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

Abstract

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.

HIV signaling through CD4 and CCR5 activates Rho family GTPases that are required for optimal infection of primary CD4+ T cells

“…Parallel plates were prepared for analysis of viral fusion and LTR-driven EGFP expression as previously described [24]. Following incubation with small molecule inhibitors, cells were infected with HIV-1 reporter virus strain NL4-3-deltaE-EGFP (obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pNL4-3-delta-E-EGFP (Cat #11100) from Drs.…”

“…To achieve this, we selected a commercially available panel of small molecule inhibitors tailored to enzymes involved in epigenetic and PTM pathways and analyzed them using a combination reporter virus system that measures both viral fusion and LTR-driven EGFP expression, which requires not only fusion but also post-entry steps including uncoating, reverse transcription, nuclear import, integration, Tat-dependent transcription and Rev-dependent mRNA export, and translation to occur successfully [24]. While the majority of compounds had no effect at the level of fusion, 39 compounds yielded greater than 0.5 log 2 fold change in viral infection as measured by LTR-driven EGFP expression.…”

The Histone Deacetylase Inhibitor Vorinostat (SAHA) Increases the Susceptibility of Uninfected CD4 + T Cells to HIV by Increasing the Kinetics and Efficiency of Postentry Viral Events

“…We reasoned that spinoculation might mask the effects of vorinostat on fusion and therefore repeated the experiment in the absence of spinoculation. A 10-to 15-fold-higher viral inoculum was used in these studies, as fusion is significantly diminished in the absence of spinoculation in the combination reporter virus assay (31). Again, no enhancement of viral fusion was observed for reporter viruses pseudotyped with either R5-or X4-tropic Envs (Fig.…”

“…Combination reporter viruses were produced as previously detailed (31). Briefly, 293 T cells were transfected with 10 g pNL4-3-delta-E-enhanced green fluorescent protein (EGFP) (obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH [catalog number 11100] from Haili Zhang, Yan Zhou, and Robert Siliciano), 7.5 g virion-associated ␤-lactamaseVpr (bla-Vpr) plasmid, and 6.0 g of HIV Env REJO.D12.1972 (32) (CCR5 tropic) or JOTO.TA1.2247 (33) (CXCR4 tropic), using calcium phosphate methods.…”

CD4 + Memory Stem Cells Are Infected by HIV-1 in a Manner Regulated in Part by SAMHD1 Expression

“…We developed an HIV reporter virus system that allows measurement of both viral fusion and LTR-driven gene expression, providing readouts of two stages of the viral life cycle using a single viral construct ( Fig. 1A) (21). Briefly, virions that incorporate Bla-Vpr protein and package the HIV genome containing the egfp-env gene are produced.…”