Tailored HIV-1 Vectors for Genetic Modification of Primary Human Dendritic Cells and Monocytes

Durand, Stéphanie; Nguyen, Xuan-Nhi; Turpin, Jocelyn; Cordeil, Stéphanie; Nazaret, Nicolas; Croze, Séverine; Mahieux, Renaud; Lachuer, Joël; Legras-Lachuer, Catherine; Cimarelli, Andrea

doi:10.1128/jvi.01459-12

Cited by 15 publications

(16 citation statements)

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“…The plasmids pCMVdR8.2, the VSV-G envelope expressing pMD.G plasmid and the pTY2-CMV-GFP-W transfer vector have been already described [ 87 - 89 ]. For construction of SIV-Vpx expressing plasmid, Vpx derived from the SIVMAC251 strain was modified [ 90 ] and 10 μg of this plasmid were included in the transfection. 293 T cells were transiently transfected with the calcium phosphate-based protocol using the Calcium Phosphate-based Profection Mammalian Transfection System (Promega Corporation) and the supernatants were clarified and concentrated by ultracentrifugation as previously described [ 88 ].…”

Section: Methodsmentioning

confidence: 99%

Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation

et al. 2015

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BackgroundMacrophages are key targets of HIV-1 infection. We have previously described that the expression of CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is further up-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication in infected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibits HIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restriction factors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members.ResultsCCL2 neutralization potently reduced the number of p24 Gag+ cells during the course of either productive or single cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thus demonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viral DNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of the modulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression, to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replication mediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was type I IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expression revealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in the defence response to viruses.ConclusionsNeutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primary MDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2 blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which might contribute to regulate the expression of innate intracellular viral antagonists in vivo. Thus, our study may potentially lead to the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0132-6) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation

et al. 2015

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“…The plasmid HIV-1ΔEnv IN HA (D116A)ΔNef was obtained by insertional mutagenesis using the QuikChange II XL Site-Directed Mutagenesis kit (Agilent). HIV-1 viruses were produced by cotransfection with calcium phosphate with 10 µg HIV-1 LAI (BRU) (or NL4.3) ΔEnv Virus (NIH) or with the modified versions HIV-1ΔEnvIN HA or HIV-1ΔEnv IN HA (D116A)ΔNef in combination with 1 µg of VSV-G envelope expression plasmid pHCMV-G (VSV-G) and 3 µg of SIV MAC Vpx (Durand et al, 2013). The viruses collected from 293T cells 48 h post-transfection were ultracentrifuged at 4 ºC for 1h at 22,000 rpm.…”

Section: Plasmids and Viral Productionmentioning

confidence: 99%

Clustering and reverse transcription of HIV-1 genomes in nuclear niches of macrophages

Rensen

Mueller

Scoca

et al. 2020

Preprint

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In order to replicate, the Human Immunodeficiency Virus (HIV-1) reverse transcribes its RNA genome into DNA, which subsequently integrates into host cell chromosomes.These two key events of the viral life cycle are commonly viewed as separate not only in time but also in cellular space, since reverse transcription (RT) is thought to be completed in the cytoplasm before nuclear import and integration. However, the spatiotemporal organization of the early replication cycle in macrophages, natural nondividing target cells that constitute reservoirs of HIV-1 and an obstacle to curing AIDS, remains unclear. Here, we demonstrate that infected macrophages display large nuclear foci of viral DNA and viral RNA, in which multiple genomes cluster together.These clusters form in the absence of chromosomal integration, sequester the paraspeckle protein CPSF6 and localize to nuclear speckles. Strikingly, we show that viral RNA clusters consist mostly of genomic, incoming RNA, both in cells where RT is pharmacologically suppressed and in untreated cells. We demonstrate that, after temporary inhibition of RT, RT can resume in the nucleus and lead to vDNA accumulation in these clusters. We further show that nuclear RT can result in transcription competent viral DNA. These findings change our understanding of the early HIV-1 replication cycle, and may have implications for understanding HIV-1 persistence.

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“…Membrane fusion marks the end of the HIV entry process and the beginning of a series of post-entry events that must occur successfully for progeny viruses to be produced (Figure 1). Identifying host and viral proteins that modulate post-entry efficiency is critical to developing strategies to restrict HIV replication (Busschots et al, 2009) and improve transduction efficiency of HIV-based gene therapy vectors (Bobadilla et al, 2012; Durand et al, 2012). Not all cells that fuse with HIV progress to productive infection; many cells fail to express viral RNAs and proteins.…”

Section: Introductionmentioning

confidence: 99%

A combination HIV reporter virus system for measuring post-entry event efficiency and viral outcome in primary CD4+ T cell subsets

Tilton

Tabler

Lucera

et al. 2014

Journal of Virological Methods

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Fusion between the viral membrane of human immunodeficiency virus (HIV) and the host cell marks the end of the HIV entry process and the beginning of a series of post-entry events including uncoating, reverse transcription, integration, and viral gene expression. The efficiency of post-entry events can be modulated by cellular factors including viral restriction factors and can lead to several distinct outcomes: productive, latent, or abortive infection. Understanding host and viral proteins impacting post-entry event efficiency and viral outcome is critical for strategies to reduce HIV infectivity and to optimize transduction of HIV-based gene therapy vectors. Here, we report a combination reporter virus system measuring both membrane fusion and viral promoter-driven gene expression. This system enables precise determination of unstimulated primary CD4+ T cell subsets targeted by HIV, the efficiency of post-entry viral events, and viral outcome and is compatible with high-throughput screening and cell-sorting methods.

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Tailored HIV-1 Vectors for Genetic Modification of Primary Human Dendritic Cells and Monocytes

Cited by 15 publications

References 50 publications

Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation

Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation

Clustering and reverse transcription of HIV-1 genomes in nuclear niches of macrophages

A combination HIV reporter virus system for measuring post-entry event efficiency and viral outcome in primary CD4+ T cell subsets

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