Intracellular Trafficking of BK Polyomavirus: From the ER to the Nucleus

[摘要] BK Polyomavirus (BKPyV) is a widespread human pathogen that establishes a lifelong persistent infection in the kidneys and can cause severe disease in immunosuppressed patients. BKPyV is a nonenveloped DNA virus whose productive intracellular trafficking pathway requires trafficking through the endoplasmic reticulum (ER); however, it is unclear how BKPyV exits the ER and undergoes nuclear entry. In this dissertation, I elucidated the role of the ER-associated degradation (ERAD) pathway and show that BKPyV traffics through the cytosol en route to the nucleus. Furthermore, I determined the importance of the nuclear localization signal located on the minor capsid proteins VP2 and VP3 during BKPyV trafficking. These studies were performed in renal proximal tubule epithelial (RPTE) cells, a natural host cell, and provide a relevant model for BKPyV infection. Using ERAD inhibitors, I showed that ERAD is required for productive entry. Altered trafficking and accumulation of uncoating intermediates was detected in the presence of inhibitor.Additionally, there was a change in localization of uncoated virus within the ER during proteasome inhibition to a calnexin-rich subregion, indicating virus is accumulating in an ER subcompartment. By comparing the cytosolic entry of the related polyomavirus SV40, I found that trafficking varied between immortalized CV-1 cells and primary RPTE cells. Additionally, by measuring viral DNA in the cytosol I found that ERAD inhibition led to a decrease in cytosolic viral DNA, supporting a role for ERAD in exit into the cytosol.To elucidate the nuclear entry mechanism, a nuclear localization signal (NLS) in the C-terminus of both minor capsid proteins was first shown to be important for nuclear localization by site directed mutagenesis. The analogous mutation in the genome caused attenuation of infectivity based on an entry defect post-ER trafficking. The role of the NLS in the individual minor capsid proteins was addressed using pseudovirus containing mutations in one or both minor capsid proteins. Only mutation of the VP3-NLS led to an attenuated phenotype in 293TT cells. Overall these data are the first to show that BKPyV traffics through the cytosol and the minor capsid protein NLS is important during BKPyV cellular entry in RPTE cells.

[发布机构] University of Michigan

[效力级别] Microbiology and Immunology

[关键词] BK Polyomavirus;Microbiology and Immunology;Science;Cellular & Molecular Biology