Antiviral activity of 3,4'-dihydroxyflavone on influenza a virus.

Influenza virus infection causes thousands of deaths and millions of hospitalizations worldwide every year and the emergence of resistance to anti-influenza drugs has prompted scientists to seek new natural antiviral materials. In this study, we screened 13 different flavonoids from various flavonoid groups to identify the most potent antiviral flavonoid against human influenza A/PR/8/34 (H1N1). The 3-hydroxyl group flavonoids, including 3,2᾿dihydroxyflavone (3,2᾿DHF) and 3,4᾿dihydroxyflavone (3,4᾿DHF), showed potent anti-influenza activity. They inhibited viral neuraminidase activity and viral adsorption onto cells. To confirm the anti-influenza activity of these flavonoids, we used an in vivo mouse model. In mice infected with human influenza, oral administration of 3,4᾿DHF significantly decreased virus titers and pathological changes in the lung and reduced body weight loss and death. Our data suggest that 3-hydroxyl group flavonoids, particularly 3,4᾿DHF, have potent antiviral activity against human influenza A/PR/8/34 (H1N1) in vitro and in vivo. Further clinical studies are needed to investigate the therapeutic and prophylactic potential of the 3-hydroxyl group flavonoids in treating influenza pandemics.

Susceptibilities of human cytomegalovirus clinical isolates and other herpesviruses to new acetylated, tetrahalogenated benzimidazole D-ribonucleosides.

Recently we characterized two inhibitors targeting the human cytomegalovirus (HCMV) terminase, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) benzimidazole (Cl(4)RB). The terminase consists of the ATP-hydrolyzing subunit pUL56 and the subunit pUL89 required for duplex nicking. Because mammalian cell DNA replication does not involve cleavage of concatemeric DNA by a terminase, these compounds represent attractive alternative HCMV antivirals. We now have tested these previously identified benzimidazole ribonucleosides in order to determine if they are active against HCMV clinical isolates as well as those of herpes simplex virus type 1, mouse cytomegalovirus, rat cytomegalovirus (RCMV), and varicella-zoster virus (VZV). Antiviral activity was quantified by measurement of viral plaque formation (plaque reduction) and by viral growth kinetics. Interestingly, both BTCRB and Cl(4)RB had an inhibitory effect in ganciclovir (GCV)-sensitive and GCV-resistant clinical isolates, with the best effect produced by Cl(4)RB. Electron microscopy revealed that in cells infected with GCV-sensitive or GCV-resistant isolates, B capsids and dense bodies were formed mainly. Furthermore, pulsed-field gel electrophoresis showed that cleavage of concatenated DNA was inhibited in clinical isolates. In addition, the antiviral effect on other herpesviruses was determined. Interestingly, in plaque reduction assays, BTCRB was active against all tested herpesviruses. The best effects were observed on VZV- and RCMV-infected cells. These results demonstrate that the new compounds are highly active against GCV-resistant and GCV-sensitive clinical isolates and slightly active against other herpesviruses.

Identification of acetylated, tetrahalogenated benzimidazole D-ribonucleosides with enhanced activity against human cytomegalovirus.

DNA packaging is the key step in viral maturation and involves binding and cleavage of viral DNA containing specific DNA-packaging motifs. This process is mediated by a group of specific enzymes called terminases. We previously demonstrated that the human cytomegalovirus (HCMV) terminase is composed of the large subunit pUL56 and the small subunit pUL89. While the large subunit mediates sequence-specific DNA binding and ATP hydrolysis, pUL89 is required only for duplex nicking. An excellent inhibitor targeting HCMV terminase is 2-bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB), but it was not developed as an antiviral drug due to its metabolic cleavage in experimental animals. We now have tested several new benzimidazole d-ribonucleosides in order to determine whether these compounds represent new, potent inhibitors. Analysis by bioluminometric ATPase activity assays identified two of the new compounds with a high inhibitory effect, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl benzimidazole (Cl(4)RB). By using viral plaque formation, viral yield, and viral growth kinetics, we demonstrated that the two compounds BTCRB and Cl(4)RB had antiviral activities similar to that of BDCRB. Interestingly, BTCRB retained its inhibitory activity after preincubation with HFF cells. By use of electron microscopy, we observed an increase of B capsids and a lack of cytoplasmic capsids in the presence of the compounds that correlated with the virus yield. Furthermore, cleavage of concatenated DNA was inhibited by both compounds, and inhibition by BTCRB was shown to be dose dependent. These results demonstrate that the new compounds are highly active against HCMV and act by mechanisms similar but not identical to those of BDCRB.

Identification of the interaction domain of the small terminase subunit pUL89 with the large subunit pUL56 of human cytomegalovirus.

The small terminase subunit pUL89 of human cytomegalovirus (HCMV) is thought to be required for cleavage of viral DNA into unit-length genomes in the cleavage/packaging process. Immunoprecipitations with a UL89-specific antibody demonstrated that pUL89 occurs predominantly as a monomer of approximate M(r) 75.000 together with a dimer of approximate 150.000. This was confirmed by gel permeation chromatography. In view of its putative function, pUL89 needs to be transported into the nucleus. By use of laser scanning confocal microscopy, pUL89 was found to be predominantly localized throughout the nucleus and in particular in viral replication centers of infected cells. By immunofluorescence, we demonstrated that both terminase subunits co-localized in viral replication centers. Furthermore, analysis with pUL89 GST-fusion protein mutants showed that amino acids 580-600 may represent the interaction domain with pUL56. To verify this result, a recombinant HCMV genome was constructed in which the UL89 open reading frame was disrupted. By transfection of the deletion BACmid alone, we showed that it has a lethal phenotype. Cotransfection assays demonstrated that, in contrast to pUL89 wild-type, a plasmid construct encoding a pUL89 variant without aa 580-590 as well as one encoding a variant without aa 590-600 could not complement the HCMV-pUL89 null genome, thus, suggesting that the 20 aa sequence GRDKALAVEQFISRFNSGYIK is sufficient for the interaction with pUL56 and in conclusion required for DNA packaging.

ATPase activity of the terminase subunit pUL56 of human cytomegalovirus.

Herpesviral DNA packaging is a complex process resulting in unit-length genomes packed into preformed procapsids. This process is believed to be mediated by two packaging proteins, the terminase subunits. In the case of double-stranded DNA bacteriophages, the translocation of DNA was shown to be an energy-dependent process associated with an ATPase activity of the large terminase subunit. In the case of human cytomegalovirus it was not known which protein has the ability to hydrolyze ATP. In this study we expressed human cytomegalovirus terminase subunits, pUL89 and the carboxyl-terminal half of pUL56, as GST fusion proteins and purified these by affinity chromatography. ATPase assays demonstrated that the enzymatic activity is exclusively associated with pUL56. The characterization of the ATP hydrolysis showed that the enzymatic reaction is a fast process, whereas the spontaneous ATP decay followed slow kinetics. Interestingly, although pUL89 did not show any ATPase activity, it was capable of enhancing the UL56-associated ATP hydrolysis. Furthermore, a specific association of in vitro translated pUL89 with the carboxyl-terminal half of GST-UL56C was detected. This interaction was confirmed by co-immunoprecipitations of infected cells. Our results clearly demonstrated that (i) both terminase subunits interact with each other and (ii) the subunit pUL56 has an ATPase activity.