INTERACTION BETWEEN THE HYPOXIA INDUCIBLE FACTOR 1 ALPHA AND THE HUMAN POLYOMAVIRUS BK: A RISK FACTOR FOR THE DEVELOPMENT OF POLYOMAVIRUS ASSOCIATED NEPHROPATHY

Polyomaviruses are nonenveloped viruses with an icosahedral capsid of about 40-45 nm in diameter. The human polyomavirus BK (BKV) is a member of the Polyomaviridae family detected in 1971 in the urine of an immunosuppressed renal transplant recipient who developed ureteric stenosis. BKV has a worldwide seroprevalence of about 90%. After the primary infection, BKV establishes a life-long latency within the urogenital tract. The severe immunological impairment occurring in transplant kidney recipients leads to BKV reactivation that may result in the polyomavirus associated nephropathy (PVAN). During transplantation, kidney is subjected to hypoxic conditions, driven by the action of Hypoxia Inducible Factor (HIF). It has been proved that HIF isoform-1α (HIF-1α) may interact with several viruses, but till now there are no evidences regarding the interaction between BKV and HIF-1α. In the present study, we aimed to achieve a better understanding about the relation between BKV infection and hypoxia conditions in kidney cells in case of transplantation. Firstly, 17 kidney paraffin-embedded tissue samples were collected from kidney transplant patients, who developed or not PVAN (PVAN and NOT PVAN group) and from a control group in order to evaluate HIF-1α expression levels in vivo. Total RNA was extracted from paraffin embedded tissues, reverse transcripted into cDNA and HIF-1α expression was measured by means of a qualitative Real Time PCR. Then, in vitro experiments were conducted using the VERO cell line to evaluate the possible interaction between the BKV promoter and HIF-1α. Luciferase and Chromatin Immunoprecipitation (ChIP) assays were performed on BKV transfected VERO cells, to verify the interaction between the viral promoter and HIF-1α. In parallel, to clarify the nature of the interaction between HIF-1α and the BKV promoter, the sequences were in silico analyzed using BLASTN 2.2.32+ software to find whether one or more hypoxia response elements (HRE) core sequences were present in the BKV promoters. Finally, the effect of hypoxia on BKV replication was assessed by evaluating BKV replication in BKV infected VERO cells, treated and not treated with the hypoxia mimic Cobalt Chloride (CoCl2). The HIF-1α expression level resulted 13.6 folds higher in PVAN tissues than in the control group while no differences were observed between the NOT PVAN tissues and the control group. Luciferase assay showed that the presence of HIF-1α stabilized the BKV promoter, increasing its activity from 2-folds to 6-folds (p<0.05) in transfected cells. ChIP assay showed a physical interaction between HIF-1α and the BKV promoter. BLASTN analysis showed no match between HRE sequence and the BKV promoter sequences, confirming that no binding sites for HIF-1α are present in the viral promoter. Finally, data obtained from BKV infected VERO cells revealed that BKV viral load was 5-fold increased in CoCl2 treated cells compared to not treated infected cells. These data, taken together, are significant to define the role of hypoxic stress in BKV replication after renal transplantation. In particular, it can be concluded that the replication of this opportunistic virus, mainly due to immunosuppressive therapy, is furthermore stimulated and favorite by HIF-1α activation, driven by hypoxic conditions during transplantation. The experimental results suggested a hypothetical molecular mechanism underling this thesis, which can be outlined as follows: cellular response to hypoxic environment prevents HIF-1α proteasomal degradation, allowing the creation of a HIF-1 active complex. This complex translocates to the nucleus where it binds to the BKV promoter, stimulating the transcription of viral genes and promoting the BKV replication. If confirmed by further experiments, this scenario may have important clinical implications: exposition to hypoxia during renal transplantation process should be considered as a crucial risk factor for the development of PVAN. These findings could be translated into clinical practice, replacing modulation of HIF system with ex-vivo organ preservation technologies, such as extra-corporeal membrane oxygenation, or considering HIF-1α as a target to be inhibited, perhaps using RNA interference technology.