HIV-1 latency and virus production from unintegrated genomes following direct infection of resting CD4 T cells

Abstract

BackgroundHIV-1 integration is prone to a high rate of failure, resulting in the accumulation of unintegrated viral genomes (uDNA) in vivo and in vitro. uDNA can be transcriptionally active, and circularized uDNA genomes are biochemically stable in non-proliferating cells. Resting, non-proliferating CD4 T cells are prime targets of HIV-1 infection and latently infected resting CD4 T cells are the major barrier to HIV cure. Our prior studies demonstrated that uDNA generates infectious virions when T cell activation follows rather than precedes infection.ResultsHere, we characterize in primary resting CD4 T cells the dynamics of integrated and unintegrated virus expression, genome persistence and sensitivity to latency reversing agents. Unintegrated HIV-1 was abundant in directly infected resting CD4 T cells. Maximal gene expression from uDNA was delayed compared with integrated HIV-1 and was less toxic, resulting in uDNA enrichment over time relative to integrated proviruses. Inhibiting integration with raltegravir shunted the generation of durable latency from integrated to unintegrated genomes. Latent uDNA was activated to de novo virus production by latency reversing agents that also activated latent integrated proviruses, including PKC activators, histone deacetylase inhibitors and P-TEFb agonists. However, uDNA responses displayed a wider dynamic range, indicating differential regulation of expression relative to integrated proviruses. Similar to what has recently been demonstrated for latent integrated proviruses, one or two applications of latency reversing agents failed to activate all latent unintegrated genomes. Unlike integrated proviruses, uDNA gene expression did not down modulate expression of HLA Class I on resting CD4 T cells. uDNA did, however, efficiently prime infected cells for killing by HIV-1-specific cytotoxic T cells.ConclusionsThese studies demonstrate that contributions by unintegrated genomes to HIV-1 gene expression, virus production, latency and immune responses are inherent properties of the direct infection of resting CD4 T cells. Experimental models of HIV-1 latency employing directly infected resting CD4 T cells should calibrate the contribution of unintegrated HIV-1.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0234-9) contains supplementary material, which is available to authorized users.

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

“…Integrase inhibitors effectively suppress the integration of these new HIV DNA forms, but cannot prevent the production of viral proteins derived from the episomal HIV DNA forms which accumulate due to the blockage of viral integration. Our data are supported by recent reports that also suggest a contribution of unintegrated DNA to viral gene expression in resting CD4+ T cells1923.…”

“…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.…”

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

“…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.…”

Targeting the Latent Reservoir for HIV-1

“…Thus monocytes cannot serve as a long-lived reservoir. Tissue-resident macrophages derived from monocytes have a relatively short half-life on the order of weeks, depending on the tissue (Ginhoux & Guilliams 2016), while those derived from embryonic hematopoetic precursors have a longer half-life of 24–36 months (Kandathil et al 2016). Integrated proviral DNA has been observed in brain astrocytes and microglia (Churchill et al 2006), which have half-lives on the order of months and years, respectively (Marban et al 2016).…”