Experimental And Computational Investigations Into Interactions Between Hiv 1 And The Rna Interference Pathway PDF Download

"As an obligate intracellular pathogen, the human immunodeficiency virus type 1 (HIV-1) targets and co-opts a diverse set of host processes to overcome cellular barriers to infection, including pathways involved in the regulation of gene expression. It remains controversial whether HIV-1 affects the RNA interference (RNAi) pathway, a key post-transcriptional regulatory mechanism that is being used to develop a new class of gene therapies. To better understand the replication cycle of the virus and inform the development of future antiviral therapies, we investigated the hypothesis that HIV-1 changes the substrates and functionality of the RNAi system in specific contexts. To investigate our hypothesis, we first aimed to identify regulatory networks associated with blocks in virus replication during latency. We characterized a novel cellular model for HIV-1 infection that was designed to study events in HIV-1 latency reversion that follow transcription initiation. We then reactivated this model using differently acting latency reversing agents, to then sequence long and short RNA transcriptomes associated with latency maintenance and reversion. These data contributed new genes and regulatory RNA networks to our understanding of latency, and possibly new targets for RNA therapies. We next analyzed an interaction between the HIV-1 protein Gag and the RNAi enzyme Dicer, which leads to the specific enrichment of three microRNAs (miRNAs) on Dicer. A combination of bioinformatic analyses were used to identify the targets of these miRNAs and to define a target-specific hypothesis for the function of this interaction. Using gene reporter assays, Western blots and reverse transcription quantitative polymerase chain reactions, we explored one miRNA-target interaction in depth, showing that this miRNA inhibits HIV-1 expression and that Gag promotes viral expression by increasing expression of the target, possibly by inhibiting the antiviral miRNA. Finally, we sought to develop a testing platform to distinguish between RNAi substrates that could be used in combination therapies against HIV-1. We designed a novel protocol that can score RNAi substrates with four primary measures: cellular toxicity, inhibition of HIV expression, inhibition of HIV replication, and ability to lock proviruses in a latent state. Due to delays related to cloning, this protocol could not be fully employed in this manuscript. However, we individually tested the first three endpoints and used a surrogate fourth endpoint to make preliminary assessments of the therapeutic potential of several molecules. Here, we describe two workflows used to gain better insight into interactions between HIV-1 and the RNAi pathway, and further designed a protocol for testing small RNAs for their potential to lock the HIV-1 in a latent state"--