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

Human papillomavirus (HPV) is the most common sexually transmitted pathogen in the United States, causing 99% of cervical cancers and 5% of all human cancers worldwide. HPV infection requires transport of the viral genome (vDNA) into the nucleus of basal keratinocytes. During this process, minor capsid protein L2 facilitates subcellular retrograde trafficking of the vDNA from endosomes to the Golgi, and accumulation at host chromosomes during mitosis for nuclear retention and localization during interphase. Here we investigated the relationship between cytosolic GSH and HPV16 infection. siRNA knockdown of GSH biosynthetic enzymes results in a partial decrease of HPV16 infection. Likewise, infection of HPV16 in GSH depleted keratinocytes is inefficient, an effect that was not seen with adenoviral vectors. Analysis of trafficking revealed no defects in cellular binding, entry, furin cleavage of L2, or retrograde trafficking of HPV16, but GSH depletion hindered post-Golgi trafficking and translocation, decreasing nuclear accumulation of vDNA. Although precise mechanisms have yet to be defined, this work suggests that GSH is required for a specific post-Golgi trafficking step in HPV16 infection.

Homeostasis of cellular thiol and disulfide redox is largely maintained by a large 91 intracellular pool of glutathione (GSH). Given natural redox gradient of the epithelium, 92 5 and the prominent role of GSH in maintaining redox balance (35) we sought to 93 investigate the role of cellular GSH in HPV16 infection. We find that siRNA knockdown 94 of key enzymes in the GSH synthesis pathway impairs HPV16 pseudovirus infection. 95 Depletion of the intracellular GSH pool caused a marked decrease in the infection of 96 HPV16 but not adenoviral vectors. GSH was not important for HPV16 binding, endocytic 97 uptake, cleavage of minor capsid protein L2, or trafficking of vDNA to the TGN, but was 98 critical for efficient post-Golgi trafficking and intranuclear delivery of HPV16 L2/vDNA. 99 Further work will be necessary to specifically define the GSH-dependent factors 100 necessary for HPV16 infection.

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forming GSH (38). Cells normally maintain GSH at high concentrations, ranging from 1-110 10 mM. Such a high concentration of reduced GSH protects cells from oxidative stress 111 and reactive oxygen species (ROS), through the actions of free GSH and GSH-112 dependent glutathione peroxidases and glutaredoxins (39,40). Glutathione reductase 113 (GR, EC 1.8.1.7) maintains a high GSH/GSSG ratio by converting oxidized GSSG back 114 6 to two molecules of reduced GSH (41). This high concentration of reduced GSH 115 maintains the cytosol in a reduced state, favoring free thiols over disulfides. 116 To understand whether GSH and a reducing cytosolic environment is important 117 for HPV16 infection, we targeted the enzymes GCL, GSS and GR for siRNA knockdown 118 and measured HPV16 infection in HaCaT cells. The heterodimeric enzyme GCL is 119 comprised of two protein subunits; the catalytic GCLc and the modulatory GCLm (42).

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Maximal catalytic activity of GCL holoenzyme is only achieved upon binding of the 121 modulatory GCLm to the catalytic GCLc subunit. GCLm is expressed at lower levels 122 than GCLc, and is therefore rate-limiting in the formation of active GCL holoenzyme (43).

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Thus, we chose to target the GCLm subunit for siRNA knockdown experiments.

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HPV16 infection dropped about 40-50% upon knockdown of either the 125 modulatory subunit of GCL or GR (Fig.1B, levels of GSH to be observed. After 72h treatment with 800µM BSO, cytosolic GSH 138 levels were drastically depleted compared to the control vehicle-treated cells (Table 1).

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BSO treated HaCaT cells displayed a slightly slower proliferation rate and cell cycle 140 anaylsis by PI staining revealed a subtle but statistically insignificant expansion of S 141 phase upon BSO treatment (Fig 2A,B). This is consistent with other studies that BSO 142 treatment does not considerably alter cell cycle kinetics (47,48 was detected by non-reducing SDS-PAGE and western blot. Similar levels of full-length 160 8 disulfide-linked L1 dimers and trimers were observed in control or BSO treated samples 161 that did not receive the high pH washes (Fig 3A,B), indicating that GSH depletion had 162 no effect on HPV16 binding and the the high pH wash does indeed remove extracellular 163 virus.

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Next we investigated viral uptake by pre-binding HPV16 to cells followed by a 2h 165 shift to 37°C to allow entry. After 2h cells were either processed for non-reducing L1 166 western blot or cleared of extracellular virus by high pH washing and returned to 37C for 167 additional times. In this manner we are able to track the population of intracellular 168 HPV16 that has entered the cells during the initial 2h incubation. Upon return to 37°C 169 this "2h wave" of incoming virus will continue trafficking through the degradative is not an obligate step of the infectious trafficking pathway, it does serve as a proxy for 182 endolysosomal trafficking (8,50,51). Exposure of the L1-7 epitope, which is buried within 183 the intact capsid (52), is a useful marker for degradative uncoating and normally occurs 184 by 6-8h post infection (53). Control and BSO treated cells were infected with HPV16 for 185 2h or 8h and total L1 or L1-7 levels were visualized by immunofluorescence staining 186 and confocal microscopy. L1-7 was undetectable in either group at the early 2h time 187 point, as expected ( Fig 3D). Both the control and BSO treated groups displayed 188 significant L1-7 signal by 8h, indicative of capsid degradation and proper endolysosomal 189 trafficking ( Fig 3D). Taken together these data suggest GSH depletion does not 190 significantly affect HPV16 binding, entry, or early subcellular trafficking events.  Although the precise mechanisms underlying the requirement for GSH remain to 258 be determined, these findings are novel and significant because they represent only the 259 second broad cellular "factor" necessary for this enigmatic process, the first being