Controlling the intracellular localization of fluorescent polyamide analogues in cultured cells.

The intracellular distribution of fluorescent-labeled polyamides was examined in live cells. We showed that BODIPY-labeled polyamides accumulate in acidic vesicles, mainly lysosomes, in the cytoplasm of HCT116 colon cancer cells and human rheumatoid synovial fibroblasts (RSF). Verapamil blocked vesicular accumulation and led to nuclear accumulation of the BODIPY-labeled polyamide in RSFs. We infer that the basic amine group commonly found at the end of synthetic polyamide chains is responsible for their accumulation in cytoplasmic vesicles in mammalian cells. Modifying the charge on a polyamide by replacing the BODIPY moiety with a fluorescein moiety on the amine tail allowed the polyamide to localize in the nucleus of the cell and bypass the cytoplasmic vesicles in HCT116 cells.

Recombinant full-length human cytomegalovirus protease has lower activity than recombinant processed protease domain in purified enzyme and cell-based assays.

Herpesviruses encode a protease that is essential for virus replication. The protease undergoes cleavage to a processed form during capsid maturation. A recombinant 75 kDa form of the protease from human cytomegalovirus was purified and compared with the recombinant 29 kDa processed form. Modification with an active site titrant suggested that most of each recombinant protease preparation was active (66 and 86%, respectively). Protease activity was compared using a low-molecular weight peptide substrate and the native substrate, capsid assembly protein. In addition, a cell-based assay for both enzymes was developed in which the target sequence of the protease has been fused inframe into the herpes simplex virus VP16 molecule. Cleavage of the fusion protein by the protease releases the carboxyl terminal transactivation domain, resulting in a decrease in the ability of the fusion molecule to transactivate a target promoter linked to a reporter gene in mammalian cells. Results suggest that the 75 kDa form of the enzyme is significantly less active than the 29 kDa form by all criteria.

Adenovirus inhibition by peptidomimetic integrin antagonists.

Many viruses and bacterial pathogens are capable of exploiting host cell surface integrins during their replication cycles. The ligands for many integrins contain an arginine-glycine-aspartic acid (RGD) amino acid sequence that is essential for protein-protein interaction. Human adenovirus particles contain this sequence in the penton base protein, and previous studies support a role for this RGD in integrin-dependent internalization of the virus by the cell. As synthetic peptidomimetics of RGD have been shown in other experimental systems to be antagonists of the activities of specific integrins both in vitro and in vivo, we sought to determine whether these small molecules are antagonists of adenovirus infection. Such compounds inhibited viral infection of cultured cells with similar rank order potency to that determined in assays utilizing purified extracellular matrix proteins as integrin ligands. The maximal level of inhibition achieved with the peptidomimetics was comparable to that of RGD-containing peptides, whereas no significant effects were apparent with an RGE-containing peptide. An engineered adenovirus having a mutated RGD sequence in the penton base was not susceptible to the inhibition. The results obtained with these synthetic antagonists, which have varied structures and potencies, suggest that integrins interact with adenoviral RGD in a manner similar to that of other protein ligands such as vitronectin. Furthermore, the results confirm the role of RGD in the replication cycle, and suggest peptidomimetic compounds may be useful antimicrobial agents in the treatment of a variety of diseases.