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.
Recent studies have demonstrated that HPV proteins target aspects of the innate immune system. Normal keratinocytes constitutively express low levels of IFN in the absence of viral infection (Bielenberg and others 1999). HPV proteins modulate the response to IFN in several ways. First, the levels of ISGs are reduced in HPV-infected cells from that seen in normal keratinocytes. Microarray analyses have demonstrated that the expression of MxA, 2-5 oligoadenylate synthetase 2, as well as STAT1 is reduced in HPV-positive cells. The addition of IFN still induces expression of these IFN-inducible genes but at initially reduced rates. Following 24 h of exposure to IFN, the levels of expression, however, increases to that seen in normal cells (Chang and Laimins 2000). Additional studies have demonstrated that HPV proteins directly target components of the innate immune system to inhibit their action. The E6 oncoprotein has been shown to bind to IFN regulatory factor-3 (IRF-3), thereby interfering with its ability to act as a transcriptional activator (Ronco and others 1998). In addition, E6 has been reported to bind the Tyk2 kinase of the Jak-Sat pathway and inhibit its function (Li and others 1999). In a similar manner, E7 has been shown to bind to IRF-1 and block its functions (Park and others 2000). E7 has also been shown to bind to p48, a component of the ISG factor 3 (ISGF3) complex, blocking the translocation of this complex to the nucleus and its ability to activate gene expression in response to interferon (Barnard and McMillan 1999). These interactions may provide a mechanism by which HPV proteins suppress expression of ISGs. Many ISGs are estimated to be the ultimate antiviral effectors of the IFN signaling (Paludan SR et al., 2016). They function either by targeting different steps of the viral life cycle or by reinforcing host defense by further driving the expression of ISG-encoding genes (Paludan SR et al., 2016). In particular, virally infected cells release type I/III IFNs, which induce the phosphorylation and activation of the STAT1 and STAT2 transcription factors. When combined with the transcriptional regulator IRF9, phosphorylated STAT1 and STAT2 form the ISGF3 complex, which drives the expression of ISGs that are important for antiviral immune reposne. While the transcription factor ISGF3 drives the first rapid response phase, the related factor un-phosphorylated ISGF3 (U-ISGF3), formed by IFN-induced high levels of IRF9 and STATs 1 and 2 without tyrosine phosphorylation, drives the second prolonged response (Cheon H et al, 2013). This response appeared to be determinant in viral reprograming of host gene expression in premalignant keratinocytes immortalized by HPV16 (Evans et al., 2017). Hence, we hypothesize that one or more of the STAT1 down-stream genes play a primary role in controlling MmuPV1 infection. Thus, it is a priority to characterize STAT1 down-stream genes involved in resistance to MmuPV1 and to validate candidate ISGs during in vivo HPV infection (e.g. HPV positive women). In particular, our project will examine among the 300 STAT1 downstream genes, a candidate set of ISGs based on current literature in the papillomavirus field, in order to identify one or more responsible for controlling MmuPV1 replication in mouse keratinocytes (Virology Unit: Scagnolari C, F. Frasca). Subsequently the expression levels of this candidate set of ISGs will be evaluated in HPV positive and negative women attending gynaecological clinic (Policlinico Umberto I hospital/ Recine N Clinical Unit, Scagnolari C, F. Frasca Virology Unit) We retain that despite this unfavorable outcome, studies on the regulation of IFN pathways by HPV infection studies at the molecular level have provided and will continue to provide important insights into complex interactions between the HPV oncoproteins and host factors that control innate immunity. The resulting knowledge of this project will be useful in the design of novel strategies that combat HPV infection and associated disease. Indeed, a better understanding of un- phosphorylated ISGF3-regulated genes and other select ISGs that mediate the downstream STAT1-dependent inhibition of papillomavirus replication and their in vivo relevance may lead to the development of therapies based on peptides that block the viral-host protein interactions and restore IFN signaling through endogenous control mechanisms. The feasibility of this project is based on ad hoc multidisciplinary approach among two major research Units: Clinical Unit (component: Prof. N. Recine), and Virology/IFN Unit (components: Prof. C. Scagnolari, Dr. F. Frasca) .