High-risk human papillomaviruses (HPVs) cause 5% of all human cancers worldwide. The HPV capsid consists of 72 disulfide-linked pentamers of major capsid protein L1 and up to 72 molecules of minor capsid protein L2. The viral genome (vDNA) is 8KB circular dsDNA, condensed with histones and complexed with L2. HPV infection requires the virion particle to get access to basal layer keratinocytes, binding and entry of the cells, uncoating, and transport of the viral genomes to the host cell nucleus. During infection, L2 is important for transport of the viral genome from membrane bound vesicular compartments, through the cytosol and into the host cell nucleus. Previous work has identified a conserved disulfide bond between Cys22 and Cys28, which is necessary for HPV16 infection. We hypothesize that endosomal reduction of this disulfide might be important for L2 conformational changes that allow a hydrophobic transmembrane-like region in L2 to span across endosomal membranes, exposing sorting adaptor binding motifs within L2 to the cytosol. Prior research suggests that cytosolic glutathione (GSH) redox potential is important for reduction of disulfide-linked proteins within the lumen of endosomes. This is achieved by endosomal influx of cytosolic reduced cysteine, where it can reduce disulfide bonds in lumenal proteins. Cytosolic GSH regenerates the pool of reduced cysteine needed to maintain endosomal redox potential. Here we studied the relationship between cytosolic GSH and HPV16 infection. siRNA knockdown of critical enzymes of the GSH biosynthesis pathway or the endosomal cystine efflux pump cystinosin caused partial abrogation of HPV16 infection. Likewise, inhibition of the GSH biosynthesis pathway with L-buthionine sulfoximine (L-BSO) blocked HPV16 infection in multiple cell types, suggesting that cytosolic GSH redox may be important for HPV16 infection. Further studies have revealed that the decrease of HPV16 infection is not because of defects in binding, entry, L2 cleavage or capsid uncoating, but rather is due to inefficient cytosolic translocation of L2/viral genome from the trans-Golgi network (TGN). Contrary to our initial hypothesis, we show that L2 is able to span the endosomal membrane and direct TGN localization in the presence of BSO. Lack of cytosolic GSH causes L2/viral genome to become trapped in the TGN lumen. This suggests that there are redox-sensitive viral or cellular factors necessary for L2/viral genome translocation at the TGN. Future research will focus on the redox state of the Cys22-Cys28 disulfide bond during infection of normal and GSH-depleted cells.

Thesis advisor: Junona Moroianu / The L2 minor capsid protein of human papillomavirus is one of two structural proteins that comprise the icosahedral shell. Two potential, leucine-rich nuclear export signals (NESs) had been identified in the HPV16 L2 sequence, one in the n-terminus (51MGVFFGGLGI60) and one in the c-terminus (462LPYFFDSVSL471). DNA primers for mutant L2 proteins were designed to specifically target these two potential NES regions. Two primers had mutations in the n-terminal located NES (nNES), while the other two primers had mutations in the c-terminal NES (cNES). L2 nuclear retention mutants, RR297AA (“MS4”) and RTR313AAA (“MS5”), served as the templates for these NES mutations. Using mutagenesis, the desired secondary mutations were introduced into the mutant L2 genes in order to create four, distinct mutants: RR297AA + P463_ (“MS4 T1”), RR297AA + V469_ (“MS4 T2), RTR313AAA + P463_ (“MS5 T1”), and RTR313AAA + V469_ (“MS5 T2”). In contrast to the pancellular localization of the MS4 and MS5 L2 mutants, the “MS4 T1,” “MS4 T2,” “MS5 T1”, and “MS5 T2” mutants were all localized nuclearly. These results suggest that deletion of the cNES inhibits nuclear export of the HPV16 L2 minor capsid protein. / Thesis (BS) — Boston College, 2011. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology Honors Program. / Discipline: Biology.