amplification-near-full-length-hiv-1-proviruses-for-next-generation

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

University of Sydney

Effect of ipilimumab on the HIV reservoir in an HIV-infected individual with metastatic melanoma.

The 'shock and kill' approach to cure human immunodeficiency virus (HIV) includes transcriptional induction of latent HIV-1 proviruses using latency-reversing agents (LRAs) with targeted immunotherapy to purge infected cells. The administration of LRAs (panobinostat or vorinostat) to HIV-1-infected individuals on antiretroviral therapy induces a significant increase in cell-associated unspliced (CA-US) HIV-1 RNA from CD4(+) T cells. However, it is important to discern whether the increases in CA-US HIV-1 RNA are due to limited or broad activation of HIV-1 proviruses. Here we use single-genome sequencing to find that the RNA transcripts observed following LRA administration are genetically diverse, indicating activation of transcription from an extensive range of proviruses. Defective sequences are more frequently found in CA HIV-1 RNA than in HIV-1 DNA, which has implications for developing an accurate measure of HIV-1 reservoir size. Our findings provide insights into the effects of panobinostat and vorinostat as LRAs for latent HIV-1.

Broad activation of latent HIV-1 in vivo.

To investigate the origin of the HIV-1 viremia induced by the latency-reversing agent romidepsin.

Romidepsin-induced HIV-1 viremia during effective antiretroviral therapy contains identical viral sequences with few deleterious mutations.

To study the effects of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy (ART) with Sezary syndrome, a rare malignancy of CD4 T cells.

Impact of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy with Sezary syndrome.

Latent replication-competent HIV-1 persists in individuals on long-term antiretroviral therapy (ART). We developed the Full-Length Individual Proviral Sequencing (FLIPS) assay to determine the distribution of latent replication-competent HIV-1 within memory CD4 T cell subsets in six individuals on long-term ART. FLIPS is an efficient, high-throughput assay that amplifies and sequences near full-length (∼9 kb) HIV-1 proviral genomes and determines potential replication competency through genetic characterization. FLIPS provides a genome-scale perspective that addresses the limitations of other methods that also genetically characterize the latent reservoir. Using FLIPS, we identified 5% of proviruses as intact and potentially replication competent. Intact proviruses were unequally distributed between T cell subsets, with effector memory cells containing the largest proportion of genetically intact HIV-1 proviruses. We identified multiple identical intact proviruses, suggesting a role for cellular proliferation in the maintenance of the latent HIV-1 reservoir.

Article	Year
Effect of ipilimumab on the HIV reservoir in an HIV-infected individual with metastatic melanoma. AIDS (London, England)\| PubMed ID: 25628259	2015
Broad activation of latent HIV-1 in vivo. Nature communications\| PubMed ID: 27605062	2016
Romidepsin-induced HIV-1 viremia during effective antiretroviral therapy contains identical viral sequences with few deleterious mutations. AIDS (London, England)\| PubMed ID: 28272134	2017
Impact of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy with Sezary syndrome. AIDS (London, England)\| PubMed ID: 28514279	2017
Identification of Genetically Intact HIV-1 Proviruses in Specific CD4 T Cells from Effectively Treated Participants. Cell reports\| PubMed ID: 29045846	2017

Bonnie Hiener

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