Effective aspects of positive semi-definite real and complex polynomials

Master'sMASTER OF SCIENC

Do mutations in the tat exonic splice enhancer contribute to HIV-1 latency?

Latent infection of long-lived memory CD4+ T cells is thought to be a major barrier to the eradication of HIV-1. Globally there is a substantial research effort aimed at disrupting latency to enable the purging of the latent reservoir. Despite maximal activation a significant proportion of viruses are not reactivated from latency (Ho et al. 2013). We examined sequences deposited by the Siliciano lab from non-induced proviruses found in patient samples. All sequences contained mutations in a recently described exonic splice enhancer involved in the regulation of tat mRNA splicing (Erkelenz et al. 2015). By comparison with over 2000 subtype B sequences deposited in the Los Alamos database we identified mutations which are highly enriched in the latent sequences. We hypothesise that mutations in this region could result in inefficient splicing of tat mRNA; preventing the establishment of the Tat-TAR feedback loop and leading to silencing. One of the mutations corresponding to a G to A mutation at position 5817 in HXB2 was found in 11/18 (60%) of the latent sequences but only 10% of subtype B sequences To investigate this further, we have cloned this mutation into NL4-3 and NL4-3 expressing GFP in env. We compared the replicative capacity of the mutant virus compared to wild type. We also studied the effect of this single mutation on viral RNA splicing pattern and the dynamics of viral gene expression in a primary cell model of latency. Results of our studies will be presented in the meeting

HIV silencing and inducibility are heterogeneous and are affected by factors intrinsic to the virus

Transcriptionally silent HIV proviruses form the major obstacle to eradicating HIV. Many studies of HIV latency have focused on the cellular mechanisms that maintain silencing of proviral DNA. Here we show that viral sequence variation affecting replicative ability leads to variable rates of silencing and ability to reactivate. We studied naturally occurring and engineered polymorphisms in a recently identified exonic splice enhancer (ESEtat) that regulates tat mRNA splicing and constructed viruses with increased (M1), reduced (M2) or completely absent (ERK) binding of splicing factors essential for optimal production of tat mRNA resulting in a corresponding change in Tat activity. The mutations affected viral replication, with M1 having wild type kinetics, M2 exhibiting reduced kinetics and with replication completely abrogated in ERK. Using single round GFP expressing viruses to study proviral gene expression, we observed progressively greater rates of silencing relating to the degree of ESEtat disruption, with WT at 53%, M2 at 69% and ERK at 94%. By stimulating infected cells with each of the latency reversal agents PMA, panobinostat and JQ1, we observed that the dose required to achieve 50% of the maximum signal was lowest in WT, intermediate in M2 and highest in ERK, indicating a progressively higher threshold for reactivation. These results suggest that the ability of silent proviruses to reactivate from latency is variable and that minor differences in the viral sequence can alter the proportion of silenced viruses as well as the threshold required to induce silenced viruses to reactivate and express.HPM is supported by the Medical Research Council, UK (MR/N02043X/1) and the Academy of Medical Sciences (UK). NJN is supported by the British Infection Association and the Medical Research Council (MR/M003515/1). This work was supported by the Evelyn Trust and the Cambridge Clinical Academic Reserve. Work in the laboratory is supported by the Cambridge NIHR Biomedical Research Centre and the MRC

Innovations in the quantitative virus outgrowth assay and its use in clinical trials.

A robust measure of the size of the latent HIV reservoir is essential to quantifying the effect of interventions designed to deplete the pool of reactivatable, replication competent proviruses. In addition to the ability to measure a biologically relevant parameter, any assay designed to be used in a clinical trial needs to be reproducible and scalable. The need to quantify the number of resting CD4+ T cells capable of releasing infectious virus has led to the development of the quantitative viral outgrowth assay (VOA). The assay as originally described has a number of features that limit its scalability for use in clinical trials; however recent developments reducing the time and manpower requirements of the assay, while importantly improving reproducibility mean that it is becoming much more practical for it to enter into more widespread use. This review describes the background to VOA development and the practical issues that they present in utilising them in clinical trials. It describes the innovations that have made their usage more practical and the limitations that still exist

No evidence of ongoing evolution in replication competent latent HIV-1 in a patient followed up for two years.

The persistence of infected T cells harbouring intact HIV proviruses is the barrier to the eradication of HIV. This reservoir is stable over long periods of time despite antiretroviral therapy. There has been controversy on whether low level viral replication is occurring at sanctuary sites periodically reseeding infected cells into the latent reservoir to account its durability. To study viral evolution in a physiologically relevant population of latent viruses, we repeatedly performed virus outgrowth assays on a stably treated HIV positive patient over two years and sequenced the reactivated latent viruses. We sought evidence of increasing sequence pairwise distances with time as evidence of ongoing viral replication. 64 reactivatable latent viral sequences were obtained over 103 weeks. We did not observe an increase in genetic distance of the sequences with the time elapsed between sampling. No evolution could be discerned in these reactivatable latent viruses. Thus, in this patient, the contribution of low-level replication to the maintenance of the latent reservoir detectable in the blood compartment is limited

Recent advances in the compound-oriented and pattern-oriented approaches to the quality control of herbal medicines

The current approaches to the quality control of herbal medicines are either compound-oriented or pattern-oriented, the former targeting specific components with some known chemical properties and the latter targeting all detectable components. The marker approach uses specific chemical compounds with known molecular structures, while the multi-compound approach uses both chemical compounds with known structures and those with partial chemical information e.g. retention times, mass spectra and ultraviolet spectra. Apart from chromatographic techniques, new techniques such as oscillating and electrochemistry fingerprints have been developed for quality control. Chemometric resolution methods are widely used for component deconvolution and data comparison. Pattern recognition techniques are used for authentication of herbal medicines