Integrase inhibitor reversal dynamics indicate unintegrated HIV-1 dna initiate de novo integration

Thierry, Sylvain; Munir, Soundasse; Thierry, Eloïse; Subra, Frédéric; Leh, Hervé; Zamborlini, Alessia; Saenz, Dyana T.; Levy, David; Lesbats, Paul; Saı̈b, Ali; Parissi, Vincent; Poeschla, Eric M.; Deprez, Eric; Delelis, Olivier

doi:10.1186/s12977-015-0153-9

Cited by 38 publications

(49 citation statements)

References 32 publications

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“…ϩ T cells (58)(59)(60)(61). To exclude the possibility that some forms of this unintegrated HIV-1 DNA become integrated upon T cell activation, resulting in productive infection and virus particle production, we next assessed the contribution of unintegrated HIV-1 LAI DNA to extracellular vRNA production from both T N and T CM cells by including the integrase inhibitor raltegravir (RAL) at the same time as anti-CD3/CD28 (Fig.…”

Section: Resting Cd4mentioning

confidence: 99%

Establishment and Reversal of HIV-1 Latency in Naive and Central Memory CD4⁺T CellsIn Vitro

Zerbato

Serrao

Lenzi

et al. 2016

J Virol

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The latent HIV-1 reservoir primarily resides in resting CD4؉ T cells which are a heterogeneous population composed of both naive (T N ) and memory cells. In HIV-1-infected individuals, viral DNA has been detected in both naive and memory CD4 ؉ T cell subsets although the frequency of HIV-1 DNA is typically higher in memory cells, particularly in the central memory (T CM ) cell subset. T N and T CM cells are distinct cell populations distinguished by many phenotypic and physiological differences. In this study, we used a primary cell model of HIV-1 latency that utilizes direct infection of highly purified T N and T CM cells to address differences in the establishment and reversal of HIV-1 latency. Consistent with what is seen in vivo, we found that HIV-1 infected T N cells less efficiently than T CM cells. However, when the infected T N cells were treated with latency-reversing agents, including anti-CD3/CD28 antibodies, phorbol myristate acetate/phytohemagglutinin, and prostratin, as much (if not more) extracellular virion-associated HIV-1 RNA was produced per infected T N cell as per infected T CM cell. There were no major differences in the genomic distribution of HIV-1 integration sites between T N and T CM cells that accounted for these observed differences. We observed decay of the latent HIV-1 cells in both T cell subsets after exposure to each of the latency-reversing agents. Collectively, these data highlight significant differences in the establishment and reversal of HIV-1 latency in T N and T CM CD4؉ T cells and suggest that each subset should be independently studied in preclinical and clinical studies. IMPORTANCE The latent HIV-1 reservoir is frequently described as residing within resting memory CD4؉ T cells. This is largely due to the consistent finding that memory CD4 ؉ T cells, specifically the central (T CM )

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Section: Resting Cd4mentioning

confidence: 99%

Establishment and Reversal of HIV-1 Latency in Naive and Central Memory CD4⁺T CellsIn Vitro

Zerbato

Serrao

Lenzi

et al. 2016

J Virol

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“…Interestingly, RNA transcription from unintegrated HIV DNA circles has been reported1516171819. It has been shown that episomal HIV DNA can lead to the production of HIV proteins and may even generate fully replication competent viruses202122. However, the implications of transcription from 2-LTR circles, especially in the context of HIV latency, have remained underestimated and incompletely understood1923.…”

mentioning

confidence: 99%

“…However, this is based on the assumption that unintegrated HIV DNA forms do not contribute to the production of viral transcription and protein production. It is already well known that 2-LTRs accumulate in the presence of INSTI in active infection as well as in treatment intensifications studies in vivo 1720212425.…”

mentioning

confidence: 99%

Protein expression from unintegrated HIV-1 DNA introduces bias in primary in vitro post-integration latency models

Bonczkowski

Scheerder

Malatinková

et al. 2016

Sci Rep

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To understand the persistence of latently HIV-1 infected cells in virally suppressed infected patients, a number of in vitro models of HIV latency have been developed. In an attempt to mimic the in vivo situation as closely as possible, several models use primary cells and replication-competent viruses in combination with antiretroviral compounds to prevent ongoing replication. Latency is subsequently measured by HIV RNA and/or protein production after cellular activation. To discriminate between pre- and post-integration latency, integrase inhibitors are routinely used, preventing novel integrations upon cellular activation. Here, we show that this choice of antiretrovirals may still cause a bias of pre-integration latency in these models, as unintegrated HIV DNA can form and directly contribute to the levels of HIV RNA and protein production. We further show that the addition of reverse transcriptase inhibitors effectively suppresses the levels of episomal HIV DNA (as measured by 2-LTR circles) and decreases the levels of HIV transcription. Consequently, we show that latency levels described in models that only use integrase inhibitors may be overestimated. The inclusion of additional control conditions, such as 2-LTR quantification and the addition of reverse transcriptase inhibitors, is crucial to fully elucidate the actual levels of post-integration latency.

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“…However, Thierry et al recently provided new insight into the role of 2-LTRs for the viral replication cycle; where accumulated 2-LTR forms by RAL treatment in vitro could be converted to linear forms by RAL withdrawal, which were then integrated (Thierry et al, 2015). Although our observations that accumulation of cHIV DNAs by INSTIs were especially emphasized under cell-to-cell transmission, they suggest a cryptic role for unintegrated cHIV DNA in viral replication.…”

Section: Discussionmentioning

confidence: 59%

Impact of HIV-1 infection pathways on susceptibility to antiviral drugs and on virus spread

et al. 2015

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The infection routes of HIV-1 can affect several viral properties, including dissemination, pathogenesis, and immune evasion. In this study, we evaluated the inhibitory activity of a wide variety of anti-HIV drugs, focusing on the impact that different infection pathways have on their efficacy. Compared to cell-free infection, inhibitory activities were reduced in cell-to-cell productive transmission for all drugs tested. We detected weak reporter-expressing target cells after cell-to-cell transmission in the presence of integrase strand transfer inhibitors (INSTIs). Further analysis revealed that this expression was mainly due to unintegrated circular HIV (cHIV) DNAs, consisting of 1-LTR and 2-LTR circles. When in vitro-constructed cHIV DNAs were introduced into cells, the production of infectious and intercellular transmittable virions was observed, suggesting that cHIV DNA could be a source of infectious virus. These results highlight some advantages of the cell-to-cell infection mode for viral expansion, particularly in the presence of anti-retroviral drugs.

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Integrase inhibitor reversal dynamics indicate unintegrated HIV-1 dna initiate de novo integration

Cited by 38 publications

References 32 publications

Establishment and Reversal of HIV-1 Latency in Naive and Central Memory CD4⁺T CellsIn Vitro

Establishment and Reversal of HIV-1 Latency in Naive and Central Memory CD4⁺T CellsIn Vitro

Protein expression from unintegrated HIV-1 DNA introduces bias in primary in vitro post-integration latency models

Impact of HIV-1 infection pathways on susceptibility to antiviral drugs and on virus spread

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Integrase inhibitor reversal dynamics indicate unintegrated HIV-1 dna initiate de novo integration

Cited by 38 publications

References 32 publications

Establishment and Reversal of HIV-1 Latency in Naive and Central Memory CD4+T CellsIn Vitro

Establishment and Reversal of HIV-1 Latency in Naive and Central Memory CD4+T CellsIn Vitro

Protein expression from unintegrated HIV-1 DNA introduces bias in primary in vitro post-integration latency models

Impact of HIV-1 infection pathways on susceptibility to antiviral drugs and on virus spread

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Establishment and Reversal of HIV-1 Latency in Naive and Central Memory CD4⁺T CellsIn Vitro

Establishment and Reversal of HIV-1 Latency in Naive and Central Memory CD4⁺T CellsIn Vitro