Parvovirus B19 NS1 protein nuclear transport: a new potential therapeutic target

Parvovirus B19 (B19V) is a human pathogenic virus whose clinical manifestations include transient aplastic crisis, pure red cells aplasia and persistent viremia in adults, fifth disease in children and in fetuses hydrops fetalis and death. Neither effective antiviral therapy nor a vaccine is currently available for this clinically relevant virus. As other parvoviruses, B19 virion contains a ssDNA genome encoding structural and non-structural proteins. Among the non-structural proteins there is NS1 which is multifunctional and essential for virus replication, being involved in interaction with host cell components, as well as transcription and replication of the viral genome in the host cell nucleus. Since the nuclear import pathway of NS1 is not characterized, in this thesis it has been investigated from a functional point of view via quantitative confocal laser scanning microscopy (CLSM), Bioluminescence Resonance Energy Transfer (BRET) as well as upon infection of UT7/EpoS1 cells with B19V. Data analysis showed that the NS1 protein presents a classical nuclear localization signal that allows nuclear import in an energy dependent manner, via the importin (IMP) αβ pathway; to this purpose, the lysine 177 appeared to be essential, since its mutation into threonine abrogates interaction with IMPα and nuclear accumulation of NS1 when transiently expressed in Mammalian cells, or in the context of a B19V minigenome. BRET and CLSM assays showed that the protein can multimerize at the level of cytoplasm before nuclear entry. Treatment with the well-known IMP/ inhibitor Ivermectin reduced viral replication in UT7/EpoS1 infected cells. Therefore, since NS1 is crucial for viral life cycle, and it must be imported into the nucleus to exert its activities, its nuclear transport could be a promising therapeutic target.