Viral complementation allows HIV-1 replication without integration

<p>Abstract</p> <p>Background</p> <p>The integration of HIV-1 DNA into cellular chromatin is required for high levels of viral gene expression and for the production of new virions. However, the majority of HIV-1 DNA remains unintegrated and is generally considered a replicative dead-end. A limited amount of early gene expression from unintegrated DNA has been reported, but viral replication does not proceed further in cells which contain only unintegrated DNA. Multiple infection of cells is common, and cells that are productively infected with an integrated provirus frequently also contain unintegrated HIV-1 DNA. Here we examine the influence of an integrated provirus on unintegrated HIV-1 DNA (uDNA).</p> <p>Results</p> <p>We employed reporter viruses and quantitative real time PCR to examine gene expression and virus replication during coinfection with integrating and non-integrating HIV-1. Most cells which contained only uDNA displayed no detected expression from fluorescent reporter genes inserted into early (Rev-independent) and late (Rev-dependent) locations in the HIV-1 genome. Coinfection with an integrated provirus resulted in a several fold increase in the number of cells displaying uDNA early gene expression and efficiently drove uDNA into late gene expression. We found that coinfection generates virions which package and deliver uDNA-derived genomes into cells; in this way uDNA completes its replication cycle by viral complementation. uDNA-derived genomes undergo recombination with the integrated provirus-derived genomes during second round infection.</p> <p>Conclusion</p> <p>This novel mode of retroviral replication allows survival of viruses which would otherwise be lost because of a failure to integrate, amplifies the effective amount of cellular coinfection, increases the replicating HIV-1 gene pool, and enhances the opportunity for diversification through errors of polymerization and recombination.</p

HIV-1 Infection Results in Sphingosine-1-Phosphate Receptor 1 Dysregulation in the Human Thymus

Regeneration of functional naïve T lymphocytes following the onset of human immunodeficiency virus (HIV) infection remains a crucial issue for people living with HIV (PLWH), even when adhering to antiretroviral therapy (ART). Thus far, reports on the impact of HIV-1 infection on the entry of thymic precursors and the egress of functional naïve T lymphocytes to and from the thymus are limited. We examined the impact of HIV-1 on Sphingosine-1-phosphate (S1P) signaling, which governs the egress of functional naïve thymocytes from the thymus to the periphery. Using in vitro experiments with primary human thymocytes and in vivo and ex vivo studies with humanized mice, we show that HIV-1 infection results in upregulation of the expression of S1P receptor 1 (S1PR1) in the human thymus. Intriguingly, this upregulation occurs during intrathymic infection (direct infection of the human thymic implant) as well as systemic infection in humanized mice. Moreover, considering the dysregulation of pro- and anti-inflammatory cytokines in infected thymi, the increased expression of S1PR1 in response to in vitro exposure to Interferon-Beta (IFN-β) and Tumor Necrosis Factor-Alpha (TNF-α) indicates that cytokine dysregulation following HIV infection may contribute to upregulation of S1PR1. Finally, an increased presence of CD3hiCD69− (fully mature) as well as CD3hiCD69+ (less mature) T cells in the spleen during HIV infection in humanized mice, combined with earlier expression of S1PR1 during thymocyte development, suggests that upregulation of S1PR1 may translate to increased or accelerated egress from the thymus. The egress of thymocytes that are not functionally mature from the thymus to peripheral blood and lymphoid organs may have implications for the immune function of PLWH

Genome-Wide Binding Map of the HIV-1 Tat Protein to the Human Genome

The HIV-1 Trans-Activator of Transcription (Tat) protein binds to multiple host cellular factors and greatly enhances the level of transcription of the HIV genome. While Tat's control of viral transcription is well-studied, much less is known about the interaction of Tat with the human genome. Here, we report the genome-wide binding map of Tat to the human genome in Jurkat T cells using chromatin immunoprecipitation combined with next-generation sequencing. Surprisingly, we found that ∼53% of the Tat target regions are within DNA repeat elements, greater than half of which are Alu sequences. The remaining target regions are located in introns and distal intergenic regions; only ∼7% of Tat-bound regions are near transcription start sites (TSS) at gene promoters. Interestingly, Tat binds to promoters of genes that, in Jurkat cells, are bound by the ETS1 transcription factor, the CBP histone acetyltransferase and/or are enriched for histone H3 lysine 4 tri-methylation (H3K4me3) and H3K27me3. Tat binding is associated with genes enriched with functions in T cell biology and immune response. Our data reveal that Tat's interaction with the host genome is more extensive than previously thought, with potentially important implications for the viral life cycle

HIV integration and the establishment of latency in CCL19-treated resting CD4(+) T cells require activation of NF-κB

BACKGROUND: Eradication of HIV cannot be achieved with combination antiretroviral therapy (cART) because of the persistence of long-lived latently infected resting memory CD4(+) T cells. We previously reported that HIV latency could be established in resting CD4(+) T cells in the presence of the chemokine CCL19. To define how CCL19 facilitated the establishment of latent HIV infection, the role of chemokine receptor signalling was explored. RESULTS: In resting CD4(+) T cells, CCL19 induced phosphorylation of RAC-alpha serine/threonine-protein kinase (Akt), nuclear factor kappa B (NF-&kappa;B), extracellular-signal-regulated kinase (ERK) and p38. Inhibition of the phosphoinositol-3-kinase (PI3K) and Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/ERK signalling pathways inhibited HIV integration, without significant reduction in HIV nuclear entry (measured by Alu-LTR and 2-LTR circle qPCR respectively). Inhibiting activation of MEK1/ERK1/2, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1) and NF-&kappa;B, but not p38, also inhibited HIV integration. We also show that HIV integrases interact with Pin1 in CCL19-treated CD4(+) T cells and inhibition of JNK markedly reduced this interaction, suggesting that CCL19 treatment provided sufficient signals to protect HIV integrase from degradation via the proteasome pathway. Infection of CCL19-treated resting CD4(+) T cells with mutant strains of HIV, lacking NF-&kappa;B binding sites in the HIV long terminal repeat (LTR) compared to infection with wild type virus, led to a significant reduction in integration by up to 40-fold (range 1-115.4, p&nbsp;=&nbsp;0.03). This was in contrast to only a modest reduction of 5-fold (range 1.7-11, p&nbsp;&gt;&nbsp;0.05) in fully activated CD4(+) T cells infected with the same mutants. Finally, we demonstrated significant differences in integration sites following HIV infection of unactivated, CCL19-treated, and fully activated CD4(+) T cells. CONCLUSIONS: HIV integration in CCL19-treated resting CD4(+) T cells depends on NF-&kappa;B signalling and increases the stability of HIV integrase, which allow subsequent integration and establishment of latency. These findings have implications for strategies needed to prevent the establishment, and potentially reverse, latent infection

HIV restriction in quiescent CD4+ T cells

Abstract The restriction of the Human Immunodeficiency Virus (HIV) infection in quiescent CD4+ T cells has been an area of active investigation. Early studies have suggested that this T cell subset is refractory to infection by the virus. Subsequently it was demonstrated that quiescent cells could be infected at low levels; nevertheless these observations supported the earlier assertions of debilitating defects in the viral life cycle. This phenomenon raised hopes that identification of the block in quiescent cells could lead to the development of new therapies against HIV. As limiting levels of raw cellular factors such as nucleotides did not account for the block to infection, a number of groups pursued the identification of cellular proteins whose presence or absence may impact the permissiveness of quiescent T cells to HIV infection. A series of studies in the past few years have identified a number of host factors implicated in the block to infection. In this review, we will present the progress made, other avenues of investigation and the potential impact these studies have in the development of more effective therapies against HIV

Differentially Stimulated CD4+ T Cells Display Altered Human Immunodeficiency Virus Infection Kinetics: Implications for the Efficacy of Antiviral Agents▿

The activation state of CD4+ T cells plays a crucial role in the establishment of a productive human immunodeficiency virus infection. Here, we show that T cells stimulated for 1 day demonstrated delayed kinetics of viral reverse transcription and integration compared to cells stimulated for 2 days prior to infection. As a result, the efficiency of reverse transcription and integration inhibitors differs in these differentially stimulated cells. These studies increase our understanding of how T cells support viral replication and provide insight regarding the efficiency of antiretroviral therapy in lymphoid compartments

Immediate Activation Fails To Rescue Efficient Human Immunodeficiency Virus Replication in Quiescent CD4(+) T Cells

Unlike activated T cells, quiescent CD4(+) T cells have shown resistance to human immunodeficiency virus (HIV) infection due to a block in the early events of the viral life cycle. To further investigate the nature of this block, we infected quiescent CD4(+) T cells with HIV-1(NL4-3) and immediately stimulated them. Compared to activated (prestimulated) cells, these poststimulated cells showed slightly decreased viral entry and delays in the completion of reverse transcription. However, the relative efficiency of integration was similar to that of prestimulated cells. Together, this resulted in decreased expression of tat/rev mRNA and synthesis of viral protein. Furthermore, based on cell cycle staining and BrdU incorporation, poststimulated cells expressing viral protein failed to initiate a second round of their cell cycle, independently of Vpr-mediated arrest. Together, these data demonstrate that the early stages of the HIV life cycle are inefficient in these poststimulated cells and that efficient replication cannot be induced by subsequent activation

Noninvasive detection of tumor-infiltrating T cells by PET reporter imaging.

Adoptive transfer of tumor-reactive T cells can successfully reduce tumor burden; however, in rare cases, lethal on-target/off-tumor effects have been reported. A noninvasive method to track engineered cells with high sensitivity and resolution would allow observation of correct cell homing and/or identification of dangerous off-target locations in preclinical and clinical applications. Human deoxycytidine kinase triple mutant (hdCK3mut) is a nonimmunogenic PET reporter that was previously shown to be an effective tool to monitor whole-body hematopoiesis. Here, we engineered a construct in which hdCK3mut is coexpressed with the anti-melanoma T cell receptor F5, introduced this construct into human CD34 cells or PBMCs, and evaluated this approach in multiple immunotherapy models. Expression of hdCK3mut allowed engrafted cells to be visualized within recipient bone marrow, while accumulation of [18F]-L-FMAU in hdCK3mut-expressing T cells permitted detection of intratumoral homing. Animals that received T cells coexpressing hdCK3mut and the anti-melanoma T cell receptor had demonstrably higher signals in HLA-matched tumors compared with those in animals that received cells solely expressing hdCK3mut. Engineered T cells caused cytotoxicity in HLA/antigen-matched tumors and induced IFN-γ production and activation. Moreover, hdCK3mut permitted simultaneous monitoring of engraftment and tumor infiltration, without affecting T cell function. Our findings suggest that hdCK3mut reporter imaging can be applied in clinical immunotherapies for whole-body detection of engineered cell locations