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

• Published in: Retrovirology Vol. 18; no. 1; p. 27
• Main Authors: Ali, Haider, Bhange, Disha, Mehta, Kavita, Gohil, Yuvrajsinh, Prajapati, Harshit Kumar, Byrareddy, Siddappa N, Buch, Shilpa, Ranga, Udaykumar
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 19.09.2021; BioMed Central; BMC
• Subjects: Activator protein 1; Binding Sites; CCR5 protein; Cell activation; Comparative analysis; Gene expression; Gene Expression Regulation, Viral; Genes; Genes, Reporter; Genetic diversity; Genetic transcription; Genetic Variation; High-throughput screening; HIV; HIV (Viruses); HIV Infections - virology; HIV Long Terminal Repeat; HIV-1; HIV-1 - classification; HIV-1 - genetics; HIV-1 - isolation & purification; HIV-1 - physiology; Human immunodeficiency virus; Humans; Latency; Latency reversal; LTR; NF-kappa B; NF-κB protein; Promoter Regions, Genetic; Proteins; Reporter gene; rev Gene Products, Human Immunodeficiency Virus - genetics; rev Gene Products, Human Immunodeficiency Virus - metabolism; Sequence duplication; Strains (organisms); Subtype C; tat Gene Products, Human Immunodeficiency Virus - genetics; tat Gene Products, Human Immunodeficiency Virus - metabolism; Tat protein; Transcription factors; Transcription, Genetic; Transcriptional silence; Tumor necrosis factor-α; Virus Replication; Missouri; India; HIV-1; Latency reversal; LTR; Transcriptional silence; Sequence duplication; Subtype C
• ISSN: 1742-4690; 1742-4690
• DOI: 10.1186/s12977-021-00572-2

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-CCR5 reporter cells) and evaluated reporter gene expression under different conditions of cell activation. 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. 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.