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Genetic and biochemical data have identified at least four viral proteins essential for vaccinia virus (VACV) DNA synthesis: the DNA polymerase E9, its processivity factor (the heterodimer A20/D4) and the primase/helicase D5. These proteins are part of the VACV replication complex in which A20 is a central subunit interacting with E9, D4 and D5. We hypothesised that molecules able to modulate protein-protein interactions within the replication complex may represent a new class of compounds with anti-orthopoxvirus activities. In this study, we adapted a forward duplex yeast two-hybrid assay to screen more than 27,000 molecules in order to identify inhibitors of A20/D4 and/or A20/D5 interactions. We identified two molecules that specifically inhibited both interactions in yeast. Interestingly, we observed that these compounds displayed a similar antiviral activity to cidofovir (CDV) against VACV in cell culture. We further showed that these molecules were able to inhibit the replication of another orthopoxvirus (i.e. cowpox virus), but not the herpes simplex virus type 1 (HSV-1), an unrelated DNA virus. We also demonstrated that the antiviral activity of both compounds correlated with an inhibition of VACV DNA synthesis. Hence, these molecules may represent a starting point for the development of new anti-orthopoxvirus drugs.

International audience; A20 protein is a major component of the vaccinia virus replication complex. It binds to the DNA polymerase E9, the uracil DNA glycosylase D4 and the primase/helicase D5, three proteins that are essential for viral DNA synthesis. The identification of molecules able to interact with the replication complex and inhibit its activity is a promising strategy for the design of new anti-orthopoxvirus drugs. In this study, we used a yeast genetic approach to select, from combinatorial libraries, 8-mers peptide aptamers that specifically interact with A20. From this screen, we isolated five peptide aptamers whose binding to A20 was confirmed by a glutathione S-transferase (GST) pull-down assay. Among those, we determined that peptide aptamer 72 binds to a central domain on A20. Interestingly, this region of A20 was previously shown to be important for its function in DNA replication. We next showed that vaccinia virus DNA synthesis was impaired in cells constitutively expressing peptide aptamer 72 and that virus production was inhibited in those cells. Thus, peptide aptamer 72 may be a useful tool for the development of new compounds specifically targeting poxvirus replication.