Ricerc@Sapienza

It is clear that the IFN system did not evolve to have only one potent HPV-specific IFN stimulated genes ISG, but rather to work in a combinatorial fashion. A better mechanistic understanding of individual HPV-ISGs may lead to the development of novel therapeutics, but from a practical standpoint the most powerful ISG-based therapeutics in the near future may be those that harness the collective power of ISGs similar to IFN itself. Hence, we exploited in collaboration with Prof. R. Viscidi (Johns Hopkins University, USA) the resistance of C57BL6 mice to mouse papillomavirus (MmuPV1) infection to ask the question what innate immune defenses protect these mice from infection. In preliminary studies, we found expression of early E7^E4 gene transcripts in genital tract tissue 3-months post infection of mice deficient in MyD88 and STAT1. We will follow up our initial observations investigating the downstream ISGs that control papillomavirus viral replication. In particular, we will determine in vitro the un-phosphorylated (U) ISGF3-regulated genes and other select ISGs that mediate the downstream STAT1-dependent inhibition of MmuPV1 replication in mouse keratinocytes using siRNA technology. Keratinocytes derived from C57BL/6 (MPEK-BL6) and BALB/c mice (K38) will be stimulated with MmuPV1. Then, ISGs expression targeting U-ISGF3 complex and those of early (E7^E4) viral genes will be evaluated RT/Real Time PCR assays. As a functional screen for anti-MmuPV1 ISGs, ISGs identified in the Real Time PCR experiments, will be individually or simultaneously knocked down using RNA interference (RNAi) followed by infection with MmuPV1. In order to validate the importance of candidate anti-MmuPV1 ISGs, the expression of ISGs will be measured during in vivo HPV infection according to HPV status , the genotype of HPVs (high-risk or low-risk), HPV history, HPV E6 and E7 levels, and cervical cytology.