Early-stage inhibition of HIV-1 subtypes A, B, and C gag- protease chimeras by Penicillium chrysogenum P03MB2 metabolites and activity against cell-to-cell spread

Despite the success of antiretroviral therapy, human immunodeficiency virus type 1 (HIV-1) cell-to-cell spread remains a major barrier to viral eradication. This study evaluated the antiviral activity of secondary metabolite fractions derived from Penicillium chrysogenum P03MB2 against cell-free HIV-1 infection of patient-derived Gag-protease chimeric viruses representing HIV-1 subtypes A, B, and C, and investigated its impact on cell-to-cell spread. Crude fungal extracts from oat-grown cultures were fractionated using mixed-mode cation exchange (MCX) solid-phase extraction. Cytotoxicity was assessed in TZM-bl cells by MTT assay, antiviral activity was evaluated using a luciferase reporter assay. In silico molecular docking explored predicted interactions between five identified metabolites, including Cytochalasin J, and entry-associated targets (CCR5, CXCR4, and gp120). Cell- to cell transmission was assessed using Jurkat co-culture assays and flow cytometry. The 45% MCX Oats fraction emerged as the most active candidate exhibiting low cytotoxicity (CC₅₀ > 300 µg/mL) and potent antiviral activity across all chimeric variants (IC₅₀: 0.221-1.007 µg/mL; Selectivity Indices: 271-1235). Time-of-addition analysis indicated that maximal inhibition during the early infection stages. Molecular docking identified favourable predicted binding compatibility for Cytochalasin J against CCR5 (reaching -10.9 kcal/mol), CXCR4, and gp120 targets, sharing predicted interacting residues with reference drugs. Furthermore, the fraction significantly reduces newly infected target cells in the cell-to-cell transmission model compared to untreated controls. Collectively, these findings demonstrate that 45% MCX Oats fraction exhibits potent antiviral activity against cross-clade antiviral activity against HIV-1 Gag-Pro chimeras and limits viral transmission in vitro, supporting further bioassay-guided isolation of its active constituents.