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

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

A method to estimate the efficiency of gene expression from an integrated retroviral vector

BACKGROUND: Proviral gene expression is a critical step in the retroviral life cycle and an important determinant in the efficiency of retrovirus based gene therapy vectors. There is as yet no method described that can assess the efficiency of proviral gene expression while vigorously excluding the contribution from unstable species such as passively transferred plasmid and LTR circles. Here, we present a method that can achieve this. RESULTS: Proviral gene expression was detected by the activity of the puromycin resistance gene encoded in the viral vector, and quantified by comparing the growth curve of the sample under puromycin selection to that of a series of calibration cultures. Reproducible estimates of the efficiency of proviral gene expression could be derived. We confirm that contamination from unstable species such as passively transferred plasmid used in viral vector production and unintegrated viral DNA can seriously confound estimates of the efficiency of transduction. This can be overcome using a PCR based on limiting dilution analysis. CONCLUSION: A simple, low cost method was developed that should be useful in studying the biology of retroviruses and for the development of expression systems for retrovirus based gene therapy

Waking Up the Sleepers: HIV Latency and Reactivation

In a patient infected with HIV-1, the presence of latently infected cells from which the virus can be reactivated and rekindle HIV infection in the patient necessitates lifelong administration of antiretroviral treatment. The biology of HIV latency and viral silencing is now becoming clearer at a molecular and cellular level. However, our understanding of HIV-1 latency in vivo is still inadequate. Attempts to therapeutically reactivate the virus in infected patients have yielded disappointing results. This article reviews the research and clinical findings and discusses current thinking on the subject of HIV latency and reactivation