The isoquinoline alkaloid berberine inhibits human cytomegalovirus replication by interfering with the viral Immediate Early-2 (IE2) protein transactivating activity.

Abstract

The identification and validation of new small molecules able to inhibit the replication of human cytomegalovirus (HCMV) remains a priority to develop alternatives to the currently used DNA polymerase inhibitors, which are often burdened by long-term toxicity and emergence of cross-resistance. To contribute to this advancement, here we report on the characterization of the mechanism of action of a bioactive plant-derived alkaloid, berberine (BBR), selected in a previous drug repurposing screen expressly devised to identify early inhibitors of HCMV replication. Low micromolar concentrations of BBR were confirmed to suppress the replication of different HCMV strains, including clinical isolates and strains resistant to approved DNA polymerase inhibitors.Analysis of the HCMV replication cycle in infected cells treated with BBR then revealed that the bioactive compound compromised the progression of virus cycle at a stage prior to viral DNA replication and Early (E) genes expression, but after Immediate-Early (IE) proteins expression.Mechanistic studies in fact highlighted that BBR interferes with the transactivating functions of the viral IE2 protein, thus impairing efficient E gene expression and the progression of HCMV replication cycle. Finally, the mechanism of the antiviral activity of BBR appears to be conserved among different CMVs, since BBR suppressed murine CMV (MCMV) replication and inhibited the transactivation of the prototypic MCMV E1 gene by the IE3 protein, the murine homolog of IE2.Together, these observations warrant for further experimentation to obtain proof of concept that BBR could represent an attractive candidate for alternative anti-HCMV therapeutic strategies.

Antiviral activity of berberine

“…Lunganini et al showed that BBR interferes with the transactivating function of the HCMV IE2 protein. IE2 plays a critical role in the progression of HCMV replication and in viral pathogenesis and reactivation from latency [74]. It is the most important HCMV regulatory protein and a strong transcriptional activator of viral and cellular gene expression.…”

Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function

“…Assay optimization identified conditions using the stable cell-line that expresses EGFP under the control of the IE2-dependent UL54 early promoter as suitable for screening purposes [364]. A 2320 bioactive compound library including all FDA-approved drugs was screened and six hit compounds have so far been selected for further study [364,381,382]. These hit compounds are deguelin (DGN), nitazoxanide (NTZ), thioguanosine (TGN), alexidine dihydrochloride (AXN), manidipine dihydrochloride (MND) and berberine (BBR).…”

“…Identified inhibitors of IE2-dependent transactivation do not inhibit IE1-dependent transactivation and are thus specific to IE2 not IE1 function [364,382,383]. IE1 s function as an IFN antagonist has been targeted for drug discovery via a modular cell-based screening platform designed to identify compounds that inhibit a viral IFN antagonist choice [373].…”

Chemistry of Secondary Metabolites

“…Throughout the years, distinctive techniques have been accounted for the development of the tetrahydroisoquinoline unit [91]. The isoquinoline alkaloid berberine represses human cytomegalovirus replication by meddling with the viral Immediate Early-2 (IE2) protein transactivating action [92]. Isoquinoline alkaloids and indole alkaloids seem to have an immediate enemy of atherosclerotic impact in ApoE−/− mice [93].…”