The antiviral effects of Alternaria alternata PO4PR2 crude extracts on integration stage and integrase drug-resistant variants.

Abstract

Background: HIV-1 antiretroviral drugs have reduced the burden of people living with HIV-1. However, the development of drug resistance has reduced the effectiveness of current therapies, including HIV integrase inhibitors, highlighting the need for novel agents targeting viral integration. Natural products derived from endophytic fungi have emerged as a promising source for the development of new antiviral drugs. Hence, this study investigates the anti-HIV potential of the Alternaria alternata PO4PR2 crude extract against integrase drug-resistant viruses and explores its mechanism of action in inhibiting the viral integration stage. Materials and Methods: Major drug-resistant mutations (DRM) in HIV-1 integrase (G118R, N155H, R263K, and Y143R) were identified using the Stanford HIV Drug Resistance Database and introduced into the HIV-1 pNL4.3 molecular clone via site-directed mutagenesis. Successful introduction of these mutations was confirmed by Sanger sequencing. Integrase-resistant HIV-1 variants were generated by transfection in HEK293T cells, and their infectivity was tested on TZM-bl cell lines. The cytotoxicity of the Alternaria alternata PO4PR2 was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-d iphenyltetrazolium bromide (MTT) assay. A luciferase-based antiviral assay was used to measure the antiviral activity of A. alternata PO4PR2 in TZM-bl cells infected with the pNL4.3 virus and integrase mutant viruses. HIV-1 integrase activity assay and Alu-gag PCR were used to confirm the inhibitory effects of A. alternata PO4PR2 on the integration step and its impact on the integration phase. Molecular docking was performed to predict the binding affinity and interaction profiles of the A. alternata-derived secondary metabolites against HIV-1 integrase. Results: The A. alternata PO4PR2 crude extract showed potent inhibition of the G118R, N155H, Y143R, and R263K mutants while maintaining low toxicity and high cell viability in TZM-bl cell lines. The extract also demonstrated direct inhibition of HIV-1 integrase enzymatic activity, as confirmed by the HIV-1 integrase activity assay, with effects comparable to those of the established integrase strandtransfer inhibitor Raltegravir. Real-time Alu-gag PCR further confirmed a significant reduction in integrated HIV-1 DNA in PBMCs treated with the crude extract, with inhibition levels comparable to. Dolutegravir and approaching those of Raltegravir, indicating direct interference with the integrationstep. Docking analysis revealed that A. alternata PO4PR2 metabolites bind strongly to the HIV-1 integrase catalytic pocket across both wild-type and resistant variants, demonstrating moderate binding affinities and maintaining interactions with key catalytic residues, Mg²⁺ cofactors, and DNA bases, even in the presence of resistance-associated mutations. Discussion and Conclusion: A. alternata PO4PR2 is a promising source of antiretroviral compounds that target drug-resistant HIV-1 integrase mutations, thereby supporting its potential as a source of novel antiretroviral agents against resistant HIV-1 integrase. Several metabolites within the A. alternata crude extract interact directly with the HIV-1 integrase catalytic pocket, consistently engaging the key catalytic residues, and positioning themselves near the Mg²⁺ cofactors in both wildtype and resistant variants, mirroring the interaction patterns of clinically approved strand-transfer inhibitors such as Raltegravir and Dolutegravir. The Alu-gag PCR showed a marked reduction in proviral DNA integration, and the integrase activity assay demonstrated direct enzymatic inhibition of HIV-1 integrase, strongly suggesting that the A. alternata metabolites primarily inhibit the strandtransfer step of integration. These observations indicate that the A. alternata extract contains natural compounds with INSTI-like properties capable of retaining activity against key drug-resistant integrase mutations. Building on these findings, future studies should focus on isolating and characterizing the active compounds responsible for the observed activity to further validate the therapeutic potential of A. alternata.