UZH-Logo

Maintenance Infos

Interplay between HIV-1 transcription and the pathways of intracellular innate defense


Althaus, C. Interplay between HIV-1 transcription and the pathways of intracellular innate defense. 2011, University of Zurich, Faculty of Science.

Abstract

Human immunodeficiency virus type 1 (HIV-1) infection causes a life-long chronic disease despite the tremendous success of combination antiretroviral therapy (cART). This is due to the ability of HIV-1 to persist in latent forms in long-lived infected cells. Currently approved antiretroviral drugs target multiple steps in the viral life cycle. Thus, latent cellular reservoirs of HIV-1 remain untouched and represent one of the major obstacles towards the goal of curing HIV-1 infection. The mechanisms of induction and maintenance of HIV-1 latency have not been fully resolved so far. Besides regulation by transcription factors and/or epigenetic modulation of the proviral DNA, one hypothesis suggests that HIV-1 latency may be mediated by the intracellular machinery governing innate antiviral defences, such as RNA-interference. This mechanism is mediated by small noncoding RNAs, which bind to complementary mRNA and finally lead to cleavage of the mRNA or suppression of translation. HIV-1 latency could be induced by targeting viral or cellular RNA necessary for viral replication. Chapter 1 describes the establishment of a method to measure viral RNAs with single copy sensitivity. Quantitative PCR (qPCR) using fluorescent hydrolysis probes (FH-probes) of variable HIV-1 genomes can result in underestimation of viral copy numbers due to mismatches in the FH-probe’s target sequences. Particularly for detection of viral RNA in patients, successfully treated with cART, or in latently infected cells, highly sensitive qPCRs are essential. The study resulted in empirically validated novel principles of FH-probe design regarding conservation and qPCR performance. Several FH-probes used to quantify HIV-1 DNA over 6 orders of magnitude approached single copy sensitivity. Moreover, application of an algorithm to scan sequence databases for FH-probes with optimal phylogenetic conservation, allowed to identify functional FH-probes in various regions of the HIV-1 genome approaching coverage of the global HIV-1 pandemic. The second chapter illustrates the development of a method to efficiently select and sequence small noncoding RNAs (sncRNAs), the key molecules of RNA interference. Since HIV-1 encoded sncRNAs represent only a small minority of sncRNAs in an infected cell, their detection remains difficult and reported frequencies of HIV-1 sncRNAs are usually <0.5%, if at all. Our approach was to enrich sequences homologous to HIV-1 by hybridization capture using HIV-1 ssDNA hybridization probes attached to Streptavidin beads. With this optimized selection method we were able to enrich low abundant HIV-1 sncRNAs more than 100-fold, i.e., >70% of captured sncRNAs were derived from HIV-1. This method was further applied to characterize the scope of viral sncRNA expression in primary HIV-1 infected cells. A multitude of HIV-1 sncRNAs was identified, distributed throughout the HIV-1 genome, yet, they clustered in contigs with hot-spots at distinct sites. Furthermore, hybrids of sense and antisense sncRNAs of one contig inhibit HIV-1 replication in primary macrophages demonstrating one possible function of these sncRNAs. The third section refers to a study published in collaboration with colleagues from our research group. The study focused on monitoring HIV-1 RNA transcription patterns in peripheral blood mononuclear cells (PBMC) during cART in acutely HIV-1 infected patients to assess the effect of early treatment on cellular viral reservoirs (Schmid et al., 2010). I developed the appropriate qPCR protocols (please compare first chapter), assisted with the search for optimal patient matched primers and FHprobes for highly sensitive qPCRs, supervised the qPCR procedures, and helped in analyzing and interpreting the qPCR data. This study showed that early cART in acute HIV-1 infection significantly depleted the number of transcriptionally active proviruses when compared to levels detected in patients starting ART during chronic HIV-1 infection. Therefore, future studies aiming at HIV-1 eradication should be initiated in patients during acute infection. In conclusion, my thesis mainly focused on the establishment of highly sensitive and improved methods for the detection of HIV-1 genomes and HIV-1 derived small noncoding RNAs, respectively, to enable detailed studies of various aspects of HIV-1 latency. Thus, quantitative PCR protocols were developed to detect a broad range of highly diverse HIV-1 variants with single copy sensitivity, and a novel approach for very efficient enrichment of low abundant HIV-1 sncRNAs was established. Both methods lay the foundation for further studies, not only in the field of HIV-1, but also in other research areas where highly sensitive methods for the detection of RNA and sncRNAs are needed.

Human immunodeficiency virus type 1 (HIV-1) infection causes a life-long chronic disease despite the tremendous success of combination antiretroviral therapy (cART). This is due to the ability of HIV-1 to persist in latent forms in long-lived infected cells. Currently approved antiretroviral drugs target multiple steps in the viral life cycle. Thus, latent cellular reservoirs of HIV-1 remain untouched and represent one of the major obstacles towards the goal of curing HIV-1 infection. The mechanisms of induction and maintenance of HIV-1 latency have not been fully resolved so far. Besides regulation by transcription factors and/or epigenetic modulation of the proviral DNA, one hypothesis suggests that HIV-1 latency may be mediated by the intracellular machinery governing innate antiviral defences, such as RNA-interference. This mechanism is mediated by small noncoding RNAs, which bind to complementary mRNA and finally lead to cleavage of the mRNA or suppression of translation. HIV-1 latency could be induced by targeting viral or cellular RNA necessary for viral replication. Chapter 1 describes the establishment of a method to measure viral RNAs with single copy sensitivity. Quantitative PCR (qPCR) using fluorescent hydrolysis probes (FH-probes) of variable HIV-1 genomes can result in underestimation of viral copy numbers due to mismatches in the FH-probe’s target sequences. Particularly for detection of viral RNA in patients, successfully treated with cART, or in latently infected cells, highly sensitive qPCRs are essential. The study resulted in empirically validated novel principles of FH-probe design regarding conservation and qPCR performance. Several FH-probes used to quantify HIV-1 DNA over 6 orders of magnitude approached single copy sensitivity. Moreover, application of an algorithm to scan sequence databases for FH-probes with optimal phylogenetic conservation, allowed to identify functional FH-probes in various regions of the HIV-1 genome approaching coverage of the global HIV-1 pandemic. The second chapter illustrates the development of a method to efficiently select and sequence small noncoding RNAs (sncRNAs), the key molecules of RNA interference. Since HIV-1 encoded sncRNAs represent only a small minority of sncRNAs in an infected cell, their detection remains difficult and reported frequencies of HIV-1 sncRNAs are usually <0.5%, if at all. Our approach was to enrich sequences homologous to HIV-1 by hybridization capture using HIV-1 ssDNA hybridization probes attached to Streptavidin beads. With this optimized selection method we were able to enrich low abundant HIV-1 sncRNAs more than 100-fold, i.e., >70% of captured sncRNAs were derived from HIV-1. This method was further applied to characterize the scope of viral sncRNA expression in primary HIV-1 infected cells. A multitude of HIV-1 sncRNAs was identified, distributed throughout the HIV-1 genome, yet, they clustered in contigs with hot-spots at distinct sites. Furthermore, hybrids of sense and antisense sncRNAs of one contig inhibit HIV-1 replication in primary macrophages demonstrating one possible function of these sncRNAs. The third section refers to a study published in collaboration with colleagues from our research group. The study focused on monitoring HIV-1 RNA transcription patterns in peripheral blood mononuclear cells (PBMC) during cART in acutely HIV-1 infected patients to assess the effect of early treatment on cellular viral reservoirs (Schmid et al., 2010). I developed the appropriate qPCR protocols (please compare first chapter), assisted with the search for optimal patient matched primers and FHprobes for highly sensitive qPCRs, supervised the qPCR procedures, and helped in analyzing and interpreting the qPCR data. This study showed that early cART in acute HIV-1 infection significantly depleted the number of transcriptionally active proviruses when compared to levels detected in patients starting ART during chronic HIV-1 infection. Therefore, future studies aiming at HIV-1 eradication should be initiated in patients during acute infection. In conclusion, my thesis mainly focused on the establishment of highly sensitive and improved methods for the detection of HIV-1 genomes and HIV-1 derived small noncoding RNAs, respectively, to enable detailed studies of various aspects of HIV-1 latency. Thus, quantitative PCR protocols were developed to detect a broad range of highly diverse HIV-1 variants with single copy sensitivity, and a novel approach for very efficient enrichment of low abundant HIV-1 sncRNAs was established. Both methods lay the foundation for further studies, not only in the field of HIV-1, but also in other research areas where highly sensitive methods for the detection of RNA and sncRNAs are needed.

Downloads

449 downloads since deposited on 16 Jan 2012
66 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Dissertation
Referees:Trkola A, Fischer M
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Clinic for Infectious Diseases
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2011
Deposited On:16 Jan 2012 21:36
Last Modified:05 Apr 2016 15:24
Number of Pages:132
Related URLs:http://opac.nebis.ch/F/?local_base=NEBIS&CON_LNG=GER&func=find-b&find_code=SYS&request=006750567
Permanent URL: https://doi.org/10.5167/uzh-55511

Download

[img]
Preview
Content: Published Version
Filetype: PDF
Size: 4MB

TrendTerms

TrendTerms displays relevant terms of the abstract of this publication and related documents on a map. The terms and their relations were extracted from ZORA using word statistics. Their timelines are taken from ZORA as well. The bubble size of a term is proportional to the number of documents where the term occurs. Red, orange, yellow and green colors are used for terms that occur in the current document; red indicates high interlinkedness of a term with other terms, orange, yellow and green decreasing interlinkedness. Blue is used for terms that have a relation with the terms in this document, but occur in other documents.
You can navigate and zoom the map. Mouse-hovering a term displays its timeline, clicking it yields the associated documents.

Author Collaborations