Stable Transcriptional Repression and Parasitism of HIV-1

Surya Shrivastava,Paige Charlins,Amanda Ackley,Heather Embree,Boro Dropulic,Ramesh Akkina,Marc S Weinberg,Kevin V Morris

doi:10.1016/j.omtn.2018.04.011

Abstract

Gene-based therapies represent a promising treatment for HIV-1 infection, as they offer the potential for sustained viral inhibition and reduced treatment interventions. One approach developed here involves using conditionally replicating vectors (CR-vectors). CR-vectors utilize HIV-expressed proteins to replicate and disseminate along with HIV into the budding viral particles, thereby co-infecting target cellular reservoirs. We generated and characterized several CR-vectors carrying various therapeutic payloads of non-coding RNAs targeted to HIV-1, both transcriptionally and post-transcriptionally. Both virus and vector expression was followed in cell culture systems and T cells in the presence and absence of mycophenolic acid (MPA) selection. We find here that CR-vectors functionally suppress HIV expression in a long-term stable manner and that transcriptional targeting of and epigenetic silencing of HIV can be passaged to newly infected cells by the action of the CR-vector, ultimately establishing a sustained parasitism of HIV. Our findings suggest that CR-vectors with modulatory non-coding RNAs may be a viable approach to achieving long-term sustained suppression of HIV-1, leading ultimately to a functional cure.

Highlights

The CR-vectors generated and tested here are based on HIV-1 and contain shRNAs targeted to the LTR362 site in the LTR5,12–14 and the Tat/Rev transcript.[21]
The long terminal repeat (LTR)-362 shRNA target site is a locus in the LTR of HIV-1 containing a unique nuclear factor kB (NF-kB) doublet, not found elsewhere in the human genome, which has been observed
The pMoHIV CR-vectors, containing the sh[362] target site in the LTR, appeared to exhibit an increased vector packaging potential relative to virus compared to pD362 CR-vectors, lacking the sh[362] target site in the LTR (Figures 1F, 1G, and S3), presumably because the 362 site is an NF-kB doublet involved in viral transcription[19] and may facilitate packaging of the CR-vectors containing the NF-kB doublet

Summary

Introduction

The pathological effect on viral-infected cells stems from the virus itself replicating and interacting with the infected cell. The virus was found to elude the selective pressure of the CR-vectors due to A/ G mutations, suggesting that CR-vectors containing smaller therapeutic payloads may avoid A-G mutations and prove more fit at longterm stable suppression of HIV,[7] as has been predicted previously.[1]

Methods

Results

Conclusion