An emerging and variant viral promoter of HIV-1 subtype C exhibits low-level gene expression noise

Abstract

Background We observe the emergence of several promoter-variant viral strains in India during recent years. The variant viral promoters contain additional copies of transcription factor binding sites present in the viral modulatory region or enhancer, including RBEIII, LEF-1, Ap-1 and/or NF-κB. These sites are crucial for governing viral gene expression and latency. Here, we infer that one variant viral promoter R2N3-LTR containing two copies of RBF-2 binding sites (an RBEIII site duplication) and three copies of NF-κB motifs may demonstrate low levels of gene expression noise as compared to the canonical RN3-LTR or a different variant R2N4-LTR (a duplication of an RBEIII site and an NF-κB motif). To demonstrate this, we constructed a panel of sub-genomic viral vectors of promoter-variant LTRs co-expressing two reporter proteins (mScarlet and Gaussia luciferase) under the dual-control of Tat and Rev. We established stable pools of CEM.NKR-CCR5 cells (CEM-CCR5RL reporter cells) and evaluated reporter gene expression under different conditions of cell activation. Results The R2N3-LTR established stringent latency that was highly resistant to reversal by potent cell activators such as TNF-α or PMA, or even to a cocktail of activators, compared to the canonical RN3- or the variant R2N4-LTR. The R2N3-LTR exhibited low-level basal gene expression in the absence of cell activation that enhanced marginally but significantly when activated. In the presence of Tat and Rev, trans-complemented in the form of an infectious virus, the R2N3-LTR demonstrated gene expression at levels comparable to the wild-type viral promoter. The R2N3-LTR is responsive to Tat and Rev factors derived from viral strains representing diverse genetic subtypes. Conclusion With extremely low-level transcriptional noise, the R2N3-LTR can serve as an excellent model to examine the establishment, maintenance, and reversal of HIV-1 latency. The R2N3-LTR would also be an ideal viral promoter to develop high-throughput screening assays to identify potent latency-reversing agents since the LTR is not affected by the usual background noise of the cell.

The nuclear pore protein NUP98 impedes LTR-driven basal gene expression of HIV-1, viral propagation, and infectivity

“…The plasmids including the HIV-1 molecular clone pNL4.3 and pHEF VSV-G were a kind gift from Dr. Udaykumar Ranga (Jawaharlal Nehru Centre for Advanced Scientific and Research, Bangalore, India) ( 47 ). The plasmids pLTR-Luc and pIndie-C1 were a kind gift from Dr. Debashis Mitra (National Centre for Cell Science, Pune, India) ( 48 – 52 ).…”

HIV-1 subtypes and latent reservoirs

“…At least in the context of HIV-1C, the RBEIII motif duplication leading to stronger viral latency has been demonstrated [31]. The variant R2N3-LTR (containing two and three copies of the RBEIII and NF-κB motifs, respectively) demonstrated significantly higher levels of resistance to latency reversal than the RN3-LTR (the canonical viral promoter) when the cells harboring a reporter virus were induced with a cocktail of activators.…”