Evaluation of antivirals against corona- and lentiviruses in cell cultures

Abstract

Virus infections constitute a continuous health threat. As illustrated by the recent emergence of new - the SARS coronavirus - and the re-emergence of known viruses - influenza, ebola - this threat is certainly not yet decreasing. Rather, many conditions and considerations indicate that viruses will continue to pose problems and that vigilance and preparedness will continue to be needed. Antiviral strategies have clearly demonstrated their potential power in reducing the impact of virus infections. Hence, the focus of this thesis is on the development of antivirals. As targets of our intervention studies we selected lenti- and coronaviruses.

In the first part of the thesis, the possibilities for improvement of antiviral in vitro assessments were explored. We established an in vitro system that can be used to study the role of the dendritic cell (DC) in feline immunodeficiency virus (FIV) infection of T cells. The results are very similar to those described for human DC and T cells with HIV, strengthening the significance of the FIV system as a model for HIV.

Next an evaluation of the activity of antivirals towards FIV infection in various cell systems was performed. The conclusion could be drawn that antiviral agents performed dissimilar in different cell systems. This underlines the importance of using different cell systems for such screenings.

In view of the first promising observations with carbohydrate-binding agents (CBA) towards HIV, we evaluated their effects on FIV infection, as well as on members of different families of the Nidovirales order. The presence of carbohydrate binding compounds strongly inhibited the infections by coronaviruses, arteriviruses and torovirus. There are strong indications that the interaction of CBA during the infection process occurs mainly at the stage of virus entry. Our results indicate that CBA target the two glycosylated envelope proteins, the spike and the membrane protein, of mouse hepatitis virus (MHV) and feline infectious peritonitis virus (FIPV). Furthermore, CBA did not inhibit virus-cell attachment, but affected virus entry at a post-binding stage. The sensitivity of coronaviruses towards CBA was shown to be dependent on the processing of the N-linked glycans. Inhibition of mannosidases in host cells rendered the progeny viruses more sensitive to mannose binding compounds and even to the N-acetylglucosamine binding compound UDA. In addition, inhibition of coronaviruses was shown to be dependent on the cell type used to grow the virus stocks.

Finally it was necessary to prove that CBA are inhibitory to viruses and the antiviral activity is not mediated by influences on cell physiology. Therefore we generated escape mutants by exposing FIPV and MHV to escalating concentrations CBA. In contrast to observations with retroviruses in response to CBA, elimination of N-glycosylation sites from the coronavirus envelope glycoproteins was detected in resistant viruses only once. It is likely that the observed amino acid substitutions led to changes in the three dimensional structure of the spike glycoprotein, thereby rendering the lectin target sites inaccessible.

Our combined results clearly qualified CBA as candidate anti-nidovirus agents. In addition, these agents might as well be valuable tools for the study of the early host-nidovirus interplay and the role of glycosylation during a nidovirus infection.