Study of interaction between BK virus large T-antigen and agnoprotein

Abstract

Human polyomavirus BK (BKV) is a non enveloped virus with a double-stranded, circular DNA genome. BKV infects >70% of the human population world-wide. Infection occurs predominantly during childhood and the virus remains in a latent state throughout life in the immune competent individuals. In the context of immunosuppression, however, reactivation occurs and can lead to renal stenosis and interstitial nephritis in kidney transplant patients, and hemorrhagic cystitis in bone narrow transplant patients. Moreover, BKV has been associated with several human cancers, but its causal role remains disputed. One of BKV’s protein known as agnoprotein may play a role in these pathogenic processes. To develop antiviral therapy it is required to elucidate the exact biological function of this protein. One way to examine the function of agnoprotein is by identifying possible cellular interaction partners. Another way is to understand agnoprotein’s role in the viral life cycle. Thereto, we examined the interaction of agnoprotein with another viral protein, large T-antigen (LT-ag) and the functional implication of this interaction. First, we investigated the effect of agnoprotein on the transcriptional activity of LT-ag on the BKV early promoter by transient transfection studies in HEK293. Our results revealed that LT-ag affects BKV early promoter in a concentration-dependent manner with low concentrations of LT-ag inhibiting, while high concentrations stimulated BKV early promoter activity. Co-expression of agnoprotein repressed LT-ag-induced activation of the BKV early promoter, suggesting that agnoprotein may exert a negative regulatory effect on transactivation by LT-ag. To test whether agnoprotein mediates its effect through direct interaction with LT-ag, we studied a possible association between these proteins. GST pulldown, co-immunoprecipitation (in vivo and in vitro), and mammalian two hybrid studies confirmed an interaction between LT-ag and agnoprotein.