Papillomaviruses (PV) are double stranded DNA viruses with approximately an 8 kilobase genome. Both BPV1 and HPV16 (the genotypes used in this dissertation) have been shown to utilize a novel clathrin /caveolar pathway in order to establish infection. Although many studies have been done to illustrate the trafficking of the PV genome from the cell membrane to the nucleus, limited research has been done to determine what causes the sorting of the PV genome to either the "infectious" or "non-infectious" pathway.
The purpose of the work presented in this dissertation was to determine some of the factors involved in sorting BPV1 and HPV16 to either the "infectious" or the "non-infectious" pathways. Our studies were initiated by the observation that the BPV1-L2 capsid protein interacted with an E3 ligase, Smad ubiquitin regulatory factor 2 (Smurf2). We demonstrated that the BPV1L2-Smurf2 interaction led to degradation of capsid proteins which correlated with a decrease in BPV1 infection. We determined that the decrease in BPV1 infection was due to a decrease in viral transcription suggesting that insufficient viral genome delivery to the nucleus had occurred. BPV1 pseudovirions were found to accumulate in the lysosome in the presence of Smurf2 and ubiquitin and did not traffic to caveolin-1 positive vesicles (i.e. the infectious pathway). Inhibition of the lysosome with the inhibitor, leupeptin showed a decrease in infection. The endo-lysosomal cysteine proteases, cathepsins B and L were described in the literature as playing a role in the binding, entry and infection of several enveloped viruses but not in non-enveloped viruses. Our data showed that broad cysteine protease inhibitors and specific cathepsin B or L inhibitors were able to decrease infection, suggesting that cysteine proteases were in part mediating HPV16 infection. This was a novel finding because cathepsins B and L had not been shown to play a role in the processing of non-enveloped viruses. We also confirmed a loss of movement of HPV16 reporter-virions from endosomes to caveolin-1 positive vesicles that could account for the loss of infection observed when using the neutralizing agent NH4Cl. To our surprise the pre-treatment of purified viral particles with cathepsin B, but not cathepsin L, enhanced infectivity of HPV16 and overcame the block of infection observed in the presence of furin inhibitor. Furin protease had been shown to be necessary for viral infection by allowing the escape of the viral particle from an endosome.
The work in this dissertation alludes to cellular factors that affect PV viral genome sorting to the infectious or non-infectious pathways. The significance of the study is that virus infection can be blocked upon alteration of cellular proteins which affect viral trafficking. Additional studies need to be done to further identify cellular proteins that are involved in PV sorting.
|Advisor:||Meneses, Patricio I., Yu, Chao-Lan|
|Commitee:||Everly, David, Gilman-Sachs, Alice, Glucksman, Marc J., Meneses, Patricio I., Yu, Chao-Lan|
|School:||Rosalind Franklin University of Medicine and Science|
|Department:||Microbiology & Immunology|
|School Location:||United States -- Illinois|
|Source:||DAI-B 72/08, Dissertation Abstracts International|
|Subjects:||Molecular biology, Microbiology, Virology|
|Keywords:||Cathepsin b, Papillomavirus, Smurf2, Sorting|
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