Tailored enrichment strategy detects low abundant small noncoding RNAs in HIV-1 infected cells.

BACKGROUND: The various classes of small noncoding RNAs (sncRNAs) are important regulators of gene expression across divergent types of organisms. While a rapidly increasing number of sncRNAs has been identified over recent years, the isolation of sncRNAs of low abundance remains challenging. Virally encoded sncRNAs, particularly those of RNA viruses, can be expressed at very low levels. This is best illustrated by HIV-1 where virus encoded sncRNAs represent approximately 0.1-1.0% of all sncRNAs in HIV-1 infected cells or were found to be undetected. Thus, we applied a novel, sequence targeted enrichment strategy to capture HIV-1 derived sncRNAs in HIV-1 infected primary CD4+ T-lymphocytes and macrophages that allows a greater than 100-fold enrichment of low abundant sncRNAs. RESULTS: Eight hundred and ninety-two individual HIV-1 sncRNAs were cloned and sequenced from nine different sncRNA libraries derived from five independent experiments. These clones represent up to 90% of all sncRNA clones in the generated libraries. Two hundred and sixteen HIV-1 sncRNAs were distinguishable as unique clones. They are spread throughout the HIV-1 genome, however, forming certain clusters, and almost 10% show an antisense orientation. The length of HIV-1 sncRNAs varies between 16 and 89 nucleotides with an unexpected peak at 31 to 50 nucleotides, thus, longer than cellular microRNAs or short-interfering RNAs (siRNAs). Exemplary HIV-1 sncRNAs were also generated in cells infected with different primary HIV-1 isolates and can inhibit HIV-1 replication. CONCLUSIONS: HIV-1 infected cells generate virally encoded sncRNAs, which might play a role in the HIV-1 life cycle. Furthermore, the enormous capacity to enrich low abundance sncRNAs in a sequence specific manner highly recommends our selection strategy for any type of investigation where origin or target sequences of the sought-after sncRNAs are known.

Profound depletion of HIV-1 transcription in patients initiating antiretroviral therapy during acute infection.

BACKGROUND: Although combination antiretroviral therapy (cART) initiated in the acute phase of HIV-1 infection may prevent expansion of the latent reservoir, its benefits remain controversial. In the current study, HIV-1 RNA transcription patterns in peripheral blood mononuclear cells (PBMC) were monitored during acute cART to assess the effect of early treatment on cellular viral reservoirs. METHODOLOGY/PRINCIPAL FINDINGS: Acutely HIV-1 infected patients (n = 24) were treated within 3-15 weeks after infection. Patients elected to cease treatment after ≥1 year of therapy. HIV-1 DNA (vDNA), HIV-1 RNA species expressed both in latently and productively infected cells, unspliced (UsRNA), multiply spliced (MsRNA-tatrev; MsRNA-nef), and PBMC-associated extracellular virion RNA (vRex), expressed specifically by productively infected cells, were quantified in PBMC by patient matched real-time PCR prior, during and post cART. In a matched control-group of patients on successful cART started during chronic infection (n = 15), UsRNA in PBMC and vDNA were measured cross-sectionally. In contrast to previous reports, PBMC-associated HIV-1 RNAs declined to predominantly undetectable levels on cART. After cART cessation, UsRNA, vRex, and MsRNA-tatrev rebounded to levels not significantly different to those at baseline (p>0.1). In contrast, MsRNA-nef remained significantly lower as compared to pretreatment (p = 0.015). UsRNA expressed at the highest levels of all viral RNAs, was detectable on cART in 42% of patients with cART initiated during acute infection as opposed to 87% of patients on cART initiated during chronic infection (Fisher's exact test; p = 0.008). Accordingly, UsRNA levels were 105-fold lower in the acute as compared to the chronic group. CONCLUSION: Early intervention resulted in profound depletion of PBMC expressing HIV-1 RNA. This is contrary to chronically infected patients who predominantly showed continuous UsRNA expression on cART. Thus, antiretroviral treatment initiated during the acute phase of infection prevented establishment or expansion of long-lived transcriptionally active viral cellular reservoirs in peripheral blood.

Rational design of HIV-1 fluorescent hydrolysis probes considering phylogenetic variation and probe performance.

Quantitative PCR (qPCR) using fluorescent hydrolysis probes (FH-probes; TaqMan-probes) of variable genomes, such as HIV-1, can result in underestimation of viral copy numbers due to mismatches in the FH-probe's target sequences. Therefore both target conservation and physical properties of FH-probes, such as melting temperature, baseline fluorescence and secondary structure, should be considered in design of FH-probes. Analysis of a database of 1242 near full-length HIV-1 sequences with a novel computational tool revealed that the probability of target and FH-probe identity decreases exponentially with FH-probe length. In addition, this algorithm allowed for identification of continuous sequence stretches of high conservation, from which FH-probes with global HIV-1 clade coverage could be chosen. To revise the prerequisites of physical FH-probe function, properties of 30 DNA and 21 chimeric DNA locked nucleic acid (DLNA) HIV-1 FH-probes were correlated with their performance in qPCR. This identified the presence of stable secondary structures within FH-probes and the base composition and thermal stability of the 5' proximal end as novel predictors of FH-probe performance. Thus, empirically validated novel principles of FH-probe design regarding conservation and qPCR-performance were identified, which complement and extend current rules for FH-probe design.