número de mulheres infectadas por esse agente retroviral, o estudo de particularidades da biologia do vírus no trato genital feminino adquiriu maior importância. Com o objetivo de avaliar a excreção genital do HIV ao longo do ciclo menstrual, coletaram-se, nas diversas fases de dois ciclos, lavados cervicovaginais de 17 mulheres soropositivas para essa infecção e acompanhadas em serviço ambulatorial especializado de São Paulo. O RNA viral livre foi quantificado por RT-PCR e o DNA proviral por PCR em tempo real, empregando sistema TaqMan. Avaliou-se ainda a carga viral plasmática de HIV, o número de células CD4+ periféricas e presença de co-infecção genital. Detectou-se excreção de RNA-HIV e de DNA proviral, respectivamente, em 18,8% e 31,3% das pacientes. Todas as pacientes que apresentaram excreção de RNA viral também exibiram a de DNA proviral, incluindo paciente com viremia de HIV indetectável. Não houve variação significativa da excreção genital do vírus durante o ciclo menstrual. Em 6 dessas pacientes, que apresentaram co-infecção genital previamente à admissão no estudo, avaliou-se também a excreção genital de HIV quando da co-infecção. Em 2 casos, a excreção genital do DNA-HIV foi superior na vigência de co-infecção causada por Streptococcus sp e Ureaplasma. Não se observou excreção de RNA viral livre nas pacientes co-infectadas. Os resultados obtidos podem contribuir para o entendimento do potencial de transmissibilidade sexual da infecção por HIV e reiteram a necessidade de adesão às práticas de sexo protegido para evitar sua transmissão inter-humana / The sexual route is the main means of transmission of the human immunodeficiency virus (HIV). With the increasing numbers of HIV-infected women, the investigation of particular biological features of HIV infection in the genital tract has become more important. To evaluate HIV genital shedding during the menstrual cycle, we collected cervicovaginal lavages (CVL) from 17 women, assisted at an HIV outpatient clinic in São Paulo, in different hormonal phases during 2 cycles. HIV-RNA and proviral DNA shedding were quantified using RT-PCR and a TaqMan real-time PCR assay, respectively. In addition, patients were screened for genital coinfections and had their HIV plasma viral loads and CD4+ cell counts assessed. Cell-free HIV-RNA and proviral DNA shedding were found in 18.8% and 31.3% of women. All patients who shed HIV-RNA were also shown to present detectable proviral DNA in their CVL, including one woman with undetectable HIV plasma viral load. No significant difference in viral shedding was seen among menstrual cycle phases. Six patients from the cohort, who exhibited genital coinfections previous to admission to the study, had their HIV genital shedding compared at time of coinfection and after its resolution. In two of them proviral DNA shedding was higher at the time of coinfection, caused by Streptococcus sp and Ureaplasma. No cell-free HIV-RNA shedding was detected in coinfected patients. Our results may contribute to the understanding of HIV sexual infectivity from women and emphasize the need for adherence to protected sexual practices in order to avoid viral transmission.

A via sexual é a principal forma de transmissão inter-humana da infecção pelo vírus da imunodeficiência humana (HIV). Com o incremento do número de mulheres infectadas por esse agente retroviral, o estudo de particularidades da biologia do vírus no trato genital feminino adquiriu maior importância. Com o objetivo de avaliar a excreção genital do HIV ao longo do ciclo menstrual, coletaram-se, nas diversas fases de dois ciclos, lavados cervicovaginais de 17 mulheres soropositivas para essa infecção e acompanhadas em serviço ambulatorial especializado de São Paulo. O RNA viral livre foi quantificado por RT-PCR e o DNA proviral por PCR em tempo real, empregando sistema TaqMan. Avaliou-se ainda a carga viral plasmática de HIV, o número de células CD4+ periféricas e presença de co-infecção genital. Detectou-se excreção de RNA-HIV e de DNA proviral, respectivamente, em 18,8% e 31,3% das pacientes. Todas as pacientes que apresentaram excreção de RNA viral também exibiram a de DNA proviral, incluindo paciente com viremia de HIV indetectável. Não houve variação significativa da excreção genital do vírus durante o ciclo menstrual. Em 6 dessas pacientes, que apresentaram co-infecção genital previamente à admissão no estudo, avaliou-se também a excreção genital de HIV quando da co-infecção. Em 2 casos, a excreção genital do DNA-HIV foi superior na vigência de co-infecção causada por Streptococcus sp e Ureaplasma. Não se observou excreção de RNA viral livre nas pacientes co-infectadas. Os resultados obtidos podem contribuir para o entendimento do potencial de transmissibilidade sexual da infecção por HIV e reiteram a necessidade de adesão às práticas de sexo protegido para evitar sua transmissão inter-humana / The sexual route is the main means of transmission of the human immunodeficiency virus (HIV). With the increasing numbers of HIV-infected women, the investigation of particular biological features of HIV infection in the genital tract has become more important. To evaluate HIV genital shedding during the menstrual cycle, we collected cervicovaginal lavages (CVL) from 17 women, assisted at an HIV outpatient clinic in São Paulo, in different hormonal phases during 2 cycles. HIV-RNA and proviral DNA shedding were quantified using RT-PCR and a TaqMan real-time PCR assay, respectively. In addition, patients were screened for genital coinfections and had their HIV plasma viral loads and CD4+ cell counts assessed. Cell-free HIV-RNA and proviral DNA shedding were found in 18.8% and 31.3% of women. All patients who shed HIV-RNA were also shown to present detectable proviral DNA in their CVL, including one woman with undetectable HIV plasma viral load. No significant difference in viral shedding was seen among menstrual cycle phases. Six patients from the cohort, who exhibited genital coinfections previous to admission to the study, had their HIV genital shedding compared at time of coinfection and after its resolution. In two of them proviral DNA shedding was higher at the time of coinfection, caused by Streptococcus sp and Ureaplasma. No cell-free HIV-RNA shedding was detected in coinfected patients. Our results may contribute to the understanding of HIV sexual infectivity from women and emphasize the need for adherence to protected sexual practices in order to avoid viral transmission.