The antiviral effects of the crude extract from endophytic fungi, Alternaria alternata, on different HIV-1 subtypes and Integrase drug-resistance strains.

Abstract

Background: Antiretroviral drugs have successfully turned HIV/AIDS into a manageable chronic condition and increased life expectancy. The development of drug-resistance, side effects, toxicity and HIV subtype have impeded drug development and cure efforts. Thus, there is a demand of new strategies for developing novel antiretrovirals with decreased toxicity, potent anti-HIV activity, inhibitory activity across HIV subtypes, and activity against drugresistant strains. This study investigated the anti-HIV effect of the fungal endophyte Alternaria alternata PO4PR2 crude extract (CE) on different HIV-1 subtypes and integrase drug-resistant strains. Materials and methods: The secondary metabolites of Alternaria alternata PO4PR2 were isolated using methanolic extraction to prepare a CE. The MTT (3-[4,5-dimethylthiazol-2- yl]2,5 diphenyl tetrazolium bromide) assay was then used to determine the cell viability and cytotoxicity of the A. alternata crude extract on treated TZM-bl cells. Then, using site-directed mutagenesis, the integrase drug-resistance mutations T66K and S230R were introduced into HIV-1 molecular clone pNL4.3. The anti-HIV activity of the A. alternata crude extract against HIV-1 subtype A, B, C, D and integrase drug-resistant strains was then assessed using a luciferase-based antiviral assay. The viral replication stages inhibited by the A. alternata crude extract were determined using luciferase-based and p24 ELISA-based time of addition assays. Results: The cell viability of TZM-bl cells treated with A. alternata crude extract was greater than 80%, and the CC50 of 300 μg/mL indicated that crude extract was not cytotoxic. The A. alternata crude extract inhibited the viral replication of HIV-1 subtypes A, B, C and D viruses with IC50’s ranging between 0.10 and 0.60 μg/mL. The calculated SI values for the different subtype viruses were between 493 and 2921 showing therapeutic potential of the crude extract. The A. alternata crude extract inhibited the integrase drug-resistant strains T66K and S230R with IC50 of 0.1628 and 0. 0.1961 μg/mL with a 0.7265 and 0. 8751-fold increase in susceptibility to the CE. The A. alternata crude extract had activity during attachment (88% viral inhibition; HIV-1 p24 titre =10.38 pg/mL), reverse transcription (81% viral inhibition; HIV-1 p24 titre =13.74 pg/mL), integration (65% viral inhibition; HIV-1 p24 titre =17.74 pg/mL), and proteolysis (9% viral inhibition; HIV-1 p24 titre =49.32 pg/mL) stages of the HIV life cycle. Conclusion: The A. alternata crude extract was not cytotoxic and had strong antiviral activity against subtype A, B, C, D and integrase drug-resistant strains. The A. alternata crude extract inhibited stages key to the life cycle of HIV. The compounds in A. alternata crude extract have potential to develop drug candidates that can inhibit different HIV subtype and drug-resistance.