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. 2024 Jun 28;13(7):546.
doi: 10.3390/pathogens13070546.

The Triterpenoid MOMORDIN-Ic Inhibits HCMV by Preventing the Initiation of Gene Expression in Eukaryotic Cells

Eleanor Bradley  1 , Emma Poole  2 , Matthew B Reeves  1
Affiliations

The Triterpenoid MOMORDIN-Ic Inhibits HCMV by Preventing the Initiation of Gene Expression in Eukaryotic Cells

Eleanor Bradley et al. Pathogens. .

Abstract

Human cytomegalovirus (HCMV) primary infection, re-infection, and reactivation from latency cause morbidity in immune-compromised patients. Consequently, potential therapeutic strategies remain of interest for the treatment of infection. Naturally occurring triterpenoids derived from plants have been demonstrated to have anti-viral activity, although their precise mechanisms of action are not always fully understood. Here, we investigate the activity of Mormordin Ic (Mc) and demonstrate that it is potently anti-viral against HCMV. Through investigation of the mechanistic basis of this anti-viral activity, we identify that it is inhibitory to both viral and host gene expression, and to highly induced genes in particular. We go on to observe that Mc impacts on RNA Pol II activity and, specifically, reduces the occupancy of elongating RNA Pol II at a viral promoter. Next, we demonstrate that Mc is inhibitory to HCMV reactivation, and in doing so identify that it has greater activity against the canonical major immediate early promoter compared to the alternative ip2 promoter located downstream. Finally, we see evidence of RNA Pol II occupancy at the ip2 promoter in undifferentiated myeloid cells. Thus, Mc is potently anti-viral and a potential tool to probe the activity of multiple promoters considered important for controlling HCMV reactivation.

Keywords: anti-virals; cytomegalovirus; latency; transcriptional regulation.

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Conflict of interest statement

The authors declare no conflicts of interest and confirm the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Momordin-Ic inhibits viral infection of permissive cells in a single round infection. (a,b) HFFs were pre-treated with serial dilutions of Mc (50 μM–100 nM) and then infected in presence of drugs and stained for IE protein expression 24 hpi (a) or assayed for cell viability 24 h later (b). Statistical analysis was performed using Kruskal-Wallis with Dunn’s multiple comparison test. * p < 0.05; ns non-significant. (c) ARPE and THP1-macrophage cells were pre-treated with Mc (2 μM), infected in presence of drugs, and then stained for IE protein expression 24 hpi. Infection was quantified by Hermes WiScan automated analysis. Statistical analysis is unpaired t test with Welch’s correction. ** p < 0.01.
Figure 2
Figure 2
Momordin Ic works post-viral entry. (a) HFFs were pre-treated with DMSO (solvent control), DIDS, Heparin, DMSO (solvent control) or 2 μM Momordin Ic (Mc) and DNA was isolated 3 hpi and amplified by qPCR and expressed relative to untreated control. Statistical analysis by ANOVA followed by Tukey’s post multiple comparisons test was performed. * p < 0.05; ** p < 0.01. (b) HFFs were pre-treated with DMSO (control) or 2 μM Momordin Ic (Mc) and cells were fractionated into nuclear and cytoplasmic fractions. DNA was isolated and analysed for viral (HCMV), nuclear (B globin) and mitochondrial (mito) DNA. Values were calculated as difference of Ct from background PCR signal (arbitrary units).
Figure 3
Figure 3
Momordin-Ic inhibits viral and eukaryotic transcription. (a) HFFs untreated (control) or pre-treated with DMSO or 2 μM Momordin Ic (Mc) were analysed for IE RNA expression by qPCR. (b) HFFs were infected with HCMV and then incubated with DMSO or 2 μM Momordin Ic (Mc) 8 hpi. IE RNA expression was then analysed 24 hpi by qPCR. (c) THP1 cells were either untreated (control) or pre-treated with DMSO or 2 μM Momordin Ic (Mc) and then stimulated with IFNb (1000 U/mL). After 8 h, RNA was analysed for CXCL10 and IFIT2 expression by qPCR and expressed relative to expression in untreated, unstimulated control cells. (d) HFFs were incubated with DMSO (control) or 2 μM Momordin Ic and then analysed for 18S, GAPDH and actin expression by qPCR after 24 h. Expression in Momordin Ic treated cells relative to DMSO is shown. n = 3 for all experiments. Statistical analysis (a–c) was performed using an unpaired t test with Welch correction. * p < 0.05; ** p < 0.01.
Figure 4
Figure 4
Momordin Ic reduces occupancy of phosphor RNA Pol II at the MIEP. HFFs were pre-treated with DMSO or 2 μM Momordin Ic (Mc), infected and then subjected to ChIP analysis 3 hpi with antibodies against RNA Pol II, phosphorylated RNA Pol II or an isotype-matched control. Samples were analysed by qPCR with MIEP primers and values expressed as %Input. n = 2.
Figure 5
Figure 5
Momordin Ic inhibits IE expression in reactivating cells. (a,b) Latently infected monocyte-derived DCs (a) or THP1 cells (b) were either untreated (control), or pre-incubated with DMSO or 2 μM Momordin Ic (Mc) for 3 h prior to IL-6 (a) or PMA (b) stimulation. After 24 h, cells were analysed for IE gene expression relative to untreated control. (c) RNA harvested in (b) was subsequently analysed for MIEP and ip2-derived IE transcripts by qPCR and Mc samples expressed relative to DMSO solvent control. Statistical analysis was performed using a Mann-Whitney comparison of independent pairs. * p < 0.05; ** p < 0.01. n = 3.
Figure 6
Figure 6
RNA Pol II binding to ip2 promoter region in latently infected cells. THP1 cells were infected with HCMV and after 5 days subjected to ChIP analysis with antibodies against RNA Pol II, histone H3 or a rabbit isotype-matched control. DNA was then analysed by qPCR with primers directed against MIEP and intron A (ip2) sequences. Statistical analysis was performed using a Mann-Whitney comparison of independent pairs. * p < 0.05. n = 3.
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