Recent spread of the promoter variant (4-κB) Human immunodeficiency virus-1 clade C (HIV-1C) strain is attributed to duplication of the Nuclear Factor Kappa B (NF-κB) binding sites and potential increased heroin consumption in India. To study the underlying biology of 4-κB HIV-1C in rhesus macaques, we engineered a promoter-chimera variant (4NF-κB) Simian Human Immunodeficiency Virus (SHIV) by substituting the HIV-1C Long Terminal Repeat (LTR) region consisting of 4 NF-κB and 3 Sp-1 sites with the corresponding segment in the LTR of SHIV AD8EO. The wild-type (3NF-κB) promoter-chimera SHIV was generated by inactivating the 5 ′ proximal NF-κB binding site in SHIV 4NF-κB. CD8-depleted rhesus macaque PBMCs (RM-PBMCs) were infected with the promoter-chimera and AD8EO SHIVs to determine the effects of opioid-exposure on inflammation, NF-κB activation, neurotoxicity in neuronal cells and viral replication. Morphine-exposure of RM-PBMCs infected with SHIVs 4NF-κB, 3NF-κB, and AD8EO altered cellular transcript levels of monocyte chemoattractant protein 1, interleukin 6, interleukin 1β, and Tumor Necrosis Factor α. Of note, divergent alteration of the cytokine transcript levels was observed with these promoter-chimera wild-type and variant SHIVs. NF-κB activation was observed during infection of all three SHIVs with morphine-exposure. Finally, we observed that SHIV AD8EO infection and exposure to both morphine and naloxone had the greatest impact on the neurotoxicity. The promoter-chimera SHIV 4NF-κB and SHIV 3NF-κB did not have a similar effect on neurotoxicity as compared to SHIV AD8EO. All SHIVs replicated efficiently at comparable levels in RM-PBMCs and morphine-exposure did not alter viral replication kinetics. Future in vivo studies in rhesus macaques will provide greater understanding of 4-κB HIV-1C viral immunopathogenesis and onset of disease in the central nervous system during morphine-exposure.
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.
In the present study, conservation status of important vascular flora found in Kalam valley was assessed. Kalam Valley represents the extreme northern part of Swat District in KPK Province of Pakistan. The valley contains some of the precious medicinal plants. 245 plant species which were assessed for conservation studies revealed that 10.20% (25 species) were found to be endangered, 28.16% (69 species) appeared to be vulnerable. Similarly, 50.6% (124 species) were rare, 8.16% (20 species) were infrequent and 2.9% (7 species) were recognized as dominant. It was concluded that Kalam Valley inhabits most important plants majority of which are used in medicines; but due to anthropogenic activities including unplanned tourism, deforestation, uprooting of medicinal plants and over grazing, majority of these plant species are rapidly heading towards regional extinction in the near future. To maintain the biodiversity of the study area, some in-situ and ex-situ conservation measures in the form of protected areas, sustainable grazing, supplying alternative energy sources to the native population, seeds preservation and growing precious medicinal plant species in nurseries established under the supervision of forest department are urgently needed.
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