Table 1 Models addressing the basal (host-driven) HIV gene-expression activity

Basal HIV Gene-Expression

A. Singh et al.²⁷

Understand the mechanisms underlying basal HIV gene-expression dynamics.

Flow cytometry analysis of GFP expression, sampled from 30 Jurkat T-cell iso-clones infected with a single LTR-GFP HIV model vector with diverse IS.

(i) Provirus-specific basal HIV gene-expression is highly variable (noisy); (ii) The observed variability is poorly explained by the constitutive LTR model. Instead, it is well reproduced by a two-state (on/off) random telegraph model underlying random bursting activity; (iii) During host phase, the LTR produces bursts with average size of 2–10 mRNA.

A. Singh et al.⁷⁵

Understand which viral gene-expression model (constitutive vs bursting) prevails during the host-phase

Flow cytometry analysis of GFP expression, sampled from Jurkat T-cells iso-clones infected with a single LTR-GFP and LTR-mCh HIV model vector with diverse IS.

(i) promoter toggling between an active and inactive state is the main source of noise in basal HIV gene-expression.

R. D. Dar et al.⁴⁵

Understand what viral gene-expression model (i.e., constitutive, bursting) better explains provirus-specific basal gene-expression activity.

Flow cytometry analysis of GFP expression, sampled from 8000 Jurkat T-cells iso-clones infected with a single LTR-GFP HIV model integrated in a different genomic locus.

(i) Promoter toggling between an active and inactive state is the main source of noise in basal HIV gene-expression; (ii) The MME at the provirus IS modulates both burst frequency and size; (iii) Below an average gene-expression level, MMEs modulates only bursting frequency, whereas above such threshold, MMEs regulate only burst size; (iv) Transcriptional activators (i.e., TSA, TNF-α) regulate burst frequency and size along a provirus-specific constrained region.

K. Tantale et al.²⁸

Understand the mechanisms underlying basal HIV transcription, and the host factors modulating those mechanisms.

Single-molecule RNA fluorescence in situ hybridization (smFISH) analysis of isogenic MCP-GFP-expressing HeLa Flp-in H9 cells infected with an MS2-labeled HIV model vector in the same IS, high Tat production.

(i) HIV mRNA is produced by closely-spaced RNAPII convoys allowed by a Mediator-driven mechanism; (ii) RNAPII convoys are spaced by ~hundred nucleotides due to DNA torsional stress; (iii) The LTR displays stochastic bursting activity on two time scales, referred to as multi-scale bursting: The first underlie the rate of RNAPII convoys, the second the LTR activation rate.

K. Tantale et al.²⁹

Understand how RNAPII pausing regulate basal transcription.

smFISH analysis of three distinct isogenic MCP-GFP-expressing HeLa Flp-in H9 cell lines infected with an 128xMS2-labeled HIV model vector, characterized by low, medium, and high Tat expression.

(I) RNAPII enter a long-lived pause (>20 min) in silent LTR, limiting viral transcription; (ii) RNAPII pausing is not obligatory but stochastic. Only a small fraction of RNAPII undergo long-lived pausing in basal regime;