Glutathione is Required for Efficient Post-Golgi Trafficking of Incoming HPV16 Genome

Abstract

24Human papillomavirus (HPV) is the most common sexually transmitted pathogen in the 25 United States, causing 99% of cervical cancers and 5% of all human cancers worldwide. 26 HPV infection requires transport of the viral genome (vDNA) into the nucleus of basal 27 keratinocytes. During this process, minor capsid protein L2 facilitates subcellular 28 retrograde trafficking of the vDNA from endosomes to the Golgi, and accumulation at 29 host chromosomes during mitosis for nuclear retention and localization during 30 interphase. Here we investigated the relationship between cytosolic GSH and HPV16 31 infection. siRNA knockdown of GSH biosynthetic enzymes results in a partial decrease 32 of HPV16 infection. Likewise, infection of HPV16 in GSH depleted keratinocytes is 33 inefficient, an effect that was not seen with adenoviral vectors. Analysis of trafficking 34 revealed no defects in cellular binding, entry, furin cleavage of L2, or retrograde 35 trafficking of HPV16, but GSH depletion hindered post-Golgi trafficking and 36 translocation, decreasing nuclear accumulation of vDNA. Although precise mechanisms 37have yet to be defined, this work suggests that GSH is required for a specific post-Golgi 38 trafficking step in HPV16 infection. 39 40 42 in the United States (1). Currently there are >300 HPV types that have been deposited 43 and annotated in the Papillomavirus Episteme (PAVE) database (2). Based on disease 44 association, HPV can be divided into low-risk HPV and cancer-associated high-risk HPV 45 (3). Most HPVs are considered as low-risk HPV, causing cutaneous and mucosal warts 46 3(4). However, 99% of cervical cancers and 5% of all human cancers worldwide are 47 caused by high-risk HPV infection (5,6). HPV16 belongs to the high-risk HPV, and 48 HPV16 alone is responsible for >50% cervical cancers (7). There are highly effective 49 vaccines being used to prevent high-risk HPV infection, but the vaccine cannot protect 50 against all types of high-risk HPV infection and the high cost prevents people from 51 developing world to get access to the vaccine (7). 52HPVs are small non-enveloped DNA viruses. The icosahedral capsid is built from 53 72 pentamers (360 molecules) of the major capsid protein L1. Within this particle, 54 variable copies (<72 molecules but typically 20-40) of minor capsid protein L2 are 55 complexed to the ~8kb dsDNA genome (vDNA). At the cellular level, HPV infection 56 begins with virion attachment to keratinocytes via heparin sulfate proteoglycans 57 (HSPGs) followed by furin-and kalleikrein-dependent cleavage of the capsid (8,9), 58 likely causing conformational changes and transfer to secondary entry receptor 59 complex(es). The nature of the entry complex(es) is still debatable but likely include 60growth factor receptor tyrosine kinases, integrins, tetraspanins, and annexin A2 (10,11). 61Asynchronous cellular uptake of virion occurs by macropinocytosis-like mechanisms 62 after this potentially prolonged residence on the cell surface (12,13). 63Internalized virions initially traf...