A natural tetranortriterpenoid with antiviral activity interferes with intracellular glycoprotein trafficking

BARQUERO, A.A.; BUENO, C.A.; COTO, C.E.; ALCHÉ, L.E.

Estoril, Portugal

Workshop; 33th International Herpesvirus Workshop; 2008

The 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM) isolated from leaf extracts of Melia azedarach L. restrains VSV and HSV-1 multiplication in vitro, with low cytotoxicity. Although the precise mechanism by which CDM exerts its effect is still unresolved, it is the first tetranortriterpenoid reported responsible for the alkalinization of intracellular compartments, thus affecting both, viral endocytic and exocytic pathways. CDM blocks VSV entry and the intracellular transport of VSV-G protein confining it to the Golgi apparatus, by pre or post-treatment, respectively. Besides, HSV-1 glycoproteins were confined to the Golgi apparatus independently of the nature of the host cell. We decided to investigate the effect of CDM on viral and cellular glycoprotein transport. A human conjunctival cell line (IOBA-NHC) was transfected with a cDNA coding for transferrin receptor fused with the enhanced green fluorescent protein (TfR-GFP). In untreated cells, TfR fluorescence appeared in a punctuate pattern widely distributed throughout the cytoplasm and in the plasma membrane, whereas a compact juxtanuclear staining pattern in the majority of treated cells was observed. To determine whether this pattern was associated with the accumulation of viral glycoproteins in the Golgi apparatus, transfected cells were infected with HSV-1 and processed for gD-protein IFI staining. Confocal microscope images evidenced that in CDM-treated infected cells, gD protein always appeared associated with the perinuclear region, while TfR fluorescence showed different patterns of localization, rarely co-localizing with the viral protein. To further investigate whether CDM was causing a temporary or permanent blockage in glycoprotein transport, we added cycloheximide (CHX) after CDM removal, to evaluate the final location of both glycoproteins. Under this condition, TfR and gD fluorescence patterns resembles to those corresponding to the respective untreated controls. These findings suggest that CDM provokes a delay in glycoprotein transport instead of a permanent blockage, enough to prevent virus multiplication without cellular cytotoxicity.