Tecnologie OMICS e Systems Biology per la definizione di nuove strategie finalizzate al controllo delle infezioni virali

Introduction: Primary infection with the human polyomavirus BK (BKV) is asymptomatic, and is followed by a life-long persistence of the virus in the reno-urinary tract. BKV is the etiologic agent of Polyomavirus Associated Nephropathy (PVAN), a complication that may occur after renal transplantation, leading to irreversible graft failure. The onset of this pathology is due to the reactivation of BKV from latency. The risk factor for PVAN development is the immunosuppressive therapy to prevent organ rejection after transplantation. In addition, it has been suggested that ischemic injury during organ implantation may play a role in creating an environment for viral reactivation leading to nephropathy. Under hypoxic conditions, like those that are determined during ischemic time, cells respond through a mechanism mediated by the Hypoxia Inducible Factors (HIFs), that under normoxia are targeted for proteosomal degradation. During hypoxia, HIF ubiquitination is inhibited and HIF translocate to the nucleus where trigger the transcription of target genes, in order to prepare the cell to hypoxic conditions. Numerous studies have shown that in particular HIF-1α may have a role in affecting the replication of some viruses, such as the polyomavirus JCV, Epstein-Barr virus and parvovirus B19. So far, however, neither the existence of a link between HIF-1α and BKV replication nor the effect of BKV replication on the HIF-1α pathway have been investigated. Objectives: To investigate the link between HIF-1α. and BKV, in order to unravel the molecular mechanisms underlying BKV reactivation after renal transplantation and the onset of PVAN. Methods:

1. To investigate the effect of HIF-1α on the BKV transcription: a1) gene reporter assays will be performed with plasmids containing the BKV promoter located upstream a reporter gene and an HIF-1α expression plasmid; a2) in vitro cellular model: cells will be infected with BKV under hypoxic conditions, and BKV replication will be assessed by Quantitative Real Time PCR (Q-PCR) and Western Blot (WB). In the same model, HIF-1α will be silenced, using siRNA technique, and BKV replication and protein expression will be verified by Q-PCR and WB.
2. To evaluate the existence of a physical binding between HIF-1α and BKV promoter, and between HIF-1α and other cell transcription factors: b1) chromatin immunoprecipitation (ChIP) assay will be performed on cells transfected with the HIF-1α expression plasmid and a plasmid containing the BKV genome; b2) reporter gene assays and co-immunoprecipitation assays will be performed to assess the effect of other transcription factors, such as Smad3/4 on BKV transcription as well as the cooperativity of the two proteins and HIF- 1α in the regulation of BKV promoter; b3) co-immunoprecipitation followed by a combination of SDS-PAGE and MALDI-TOF analysis will be performed to identify unknown proteins potentially linked to HIF-1α.
3. To evaluate the effect of BKV replication on the HIF-1α pathway: c1) in vitro infection of cells with BKV and following evaluation of the presence of HIF-1α by means of WB; c2) immunohistochemical analysis on PVAN biopsy-proven tissues will be assessed using antibodies against BKV proteins and HIF-1α.
4. To define protein profiling of serum collected from BKV+ PVAN patients, compared to BKV+ not-PVAN patients, by means of 2D gel and MALDI-TOF analysis of differentially expressed proteins.

Outcomes: The proposed project may help to understand if hypoxic stress may significantly contribute to reactivation of BKV within the transplant recipients. In addition, protein profiling of PVAN patients will help to assess the differential expression of protein characterizing the disease. The study may provide insights on the mechanisms underlying reactivation o