An HIV-encoded antisense long non-coding RNA epigenetically regulates viral transcription

pre-printThe abundance of long non-coding RNAs (lncRNAs) and their wide range of functional roles in human cells are fast becoming realized. Importantly, lncRNAs have been identified as epigenetic modulators and consequently play a pivotal role in the regulation of gene expression. A human immunodeficiency virus (HIV) encoded antisense RNA transcript has recently been reported and we sought to characterize this RNA and determine its potential role in viral transcription regulation. The intrinsic properties of this HIV-expressed lncRNA were characterized and the data presented here suggest that it functions as an epigenetic brake to modulate viral transcription. Suppression of this long antisense transcript with small single stranded antisense RNAs resulted in the activation of viral gene expression. This lncRNA was found to localize to the 5'LTR and to usurp components of endogenous cellular pathways that are involved in lncRNA directed epigenetic gene silencing. Collectively, we find that this viral expressed antisense lncRNA is involved in modulating HIV gene expression and that this regulatory effect is due to an alteration in the epigenetic landscape at the viral promoter

Inhibiting HIV-1 gene expression and replication with expressed long hairpin RNAs

PhD, Faculty of Health Sciences, University of the WitwatersrandThe vast potential of the RNA interference (RNAi) pathway as a new tool for the development of therapeutic modalities has been quickly realised since its discovery in 1998. RNAi effector mimics have been developed to successfully silence an array of disease-causing genetic elements. However, because of the rapidly mutating genome of viruses such as the human immunodeficiency virus (HIV), inhibition of replication cannot be sustained with single RNAi effector mimics. Instead, a combinatorial approach is required, analogous to the cocktail of drugs necessary for successful highly active antiretroviral therapy (HAART). Pioneering studies utilizing long hairpin RNAs (lhRNAs) showed that the long double-stranded RNA stem region acts as a Dicer substrate and is processed into multiple siRNA species. This intrinsic combinatorial property of lhRNAs was exploited in this thesis by attempting to incorporate three non-contiguous potent siRNA sequences within a single lhRNA stem expressed from an RNA Pol III promoter. Although significant knockdown of three independent HIV target sequences was possible, the limitations of this approach became apparent when it was observed that human Dicer does not function efficiently as a multiple turnover enzyme. The generation of siRNA products therefore occurred in a gradient, with higher levels of siRNA produced from the base of the hairpin stem and decreasing quantities generated towards the loop. Modifications to the configuration of integrated siRNA sequences within the stem region enabled augmented RNAi activity of siRNAs in the second position of the hairpin stem. This led to the notion that further manipulation of the structural design of the stem duplex may improve efficacy of up to two siRNAs. Dual-targeting anti-HIV lhRNAs encoding only two highly effective siRNAs targeted against non-contiguous sites within the tat, nef, LTR and int viral genes were therefore propagated. The spatial arrangement of two siRNA sequences was extensively characterised within dual-targeting lhRNAs by inserting up to three random base pairs at the junctions of siRNA encoding sequences and 5 bp preceding the terminal loop sequence. A universally optimal hairpin design was identified which contained a single mismatched base pair between two 19 bp + 2 nt siRNA sequences, as well as a terminal extension. Two powerful dual-targeting lhRNA species, lhRNA-tat-nef +1 and lhRNA-LTR-int +1, each capable of producing two potent anti-HIV siRNA products in equal quantities were selected for incorporation into a combinatorial RNAi system. These two effective dual-targeting lhRNAs were combined, adjacent to one another within a single RNA Pol III-expressed transcript to create a novel lhRNA-based combinatorial RNAi structure. This double lhRNA (dlhRNA) construct served as a precursor for four discrete highly functional RNAi effector sequences which were capable of simultaneously silencing four unique HIV target sites within the tat, nef, LTR and int genes. Furthermore, the ectopic expression of dlhRNAs did not elicit activation of the interferon response, nor did it cause saturation of the endogenous miRNA biogenesis pathway in vitro. In conclusion, the inherent combinatorial RNAi properties of long hairpin RNAs were evaluated and the detailed analysis is presented in this thesis. Structurally optimised dualtargeting lhRNAs subsequently formed the core components of a novel dlhRNA precursor which meets all the requirements for an effective combinatorial RNAi strategy and therefore holds great promise for mediating an effective and sustained gene therapy against HIV

Deriving four functional anti-HIV siRNAs from a single Pol III-generated transcript comprising two adjacent long hairpin RNA precursors

Several different approaches exist to generate expressed RNA interference (RNAi) precursors for multiple target inhibition, a strategy referred to as combinatorial (co)RNAi. One such approach makes use of RNA Pol III-expressed long hairpin RNAs (lhRNAs), which are processed by Dicer to generate multiple unique short interfering siRNA effectors. However, because of inefficient intracellular Dicer processing, lhRNA duplexes have been limited to generating two independent effective siRNA species. In this study, we describe a novel strategy whereby four separate anti-HIV siRNAs were generated from a single RNA Pol III-expressed transcript. Two optimized lhRNAs, each comprising two active anti-HIV siRNAs, were placed in tandem to form a double long hairpin (dlhRNA) expression cassette, which encodes four unique and effective siRNA sequences. Processing of the 3′ position lhRNA was more variable but effective multiple processing was possible by manipulating the order of the siRNA-encoding sequences. Importantly, unlike shRNAs, Pol III-expressed dlhRNAs did not compete with endogenous and exogenous microRNAs to disrupt the RNAi pathway. The versatility of expressed lhRNAs is greatly expanded and we provide a mechanism for generating transcripts with modular lhRNAs motifs that contribute to improved coRNAi

The biogenesis and characterization of mammalian microRNAs of mirtron origin

Mirtrons, short hairpin pre-microRNA (miRNA) mimics directly produced by intronic splicing, have recently been identified and experimentally confirmed in invertebrates. While there is evidence to suggest several mammalian miRNAs have mirtron origins, this has yet to be experimentally demonstrated. Here, we characterize the biogenesis of mammalian mirtrons by ectopic expression of splicing-dependent mirtron precursors. The putative mirtrons hsa-miR-877, hsa-miR-1226 and mmu-miR-1224 were designed as introns within eGFP. Correct splicing and function of these sequences as introns was shown through eGFP fluorescence and RT–PCR, while all mirtrons suppressed perfectly complementary luciferase reporter targets to levels similar to that of corresponding independently expressed pre-miRNA controls. Splicing-deficient mutants and disruption of key steps in miRNA biogenesis demonstrated that mirtron-mediated gene knockdown was splicing-dependent, Drosha-independent and had variable dependence on RNAi pathway elements following pre-miRNA formation. The silencing effect of hsa-miR-877 was further demonstrated to be mediated by the generation of short anti-sense RNA species expressed with low abundance. Finally, the mammalian mirtron hsa-miR-877 was shown to reduce mRNA levels of an endogenous transcript containing hsa-miR-877 target sites in neuronal SH-SY5Y cells. This work confirms the mirtron origins of three mammalian miRNAs and suggests that they are a functional class of splicing-dependent miRNAs which are physiologically active

The Efficacy of Generating Three Independent Anti-HIV-1 siRNAs from a Single U6 RNA Pol III-Expressed Long Hairpin RNA

RNA Interference (RNAi) effectors have been used to inhibit rogue RNAs in mammalian cells. However, rapidly evolving sequences such as the human immunodeficiency virus type 1 (HIV-1) require multiple targeting approaches to prevent the emergence of escape variants. Expressed long hairpin RNAs (lhRNAs) have recently been used as a strategy to produce multiple short interfering RNAs (siRNAs) targeted to highly variant sequences. We aimed to characterize the ability of expressed lhRNAs to generate independent siRNAs that silence three non-contiguous HIV-1 sites by designing lhRNAs comprising different combinations of siRNA-encoding sequences. All lhRNAs were capable of silencing individual target sequences. However, silencing efficiency together with concentrations of individual lhRNA-derived siRNAs diminished from the stem base (first position) towards the loop side of the hairpin. Silencing efficacy against HIV-1 was primarily mediated by siRNA sequences located at the base of the stem. Improvements could be made to first and second position siRNAs by adjusting spacing arrangements at their junction, but silencing of third position siRNAs remained largely ineffective. Although lhRNAs offer advantages for combinatorial RNAi, we show that good silencing efficacy across the span of the lhRNA duplex is difficult to achieve with sequences that encode more than two adjacent independent siRNAs

Northern blot analysis of RNA extracted from HEK293 cells that had been transfected with the indicated lhRNA and shRNA-expressing plasmids.

<p>A. A single blot was probed with an oligonucleotide that was complementary to putative <i>tat</i>, <i>rev</i> and <i>vif</i> guide sequences. B. The blot was probed with two 14-mer LNA oligonucleotides (LNA nucleotides underlined) which were complementary to the <i>rev</i> siRNA guide sequence and adjacent nucleotides as indicated in the illustration. Both blots (from A and B) were stripped and reprobed with an oligonucleotide complementary to U6 snRNA to control for equal RNA loading.</p

Effect of nucleotide spacing between <i>tat</i>, <i>rev</i> and <i>vif</i> siRNA-encoding sequences on silencing efficacy.

<p>A. Schematic illustration of hairpin sequences with boundaries between <i>tat</i>, <i>rev</i> and <i>vif</i> duplexes indicated. The sequences and numbers of bases inserted or deleted at the junctions of the RNAi effecter-encoding sequences are indicated for each of lhRNA <i>rev</i>-<i>vif</i>-<i>tat</i> a to lhRNA <i>rev</i>-<i>vif</i>-<i>tat</i> g. B. Average normalized <i>Renilla</i>∶Firefly luciferase activity determined 48 hours after transfecting HEK293 cells with the psiCheck <i>tat-rev-vif</i> target together with each of lhRNA <i>rev</i>-<i>vif</i>-<i>tat</i> a to lhRNA <i>rev</i>-<i>vif</i>-<i>tat</i> g. C. Average normalized <i>Renilla</i>∶Firefly luciferase activity determined 48 hours after transfecting HEK293 cells with the psiCheck <i>tat</i>, psiCheck <i>rev or</i> psiCheck <i>vif</i> target together with each of lhRNA <i>rev</i>-<i>vif</i>-<i>tat</i> a to lhRNA <i>rev</i>-<i>vif</i>-<i>tat</i> g. Results are given as the average values with standard deviations from three independent transfection experiments. (*, <i>p</i><0.05, <i>t</i>-test, relative to mock transfected control). Mock transfected cells received the empty backbone U6+1 plasmid D. Northern blot analysis of RNA extracted from HEK293 cells that had been transfected with the indicated lhRNA and shRNA-expressing plasmids. The blot was probed with an oligonucleotide that was complementary to putative <i>rev</i> guide sequence. The blot was stripped and reprobed with an oligonucleotide complementary to U6 snRNA to control for equal RNA loading.</p

Knockdown of target-reporter fusion gene expression.

<p>A. psiCheck-derived vectors that include indicated HIV-1 target sequences inserted downstream of the <i>Renilla</i> luciferase ORF. The control Firefly luciferase cassette, present on the same plasmid, is also shown. Both cassettes are under control of constitutively active transcription regulatory elements: Herpes simplex virus thymidine kinase (HSV TK) and Simian virus 40 (SV40) promoters. B. Average normalized ratios of the <i>Renilla</i>∶Firefly luciferase activity when cells were transfected with psiCheck <i>tat</i>-<i>rev</i>-<i>vif</i> dual luciferase reporter plasmid together with lhRNA- or shRNA-encoding plasmid vectors. C. Average normalized ratios of the <i>Renilla</i>∶Firefly luciferase activity when cells were transfected with psiCheck <i>tat</i>, psiCheck <i>rev</i> or psiCheck <i>vif</i> dual luciferase reporter plasmids together with lhRNA- or shRNA-encoding plasmid vectors. The average values from three independent transfection experiments, with standard deviations, are given (*, <i>p</i><0.05, <i>t</i>-test, relative to mock transfected control).</p

HIV-1 challenge assay.

<p>U87.CD₄.CCR5 cells were transfected with plasmids expressing the indicated hairpins and then subjected to infection with an equivalent of TCID₅₀ 1000 particles of the HIV-1 FV5 viral isolate. Concentrations of HIV-1 p24 in the culture supernatant and viral particle equivalents (A) were determined 6 days after infection. Results are expressed as the means with standard deviations of three independent experiments (*, <i>p</i><0.05, <i>t</i>-test, relative to mock transfected control). B. Replication kinetics of a representative experiment included in (A). C. Sequence of lhRNA- and shRNA-derived <i>tat</i>, <i>rev</i> and <i>vif</i> guide sequences and complementary regions targeted within FV5 and HXB2 HIV-1 isolates. Mismatches between the putative guide and target sequences are shown. Sequence numbering is based on isolate HXB2, accession K03455. D. Knockdown of FV5 <i>vif</i> and HXB2 <i>vif</i> target-reporter fusion gene expression by shRNA <i>vif</i> and representative lhRNAs in HEK293 cells. Results are expressed as the means with standard deviations of three independent experiments (*; p<0.05, <i>t</i>-test, between annotated samples).</p