Development of a pseudovirus assay and evaluation to screen natural products for inhibition of HIV-1 subtype C reverse transcriptase

Lufuno Grace Mavhandu; Hua Cheng; Yeou-Cheng Bor; Denis Manga Tebit; Marie-Louise Hammarskjold; David Rekosh; Pascal Obong Bessong

doi:10.1016/j.jep.2020.112931

Development of a pseudovirus assay and evaluation to screen natural products for inhibition of HIV-1 subtype C reverse transcriptase

J Ethnopharmacol. 2020 Aug 10:258:112931. doi: 10.1016/j.jep.2020.112931. Epub 2020 Apr 30.

Authors

Lufuno Grace Mavhandu¹, Hua Cheng², Yeou-Cheng Bor³, Denis Manga Tebit⁴, Marie-Louise Hammarskjold⁵, David Rekosh⁶, Pascal Obong Bessong⁷

Affiliations

¹ HIV/AIDS & Global Health Research Programme, University of Venda, PMB X5050, Thohoyandou, 0950, South Africa. Electronic address: lufuno.mavhandu@univen.ac.za.
² Myles H. Thaler Center for AIDS and Human Retrovirus Research, Department of Microbiology, Immunology and Cancer Biology, University of Virginia, P.O. Box 800734, Charlottesville, VA, 22908, USA; Shanghai Municipal Center for Disease Control and Prevention, Department of HIV, STD and Leprosy, 1380 Zhongshan Road, Shanghai, 200336, China. Electronic address: chenghua@scdc.sh.cn.
³ Myles H. Thaler Center for AIDS and Human Retrovirus Research, Department of Microbiology, Immunology and Cancer Biology, University of Virginia, P.O. Box 800734, Charlottesville, VA, 22908, USA; Ambassadors for Christ, 21 Ambassador Drive, Paradise, PA, 17562, USA. Electronic address: ybor@afcinc.org.
⁴ HIV/AIDS & Global Health Research Programme, University of Venda, PMB X5050, Thohoyandou, 0950, South Africa; Myles H. Thaler Center for AIDS and Human Retrovirus Research, Department of Microbiology, Immunology and Cancer Biology, University of Virginia, P.O. Box 800734, Charlottesville, VA, 22908, USA; Global Biomed Scientific LLC, PO Box 2368, Forest, VA, 24551, USA. Electronic address: dmt@globiomedsci.com.
⁵ HIV/AIDS & Global Health Research Programme, University of Venda, PMB X5050, Thohoyandou, 0950, South Africa; Myles H. Thaler Center for AIDS and Human Retrovirus Research, Department of Microbiology, Immunology and Cancer Biology, University of Virginia, P.O. Box 800734, Charlottesville, VA, 22908, USA. Electronic address: mh7g@virginia.edu.
⁶ HIV/AIDS & Global Health Research Programme, University of Venda, PMB X5050, Thohoyandou, 0950, South Africa; Myles H. Thaler Center for AIDS and Human Retrovirus Research, Department of Microbiology, Immunology and Cancer Biology, University of Virginia, P.O. Box 800734, Charlottesville, VA, 22908, USA. Electronic address: dr4u@virginia.edu.
⁷ HIV/AIDS & Global Health Research Programme, University of Venda, PMB X5050, Thohoyandou, 0950, South Africa. Electronic address: pascal.bessong@univen.ac.za.

PMID: 32360797
DOI: 10.1016/j.jep.2020.112931

Abstract

Ethnopharmacological relevance: Medicinal plants are used in the management of Human Immunodeficiency Virus and Acquired Immunodeficiency Syndrome (HIV/AIDS) in many developing country settings where HIV-1 subtype C drives the epidemic. Efforts to identify plant derived molecules with anti-HIV properties require reproducible assay systems for routine screening of selected plant compounds. Although a number of standardized HIV-1 pseudoviruses have been generated to assess infectivity, replicability or reproducibility, HIV-1 subtype C (HIV-1-C) pseudoviruses have not been comprehensively characterized to identify inhibitory plant substances.

Aim of the study: The current study aimed at developing an HIV-1-C pseudovirus assay, and evaluate plant substances targeting reverse transcriptase (RT) activity.

Materials and methods: HIV-1 subtype C pseudoviruses containing a luciferase reporter gene were generated by transfection of human 293T cells. HIV-1 subtype B (HIV-1-B) wild type pseudoviruses and mutants resistant to nucleoside and non-nucleoside RT inhibitors were also generated and used as controls. Selected plant substances and the RT inhibitors Zidovudine (AZT) and Nevirapine (NVP), were used to evaluate inhibition. Pseudovirus infectivity was determined by luciferase measurement in CF2/CD4⁺/CCR5 cells, and cytotoxicity was determined using the MTT assay. AZT and NVP inhibited wild type pseudoviruses in a dose dependent manner, with IC₅₀ values in the nanomolar range.

Results: Pseudoviruses harbouring RT drug resistance mutations were poorly suppressed by AZT and NVP. Catechin, obtained from Peltophorum africanum inhibited HIV-1-C and HIV-1-B pseudoviruses with selective indices of 6304 μM (IC₅₀: 0.49 μM, CC₅₀: 3089 μM) and 1343 μM (IC₅₀: 2.3 μM, CC₅₀: 3089 μM), respectively; while the methanol root crude extract of Elaeodendron transvaalense gave IC₅₀ values of 11.11 μg/ml and 16.86 μg/ml, respectively.

Conclusion: The developed HIV-1-C pseudovirus assay can be used to screen plant substances for RT inhibition, and may have utility in settings with limited access to high level biosafety facilities.

Keywords: Bioassay development; HIV-1 subtype C; Natural products; Pseudoviruses inhibition.

MeSH terms

Anti-HIV Agents / administration & dosage
Anti-HIV Agents / pharmacology*
Dose-Response Relationship, Drug
HEK293 Cells
HIV Infections / drug therapy
HIV Infections / virology
HIV Reverse Transcriptase / antagonists & inhibitors*
HIV-1 / drug effects*
HIV-1 / enzymology
Humans
Inhibitory Concentration 50
Nevirapine / administration & dosage
Nevirapine / pharmacology
Plant Preparations / administration & dosage
Plant Preparations / pharmacology*
Plants, Medicinal / chemistry
Reproducibility of Results
Reverse Transcriptase Inhibitors / administration & dosage
Reverse Transcriptase Inhibitors / pharmacology*
Zidovudine / administration & dosage
Zidovudine / pharmacology

Substances

Anti-HIV Agents
Plant Preparations
Reverse Transcriptase Inhibitors
Zidovudine
Nevirapine
reverse transcriptase, Human immunodeficiency virus 1
HIV Reverse Transcriptase