A Conserved Amino Acid in the C-Terminus of HPV E7 Mediates Binding to PTPN14 And

bioRxiv preprint doi: https://doi.org/10.1101/2020.05.22.111740; this version posted May 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

1 Title: A conserved amino acid in the C-terminus of HPV E7 mediates binding to PTPN14 and

2 repression of epithelial differentiation

4 Running Title: C-terminal arginine of HPV E7 mediates PTPN14 binding

6 Joshua Hatterschidea, Alexis C. Brantlya, Miranda Graceb, Karl Mungerb, Elizabeth A. Whitea*

8 aDepartment of Otorhinolaryngology: Head and Neck Surgery, University of Pennsylvania

9 Perelman School of Medicine, Philadelphia, PA, USA

11 bDepartment of Developmental, Molecular and Chemical Biology, Tufts University School of

12 Medicine, Boston, MA, USA

14 * Correspondence: Elizabeth A. White, [email protected]

16 Abstract word count: 390 (Abstract and Importance)

17 Text word count: 5494

18 ABSTRACT

19 The human papillomavirus (HPV) E7 oncoprotein is a primary driver of HPV-mediated

20 carcinogenesis. The E7 proteins from diverse HPV bind to the host cellular non-receptor protein

21 tyrosine phosphatase type 14 (PTPN14) and direct it for degradation through the activity of the

22 E7-associated host E3 ubiquitin ligase UBR4. Herein we show that a highly conserved arginine

23 residue in the C-terminal domain of diverse HPV E7 mediates interaction with PTPN14. We

24 found that disruption of PTPN14 binding through mutation of the C-terminal arginine did not

25 impact the ability of several high-risk HPV E7 proteins to bind and degrade the retinoblastoma

26 tumor suppressor or activate E2F target gene expression. HPVs infect human keratinocytes and

27 we previously reported that both PTPN14 degradation by HPV16 E7 and PTPN14 CRISPR

28 knockout repress keratinocyte differentiation-related genes. Now we have found that blocking

29 PTPN14 binding through mutation of the conserved C-terminal arginine rendered both HPV16

30 and HPV18 E7 unable to repress differentiation-related gene expression. We then confirmed

31 that the HPV18 E7 variant that could not bind PTPN14 was also impaired in repressing

32 differentiation when expressed from the complete HPV18 genome. Finally, we found that the

33 ability of HPV18 E7 to extend the lifespan of primary human keratinocytes required PTPN14

34 binding. CRISPR/Cas9 knockout of PTPN14 rescued keratinocyte lifespan extension in the

35 presence of the PTPN14 binding-deficient HPV18 E7 variant. These results support the model

36 that PTPN14 degradation by high-risk HPV E7 leads to repression of differentiation and

37 contributes to its carcinogenic activity.

38 39 IMPORTANCE

40 Human papillomavirus (HPV)-positive carcinomas account for nearly 5% of the global

41 human cancer burden. The E7 oncoprotein is a primary driver of HPV-mediated carcinogenesis.

42 HPV E7 binds and degrades the putative tumor suppressor, PTPN14. However, the impact of

43 PTPN14 binding by E7 on cellular processes is not well defined. Here, we show that PTPN14

44 binding is mediated by a conserved C-terminal arginine residue of HPV E7 in vivo. Additionally,

45 we found that PTPN14 binding contributes to the carcinogenic activity of HPV18 E7 (the second

46 most abundant HPV type in cancers). Finally, we determined that PTPN14 binding by HPV16

47 E7 and HPV18 E7 represses keratinocyte differentiation. HPV-positive cancers are frequently

48 poorly differentiated and the HPV life cycle is dependent upon the keratinocyte differentiation

49 program. The finding that PTPN14 binding by HPV E7 impairs differentiation has significant

50 implications for both HPV-mediated carcinogenesis and the HPV life cycle.

52 INTRODUCTION

53 Infection with oncogenic human papillomaviruses (HPVs) can cause several epithelial

54 carcinomas including essentially all cervical cancers, a subset of oropharyngeal cancers, and

55 several other anogenital cancers. These HPV-positive cancers account for approximately 5% of

56 the human cancer burden (1). HPVs are non-enveloped dsDNA viruses that infect the basal

57 keratinocytes of cutaneous or mucosal stratified squamous epithelia and couple their replication

58 to keratinocyte differentiation (2). Hundreds of genetically diverse HPV genotypes have been

59 identified to date (3). Only 13-15 of the mucosal-tropic HPVs, termed the ‘high-risk’ HPVs, are

60 significantly associated with carcinogenesis. Infection with a high-risk HPV is the first step in the

61 carcinogenic process. Cells persistently infected with high-risk HPV can accumulate secondary

62 hits such as viral genome integration into a host chromosome. This can lead to dysregulated,

63 constitutive expression of the viral E6 and E7 oncoproteins, which are required for keratinocyte

64 immortalization and for the transformed phenotype in cervical carcinoma cell lines (4–8).

65 HPV E6 and E7 are multifunctional viral proteins that lack enzymatic activity. They can

66 alter host cellular processes through interactions with cellular proteins. One activity of many

67 HPV E7 is to bind and inactivate the retinoblastoma tumor suppressor (RB1) (9, 10). This

68 results in the release of E2F transcription factors and promotes entry into the cell cycle. High-

69 risk HPV E7 additionally promote the proteasomal degradation of RB1, in the case of HPV16 E7

70 through the recruitment of a Cullin 2-Zer1 E3 ubiquitin ligase complex (11, 12). High-risk HPV

71 E6 direct the tumor suppressor p53 for proteasome-mediated degradation (13, 14), blocking the

72 induction of apoptosis that would arise from E7-mediated stabilization of p53 (15, 16).

73 Binding to RB1 is necessary but not sufficient to explain the carcinogenic activities of

74 HPV E7 (17–27). We have begun to characterize a second putative tumor suppressor, the non-

75 receptor protein tyrosine phosphatase PTPN14, that is bound and degraded by HPV E7 (11, 28,

76 29). High-risk HPV E7 direct PTPN14 for proteasome-mediated degradation dependent on the

77 interaction between E7 and the host E3 ubiquitin ligase UBR4 (28, 30). Our early experiments

78 determined that amino terminal sequences in HPV E7 were required for the interaction with

79 UBR4 whereas binding to PTPN14 mapped broadly to the C-terminus of E7. HPV E7 binds to

80 the conserved C-terminal protein tyrosine phosphatase domain of PTPN14. The

81 E7/UBR4/PTPN14 complex is genetically and biochemically separable from the E7/RB1

82 complex (28, 29).

83 PTPN14 is proposed to inhibit the HIPPO pathway transcriptional coactivators YAP1 and

84 TAZ. The inhibitory activity of PTPN14 may involve interaction(s) with the HIPPO pathway

85 regulators KIBRA and LATS1 (31–33) and/or direct binding of PTPN14 to YAP1, promoting

86 YAP1 cytoplasmic sequestration (34). PTPN14 phosphatase activity is not required for its

87 inhibition of YAP1. The consequences of PTPN14 degradation in HPV E7-expressing or HPV-

88 positive cells are incompletely understood.

89 To determine the effect of PTPN14 degradation by HPV E7, we have begun to

90 characterize E7 variants that cannot degrade PTPN14. We recently described a variant of

91 HPV16 E7, HPV16 E7 E10K, that was found among HPV16 sequences from clinical samples

92 (35). HPV16 E7 E10K could not bind to UBR4 and consequently could not direct PTPN14 for

93 degradation (29). Our experiments indicated that PTPN14 degradation by HPV16 E7 impaired

94 the expression of differentiation-related genes in keratinocytes. Our data also supported that

95 PTPN14 degradation could contribute to the carcinogenic activity of high-risk HPV E7.

96 Compared to prototypical HPV16 E7, HPV16 E7 E10K was impaired in its ability to immortalize

97 HFKs when co-expressed with HPV16 E6.

98 Yun and colleagues recently reported the crystal structure of the phosphatase domain of

99 PTPN14 in complex with the C-terminal domain of HPV18 E7 (36). The two proteins bind

100 directly and with high affinity (KD=18.2 nM). Several interactions between amino acids on

101 PTPN14 and on HPV18 E7 contribute to the protein-protein interaction, including an

102 electrostatic interaction of HPV18 E7 Arg84 with PTPN14 Glu1095. In vitro binding experiments

103 confirmed our previous observation that E7 proteins encoded by diverse HPV bind to PTPN14.

104 Using a structure-guided mutational approach, the authors designed a PTPN14-binding

105 deficient HPV18 E7 and found that it was impaired in promoting keratinocyte growth and

106 migration. The C-terminal arginine that was identified by Yun and colleagues to directly

107 contribute to PTPN14 binding is highly conserved among HPV E7. Eighty-three percent of

108 genus alpha E7 have an arginine at this position and 92% have a positively charged residue.

109 Here we show that the conserved C-terminal arginine is required for diverse HPV E7

110 proteins to bind and degrade PTPN14 in cells. Consistent with our previous results, we find that

111 PTPN14 binding contributes to the ability of high-risk HPV E7 to repress keratinocyte

112 differentiation. We extend this result to include cells that contain the complete HPV genome.

113 Finally, we find that PTPN14 binding by HPV18 E7 is required for the keratinocyte lifespan

114 extension activity of HPV18 E7.

115 RESULTS

116 Mutation of a conserved C-terminal arginine residue in diverse HPV E7 restores PTPN14

117 protein levels

118 HPV E7 proteins form a complex with PTPN14 and the host E3 ubiquitin ligase UBR4 to direct

119 PTPN14 for proteasome mediated degradation (28). We have shown that mutations in HPV16

120 E7 that abrogate UBR4 binding also impair degradation of PTPN14 and prevent HPV16 E7 from

121 repressing differentiation gene expression (29). However, we had not characterized any variant

122 of E7 that did not bind to PTPN14 nor had we extended our analysis to include genotypes other

123 than HPV16.

124 HPV E7 proteins from diverse genotypes have some conserved and some divergent

125 amino acid sequences (Figure 1A). Diverse HPV E7 proteins bind to PTPN14 (11, 28). Given

126 the connection we recently established between PTPN14 and differentiation (29), we re-

127 examined the relationship between diverse genus alpha HPV E7 and PTPN14 protein levels.

128 We transduced hTert-immortalized human foreskin keratinocyte (N/Tert-1) cells with retroviral

129 vectors encoding HPV E7 from several genus alpha HPV types. Using newly derived, passage-

130 matched keratinocyte pools stably expressing HPV E7, we found that E7 proteins from high-risk,

131 low-risk, and non-cancerous genus alpha HPV reduced steady-state PTPN14 protein levels

132 (Figure 1B). Consistent with our previous results, high-risk HPV18 E7 expression resulted in the

133 most substantial reduction in PTPN14 protein levels.

134 We and others previously determined that PTPN14 binding maps broadly to the carboxyl

135 terminus of HPV E7 (28, 30). A recent crystal structure of the C-terminus of HPV18 E7 in

136 complex with the PTP domain of PTPN14 determined that the two proteins bind directly and

137 elucidated the PTPN14-HPV18 E7 binding interface (36). Several residues of HPV18 E7 were

138 found to be critical for this interaction, including Arg84 (R84; R77 in HPV16 E7) which makes an

139 electrostatic interaction with Glu1095 of PTPN14. The corresponding arginine residue is highly

140 conserved among HPV E7 proteins. It is completely conserved among the prototypical E7 open

141 reading frames (ORFs) from high-risk HPV (Figure 1A), 83% conserved among E7 ORFs from

142 genus alpha, and 80% conserved among E7 ORFs from 193 diverse HPV genotypes. The high

143 degree of conservation of the C-terminal arginine may indicate strong selective pressure for

144 HPV E7 to maintain the ability to bind PTPN14.

145 The requirement for the C-terminus of E7 to bind PTPN14 led us to characterize a

146 naturally occurring variant of HPV16 E7 identified by Mirabello and colleagues (35). The variant,

147 HPV16 E7 R77S, has a mutation in the conserved arginine residue (Arg77 to Ser; R77S). We

148 hypothesized that this HPV16 E7 might be unable to bind and degrade PTPN14. To test if

149 mutation of the C-terminal arginine impairs PTPN14 degradation by HPV E7 proteins, we

150 engineered the arginine to serine mutation into the E7 ORFs from HPV16, HPV31, HPV18, and

151 HPV6. We then transduced N/Tert-1 cells with retroviruses encoding the E7 variants, the

152 corresponding wild-type/prototypical (WT) E7s, and several additional mutants as controls. The

153 control variants are: HPV16 E7 E10K (does not bind UBR4) (29), HPV16 E7 Δ21-24 (does not

154 bind RB1) (10), and HPV18 E7 D10K (contains the mutation analogous to HPV16 E7 E10K).

155 We found that cells expressing HPV16 E7 R77S, HPV31 E7 R77S, HPV18 E7 R84S, and HPV6

156 E7 R77S exhibited higher PTPN14 protein levels than the corresponding WT E7-expressing cell

157 lines (Figure 1C and 1D). This is consistent with the idea that the C-terminal arginine is required

158 for diverse genus alpha HPV E7 proteins to interact with PTPN14. In agreement with our

159 previous results, PTPN14 expression was restored in cells expressing the HPV16 E7 E10K

160 variant (29). HPV18 E7 D10K only partially restored PTPN14 expression (Figure 1D). These

161 results support the notion that the C-terminal HPV E7 binding interface may be highly conserved

162 to mediate PTPN14 binding.

163

164 Mutation of the C-terminal arginine in high-risk HPV E7 does not affect RB1 degradation

165 or E2F target gene activation.

166 In addition to binding PTPN14, HPV E7 proteins bind and inactivate RB1 (9, 10). High-

167 risk HPV E7 proteins additionally direct RB1 for proteasomal degradation (11, 12). We found

168 that HPV16 E7 R77S, HPV31 E7 R77S, and HPV18 E7 R84S were comparable to WT E7

169 proteins with respect to their capacity to reduce RB1 steady-state protein levels (Figure 1C). To

170 test whether HPV16 E7 R77S and HPV18 E7 R84S could induce the expression of E2F target

171 genes downstream of RB1 inactivation, we transduced primary human foreskin keratinocytes

172 (HFK) with retroviral vectors encoding a panel of HPV16 and HPV18 E7 variants. Total RNA

173 was collected from these cell lines and the expression of the E2F target genes CCNE2 and

174 MCM2 was assessed by qRT-PCR. With the exception of HPV16 E7 Δ21-24, which cannot bind

175 RB1, all HPV16 and HPV18 E7 variants including HPV16 E7 R77S and HPV18 E7 R84S were

176 able to induce E2F target gene expression (Figure 2A and B). We conclude that mutation of the

177 C-terminal arginine does not affect RB1 inactivation by high-risk HPV E7 proteins.

178

179 Mutation of the conserved C-terminal arginine disrupts PTPN14 binding by high-risk HPV

180 E7

181 To confirm that mutation of the conserved C-terminal arginine disrupted PTPN14

182 binding, we immunoprecipitated E7 from the N/Tert cell lines that stably express Flag and HA-

183 tagged HPV16 and HPV18 E7 and variant E7. We found that PTPN14 did not

184 coimmunoprecipitate with HPV16 E7 R77S or HPV18 E7 R84S (Figures 3A and 3B). In contrast

185 PTPN14 coimmunoprecipitated with HPV16 E7 WT, HPV18 E7 WT, and HPV16 E7 Δ21-24,

186 which cannot bind RB1. In agreement with the analysis of total cell protein and RNA (Figures 1

187 and 2), HPV16 E7 R77S and HPV18 E7 R84S input cell lysates contained more PTPN14

188 protein than the respective WT E7 cell lysates. HPV16 E7 R77S and HPV18 E7 R84S retained

189 the capacity to coimmunoprecipitate with RB1 and reduce steady state RB1 levels. These data

190 support the model that diverse HPV E7 require the conserved C-terminal arginine to bind to

191 PTPN14 in keratinocytes.

192

193 PTPN14 binding is required for high-risk HPV E7 to repress differentiation

194 We previously determined that PTPN14 degradation by HPV16 E7 is required for an E7-

195 mediated repression of keratinocyte differentiation (29). Therefore, we hypothesized that

196 impairing PTPN14 binding by mutating the C-terminal arginine residue would likewise render

197 high-risk HPV E7 proteins unable to repress differentiation. We used a panel of N/Tert cell lines

198 expressing WT and arginine to serine mutated HPV E7 proteins from the high-risk HPV16 and

199 HPV18. Cells were induced to differentiate by culture in growth factor-free media supplemented

200 with 1.5 mM CaCl2. We collected whole cell lysates at 0 and 72 h post-differentiation and

201 analyzed cytokeratin 1 (KRT1), cytokeratin 10 (KRT10), and involucrin (IVL) by Western blot.

202 KRT1 and KRT10 are early differentiation markers and IVL is a component of the cornified

203 envelope. We found that HPV16 E7 WT and HPV18 E7 WT were able to repress the expression

204 of all three differentiation markers, whereas HPV16 E7 R77S and HPV18 E7 R84S were not

205 (Figure 4).

206 Because HPV18 E7 leads to the greatest depletion in PTPN14 protein levels, we chose

207 to focus on HPV18 E7 for the remainder of this study. Loss of contact with the basement

208 membrane induces keratinocyte differentiation in vivo. This can be simulated in cultured cells by

209 growing keratinocytes in suspension (37–39), which tends to promote high expression of

210 markers of late differentiation. To test whether PTPN14 binding by HPV E7 also impairs

211 differentiation in response to detachment, we cultured our N/Tert cell lines expressing HPV18

212 E7 WT, HPV18 E7 R84S, and the empty vector control in ultra-low adherence plates for 12 h.

213 We collected RNA from adherent cells or cells in suspension and measured the expression of

214 the late differentiation marker IVL by qRT-PCR. IVL mRNA expression was induced in all cell

215 lines after suspension. However, HPV18 E7 R84S displayed increased IVL expression post-

216 differentiation compared to HPV18 E7 WT (Figure 5A), mirroring our results obtained from

217 CaCl2 differentiation. These data indicate that PTPN14 binding by HPV18 E7 is necessary for

218 the repression of differentiation gene expression following differentiation stimuli.

219 Keratinocytes exit the cell cycle after differentiating, which leads to decreased growth

220 capacity. To test if PTPN14 binding by HPV18 E7 affects cellular viability after suspension, we

221 re-plated 1,000 cells that were grown in ultralow adherence plates for 12 h onto standard tissue

222 culture plates and cultured them for an additional 5 days. We then fixed and stained them with

223 crystal violet and measured the area covered by viable cells. We found that HPV18 E7 WT

224 increased viability after stimulus to differentiate in suspension, while the growth of HPV18 E7

225 R84S expressing cells resembled the empty vector control (Figure 5B). These data support that

226 PTPN14 binding by high-risk HPV E7 is required for the impairment of keratinocyte

227 differentiation, promoting cellular viability following a differentiation stimulus.

228

229 Repression of differentiation is the predominant effect of PTPN14 binding by HPV18 E7

230 on the transcriptome of primary keratinocytes

231 We hypothesized that comparing HPV18 E7 and HPV18 E7 R84S in an unbiased

232 analysis of gene expression might reveal additional cellular processes that are up- or down-

233 regulated by E7-mediated PTPN14 binding and degradation. To test this, we transduced

234 primary HFKs with retroviral vectors encoding HPV18 E7 WT, HPV18 E7 R84S, or the empty

235 vector control. Triplicate primary cell populations were established and selected with puromycin.

236 RNA isolated from these cell populations was polyA-selected and analyzed by RNA-sequencing

237 (RNA-seq). We found that 271 genes were differentially regulated between HFK expressing

238 HPV18 E7 WT and HPV18 E7 R84S with cut offs of >1.5-fold change and false discovery rate

239 (FDR) adjusted p-value of <0.05. The majority of these genes (247) were found to be more

240 repressed in cells expressing HPV18 E7 WT than those expressing the HPV18 E7 R84S mutant

241 (Figure 6A). We performed a gene ontology (GO) enrichment analysis on the list of genes with

242 lower expression in wild type HPV18 E7 expressing cells and found that this list was highly

243 enriched for GO terms related to keratinocyte differentiation (Figure 6B). Further analysis

244 revealed that a high percentage of these 247 genes were related to the following GO terms:

245 Cornification, 15%; Keratinocyte Differentiation, 17%; Skin Development, 19%; and Epidermis

246 Development, 20%. Among the 24 genes that were upregulated in cells expressing HPV18 E7

247 WT versus cells expressing HPV18 E7 R84S, there were no statistically significantly enriched

248 GO terms.

249 Based on the strong differentiation signature we specifically focused on a broad array of

250 differentiation-related genes involved in early differentiation, the differentiation-associated

251 desmosome complexes, cornified envelope formation, differentiation-promoting signaling, and

252 differentiation-promoting transcription factors. Analysis of these selected genes revealed that

253 HPV18 E7 WT indeed broadly represses differentiation-related genes in comparison to the

254 empty vector control and that the expression of these genes is restored in cells that express

255 HPV18 E7 R84S (Figure 6C). Furthermore, this RNA-seq analysis confirmed that both HPV18

256 E7 WT and HPV18 E7 R84S promote the expression of cell cycle-related genes such as

257 CCNE2, MCM2, and PCNA that are activated downstream of RB1 degradation. To validate our

258 RNA-seq results, we measured the mRNA expression of several key genes using qRT-PCR.

259 These included IVL, KRT1, and MAF, which is a transcription factor that promotes early

260 differentiation (40). The expression of these three differentiation-related genes was significantly

261 lower in HFK expressing HPV18 E7 WT than those expressing HPV18 E7 R84S or the empty

262 vector control (Figure 6D). In summary, we found that repressing genes related to differentiation

263 was the predominant effect of PTPN14 binding by HPV18 E7 on keratinocyte gene expression

264 in unstimulated keratinocytes.

265

266 The HPV18 E7 C-terminal arginine contributes to differentiation repression in

267 keratinocytes harboring the complete HPV genome

268 Our data support that PTPN14 binding is required for HPV E7 to repress differentiation

269 when HPV E7 is expressed in the absence of other viral proteins. However, other HPV proteins,

270 particularly E6, have been shown to repress differentiation (41–43). To test whether PTPN14

271 binding by HPV E7 contributes to repression of differentiation when E7 is expressed from its

272 native context in the complete viral genome, we introduced the HPV18 E7 R84S mutation into

273 pNeo-loxP-HPV18 (44). We transfected the WT and mutant pNeo-loxP-HPV18 vectors along

274 with NLS-Cre into primary HFK and selected stable populations with G418 (Figure 7A). The

275 resulting cell pools are HFK-HPV18E7 WT and HFK-HPV18E7 R84S. Uptake and excision of the

276 HPV18 genome were confirmed by PCR from DNA collected at an early passage post-

277 transfection (Figure 7B). To characterize the effect of HPV18 E7 WT and R84S on PTPN14 in

278 the presence of the complete HPV genome, protein lysates were collected from each cell line

279 and compared to lysates from the untransfected parental HFKs. We found that PTPN14

280 expression was strongly diminished in HFK-HPV18E7 WT but not in HFK-HPV18E7 R84S (Figure

281 7C). Reduction in RB1 protein levels can serve as a surrogate for high-risk HPV E7 expression.

282 As expected, HFKs harboring either genome displayed reduced RB1 protein levels, suggesting

283 that E7 is expressed from both pNeo-loxP-HPV18 vectors. To test how PTPN14 binding by

284 HPV18 E7 impacts differentiation in the context of the complete genome, these cell populations

285 were stimulated to differentiate with media containing 1.5 mM CaCl2. Total RNA was collected 0,

286 2, and 6 days post differentiation and expression of the early differentiation markers KRT1 and

287 KRT10 was measured by qRT-PCR. We found that throughout the time course, HFK-HPV18E7

288 WT exhibited a 2.3- to 5.9-fold reduction in KRT1 and KRT10 expression in comparison to HFK-

E7 R84S 289 HPV18 (Figure 7D). These data indicate that PTPN14 binding by HPV18 E7 contributes to

290 the ability of HPV E7 to repress differentiation gene expression even in the context of the

291 complete HPV genome.

292

293 PTPN14 binding is required for HPV18 E7 to extend the lifespan of primary human

294 keratinocytes

295 High-risk HPV E7 extends the lifespan of primary keratinocytes in vitro (8, 45). We have

296 previously reported that the E10K mutation in HPV16 E7, which blocks PTPN14 degradation,

297 also impairs its ability to extend the lifespan of primary keratinocytes (29). To assess whether

298 PTPN14 binding by HPV18 E7 is also necessary for lifespan extension by E7, we transduced

299 primary HFK with retroviral vectors encoding HPV18 E7 WT, HPV18 E7 R84S, or the empty

300 vector control and selected for stable populations. By counting cells at each passage, we

301 tracked the number of times each cell population doubled over time. We found that HPV18 E7

302 R84S was unable to extend the lifespan of primary HFK and that these cells behaved

303 comparably to the HFK-empty vector control (Figure 8A). In contrast, HPV18 E7 WT extended

304 the lifespan of primary HFK until the experiment was terminated 49 days post-transduction. This

305 result supports that PTPN14 binding by HPV18 E7 is essential for its ability to extend the

306 lifespan of primary cells. To establish that lifespan extension by HPV18 E7 is dependent on

307 PTPN14 degradation and not on another activity of HPV18 E7 related to the C-terminal arginine

308 residue, we tested whether deletion of PTPN14 could rescue the defect of the HPV18 E7 R84S

309 mutant and extend keratinocyte life span. These experiments were conducted by co-transducing

310 HFKs from two different keratinocyte donors with the HPV18 E7 retroviral vectors and

311 LentiCRISPR v2 vectors encoding control or PTPN14 targeting guide RNAs (two of each). We

312 found that PTPN14 deletion rescued the defect in the lifespan extension capability of HPV18 E7

313 R84S to a level that resembled that of HPV18 E7 WT (Figure 8B). PTPN14 KO did not extend

314 the lifespan of cells transduced with the empty vector control and did not further extend the

315 lifespan of cells expressing HPV18 E7 WT. These results further support the model that

316 PTPN14 binding and degradation is required for the carcinogenic activity of high-risk HPV E7

317 proteins.

318 DISCUSSION

319 HPV E7 from diverse virus genotypes bind and degrade PTPN14 dependent on the host

320 E3 ubiquitin ligase UBR4 (11, 28, 30, 36). A potential tumor suppressive role of PTPN14 is

321 consistent with its mutation rates in certain cancers (basal cell carcinoma: 23%; gastrointestinal

322 cancers: 5%) (46, 47) and its role as an inhibitor of the HIPPO pathway proto-oncogenes YAP

323 and TAZ (32, 34, 47). The high degree of conservation of E7-PTPN14 binding suggests that

324 PTPN14 binding and/or degradation play an important role in HPV biology. We hypothesize that

325 one consequence of PTPN14 degradation is a contribution to high-risk HPV-mediated

326 carcinogenesis.

327 High-risk E7 is central to carcinogenesis. In a recent analysis of the HPV16 sequences

328 from >5000 patient samples, E7 was found to be the most highly conserved HPV ORF in high-

329 grade lesions. This was interpreted to reflect strong selective pressure to maintain prototypical

330 E7 sequences during carcinogenic progression. The requirement for UBR4 in PTPN14

331 degradation connects PTPN14 degradation to HPV-mediated carcinogenesis. Binding to UBR4

332 was reported by several groups to contribute to the carcinogenic activities of oncogenic

333 papillomavirus E7 (23, 48). We recently characterized the HPV16 E7 E10K variant that cannot

334 bind UBR4 and is unable to degrade PTPN14 (29). We found that HPV16 E7 E10K was

335 impaired in assays of E7 carcinogenic activity. Using HPV16 E7 E10K, we also discovered that

336 HPV16 E7 represses keratinocyte differentiation downstream of PTPN14 degradation.

337 In the current study we characterized a newly described E7 variant with a mutation in a

338 highly conserved C-terminal arginine residue. A recently published crystal structure of HPV18

339 E7 and PTPN14 determined that the conserved arginine is a central component of the E7-

340 PTPN14 binding interface (36). We found that mutation of this arginine rendered the E7 proteins

341 from four HPV genotypes across three species unable to degrade PTPN14 (Figure 1). Thus, the

342 PTPN14-binding interface appears to be highly conserved, particularly among high-risk HPV E7

343 proteins. In contrast, mutation of an acidic amino acid in the E7 N-terminus (the UBR4 binding

344 region) had a somewhat variable effect on PTPN14 levels. The HPV16 E7 E10K mutation

345 almost completely restored PTPN14 protein levels whereas the analogous HPV18 E7 D10K

346 mutation had an intermediate effect. One possibility is that HPV18 E7 D10K retains some

347 capacity to bind to UBR4.

348 Our prior work showed that the E7/PTPN14/UBR4 protein complex is distinct from the

349 E7/RB1 complex (28) and that the impacts of the two on the keratinocyte transcriptome are

350 genetically separable (29). We confirmed that HPV16 E7 R77S and HPV18 E7 R84S retain the

351 capacity to bind and degrade RB1 (Figures 1 and 3) and promote the expression of E2F target

352 genes (Figures 2 and 6). These data strongly support the model that PTPN14 binding and

353 degradation is a conserved activity of HPV E7 separate from RB1 inactivation. The rare HPV16

354 E7 R77S and E10K variants were found by Mirabelllo and colleagues in HPV16-positive

355 precancerous lesions (35). We do not know whether genetic polymorphisms or a characteristic

356 of the tumor microenvironment in the patients harboring the variants enabled their progression

357 past the initial infection nor whether the lesions would have progressed to frank carcinoma.

358 Either PTPN14 degradation by HPV16 E7 or PTPN14 CRIPSR KO represses the

359 expression of differentiation-related genes (29). In this study, we confirmed that blocking

360 PTPN14 binding through mutation of the conserved C-terminal arginine likewise renders high-

361 risk HPV16 E7 and HPV18 E7 unable to repress differentiation (Figures 4 and 5). Furthermore,

362 we confirmed by RNA-seq analysis that repression of differentiation is the primary effect of

363 PTPN14 binding by HPV18 E7 on the transcriptome of unstimulated primary keratinocytes

364 (Figure 6). Other alpha papillomavirus proteins including E6 repress differentiation, although the

365 downstream mechanisms remain to be elucidated (41–43). Mouse PTPN14 is proposed to be a

366 downstream effector of p53 in the “p53-PTPN14-YAP axis” (47). If PTPN14 is also downstream

367 of p53 in human cells, inactivation of PTPN14 by E7 could be more complex in the context of

368 p53 degradation by E6. Nonetheless, we observed substantially higher expression of

369 differentiation marker expression in HFK harboring the HPV18E7 R84S genome than those

370 harboring the HPV18E7 WT genome (Figure 7). This indicates that PTPN14 degradation

371 contributes to repression of differentiation even in the presence of E6. Beta papillomavirus E6

372 represses differentiation by binding to MAML and directly inhibiting NOTCH signaling (49–52).

373 However, alpha papillomavirus E6 proteins do not bind MAML1 (53, 54). We posit that all

374 papillomaviruses require repression of differentiation gene expression and that alpha HPV

375 depend on PTPN14 degradation by E7 to repress differentiation.

376 Although HPV E7 repress differentiation-related genes through PTPN14 degradation,

377 our experiments indicate that E7-expressing cells retain the capacity to ultimately differentiate,

378 which is consistent with the differentiation-dependent nature of the HPV life cycle (55–60).

379 Additionally, we observed that PTPN14 binding by HPV18 E7 had a much stronger effect on cell

380 viability after growth in suspension (Figure 5B) than it did on differentiation-related gene

381 expression in pools of differentiated keratinocytes (Figures 4 and 5A). These observations may

382 be related to the stage of the keratinocyte differentiation program at which PTPN14 degradation

383 has the most impact. For example, it is possible that PTPN14 affects cell fate decisions early in

384 differentiation. Our findings are consistent with PTPN14 binding by E7 delaying or impairing the

385 commitment to terminal differentiation—thereby allowing for continued proliferation of some

386 cells—as opposed to completely blocking the differentiation program.

387 High-risk HPV E7 expression extends the life span of primary keratinocytes (8, 45) and

388 lifespan extension is directly correlated with E7 carcinogenic activity. Using lifespan extension

389 experiments in primary HFK, we found that HPV18 E7 R84S was substantially impaired in its

390 ability to extend the lifespan of primary cells compared to WT HPV18 E7 (Figure 8A). Moreover,

391 CRISPR/Cas9 knockout of PTPN14 in primary HFK rescued the lifespan-extending activity of

392 HPV18 E7 R84S (Figure 8B). HPV E7 can bind to PTPN21 (11, 36), a homolog of PTPN14, but

393 the rescue experiment indicates that PTPN14, not PTPN21, is the primary target involved in

394 lifespan extension. These data and our analysis of survival after growth in suspension (Figure

395 5B) implicate PTPN14 binding and degradation as a carcinogenic activity of high-risk HPV E7.

396 Our finding that PTPN14 binding and degradation leads to repression of differentiation-

397 related genes also has implications for its role in carcinogenesis. This activity is significant

398 because it suggests that PTPN14 degradation could contribute to the tendency for HPV-positive

399 oropharyngeal carcinomas to be poorly differentiated (61, 62). It is interesting to consider that

400 targeting PTPN14 binding to promote differentiation could present an opportunity for the

401 treatment of HPV positive cancers. All-trans retinoic acid (ATRA) is a highly successful

402 differentiation therapy used to treat acute promyelocytic leukemia (63). ATRA has shown

403 promise in mouse models of non-melanoma skin cancer and in reducing proliferation of human

404 squamous cell carcinoma cells (64–66).

405 Here we show that PTPN14 binding and degradation is a highly conserved activity of

406 HPV E7 proteins. By characterizing an HPV E7 variant that cannot bind PTPN14, we have also

407 shown that inactivation of PTPN14 rather than another target of E7 or of UBR4 leads to the

408 repression of differentiation. Furthermore, we found that the PTPN14 degradation-dependent

409 repression of differentiation occurs even when E7 is expressed from the complete HPV18

410 genome. Finally, we demonstrated that PTPN14 degradation contributes to the carcinogenic

411 properties of HPV18 E7 and confirmed that PTPN14 KO can rescue this activity. Future studies

412 aimed at elucidating the mechanism by which PTPN14 degradation represses differentiation,

413 promotes carcinogenesis, and influences the differentiation-dependent HPV life cycle will further

414 our understanding of this highly conserved HPV E7 activity.

415 MATERIALS AND METHODS

416 Plasmids and cloning

417 Mutations in HPV E7 ORFs were introduced by site-directed mutagenesis into pDONR-Kozak-

418 E7 plasmids and recombined into MSCV-P C-FlagHA GAW or MSCV-Neo C-HA GAW as

419 previously described (11). LentiCRISPR v2 (Addgene # 52961) vectors were cloned according

420 to standard protocols using sgRNA sequences as contained in the Broad Institute Brunello

421 library (67). pNeo-loxP-HPV18 was the kind gift of Drs. Thomas Broker and Louise Chow (44)

422 and was used as the template to generate pNeo-loxP-HPV18 E7 R84S by PCR site-directed

423 mutagenesis of the E7-containing portion of the genome, followed by reassembly of this

424 fragment into the pNeo-loxP-HPV18 vector using HiFi DNA Assembly (NEB). Plasmids used in

425 the study are listed in Supplemental Table 1.

426

427 Cells

428 Primary human foreskin keratinocytes (HFK) from two donors were isolated and provided as de-

429 identified material by the Penn Skin Biology and Diseases Resource-based Center Core B.

430 N/Tert-1 are hTert-immortalized HFK (68). Keratinocytes were cultured as previously described

431 (29). HPV E7 expressing cell populations were established by transduction with MSCV retroviral

432 vectors followed by puromycin selection. PTPN14 knockout or nontargeting control primary HFK

433 were established by transduction with LentiCRISPR v2 vectors followed by puromycin selection.

434 HPV E7 expression in the CRISPR/Cas9-edited cells was achieved by simultaneous

435 transduction of MSCV retroviral vectors and selection with G418 and puromycin. Retroviruses

436 and lentiviruses were generated as previously described (11). To generate HFK containing

437 complete HPV genomes, cells were co-transfected with pNeo-loxP-HPV18 or pNeo-loxP-HPV18

438 E7 R84S and a Cre expression plasmid using FuGENE HD (Promega) as previously described

439 (44). HFK populations containing the HPV genomes were selected for with G418 for 4 days.

440

441 Western blotting

442 Western blots were performed using Mini-PROTEAN or Criterion (BioRad) SDS-PAGE gels and

443 transfer to PVDF. After blocking in 5% nonfat dried milk in TBS-T (Tris buffered saline [pH 7.4]

444 with 0.05% Tween-20), membranes were incubated with primary antibodies as follows: RB1

445 (Calbiochem/EMD), actin (Millipore), PTPN14 (R&D Systems or Cell Signaling Technology),

446 KRT1 (Santa Cruz), KRT10 (Santa Cruz), or involucrin (Santa Cruz)). Membranes were washed

447 in TBS-T and incubated with horseradish peroxidase (HRP)-coupled anti-mouse or anti-rabbit

448 antibodies and detected using chemiluminescent substrate. HA-tagged proteins were detected

449 and visualized using an HA antibody conjugated to HRP (Roche). For anti-HA

450 immunoprecipitations, HA-tagged proteins were immunoprecipitated and processed for Western

451 blot as previously described (28).

452

453 qRT-PCR

454 Total RNA was isolated from N/Tert or HFK cells using the NucleoSpin RNA extraction kit

455 (Macherey-Nagel). RNA was reverse transcribed using the High Capacity cDNA Reverse

456 Transcription Kit (Applied Biosystems). cDNAs were assayed by qPCR using Fast SYBR Green

457 Master Mix (Applied Biosystems) using a QuantStudio 3 (ThermoFisher). KiCqStart SYBR

458 green primers for qRT-PCR (Sigma-Aldrich) were specific for MCM2, CCNE2, KRT1, KRT10,

459 IVL, MAF, GAPDH, and G6PD. All qRT-PCR data were normalized to GAPDH or to G6PD.

460

461 Keratinocyte Differentiation Assays

462 N/Tert-E7 or empty vector cells were stimulated to differentiate by growth in high-calcium

463 medium or by culture in suspension. For calcium differentiation, cells were grown to ~30%

464 confluence in 6-well plates using standard growth conditions in Keratinocyte Serum-Free

465 Medium (K-SFM) with growth factors. To induce differentiation, growth factors were withdrawn

466 and cells were refed with Keratinocyte Basal Medium (Lonza) supplemented to achieve 1.5 mM

467 [final] CaCl2. Cells were harvested for RNA analysis 72h post-treatment. For culture in

468 suspension, cells were harvested by trypsinization and re-plated in ultra-low attachment plates

469 (Sigma-Aldrich). After 0 or 12h of culture in suspension cells were harvested for RNA analysis

470 or 1000 cells were re-plated in standard 6-well tissue culture plates. Re-plated cells were

471 stained with crystal violet 5 days post re-plating. pNeo-loxP-HPV18 transfected HFK cells were

472 also grown to high confluence and stimulated to differentiate by culturing for 0, 2, or 6 days in

473 growth factor-free Keratinocyte Basal Medium (Lonza) supplemented to achieve 1.5 mM [final]

474 CaCl2.

475

476 RNA-seq

477 Total RNA was isolated from 3 independent isolates of HFK-empty vector control, HFK-18E7, or

478 HFK 18E7 R84S cells using the RNeasy mini kit (Qiagen). PolyA selection, reverse

479 transcription, library construction, sequencing, and initial analysis were performed by Novogene.

480 Differentially expressed genes were selected based on a 1.5-fold change and FDR-adjusted p <

481 0.05 cutoff and were analyzed for enriched biological processes (BP) using the GO enrichment

482 analysis tool of the PANTHER classification system (69). All GO terms in enrichment analyses

483 are displayed in rank order by FDR-adjusted p-value. RNA-seq data have been deposited in

484 NCBI GEO with accession number GSE150201.

485

486 Keratinocyte Lifespan Extension

487 To assess the ability of HPV18 E7 variants to extend keratinocyte lifespan, primary HFK were

488 transduced with MSCV-E7 retroviruses and selected with puromycin. Cells were selected in

489 puromycin and passaged for approximately 50 days. Population doublings were calculated

490 based upon the number of cells collected and re-plated at each passage. Each experiment

491 consisted of three independent replicates conducted simultaneously.

492

493 Data Availability

494 RNA-seq data from HFK-empty, HFK-18E7, and HFK-18E7 R84S are freely accessible in NCBI

495 GEO with accession number GSE150201.

496

497 Acknowledgements

498 We thank the members of our laboratories for helpful discussions. This work was supported by

499 American Cancer Society grant 131661-RSG-18-048-01-MPC and NIH R01 AI148431 to EAW

500 and NIH R01 CA066980 to KM. JH was supported by NIH T32 AI007324. The Penn Skin

501 Biology and Diseases Resource-based Center was supported by NIH P30 AR068589.

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715

716

717 FIGURE LEGENDS

718 Figure 1. Conserved PTPN14 binding and degradation by HPV E7. (A) Protein sequence

719 alignment of 22 HPV E7 ORFs from diverse HPV genotypes. Alignments were generated using

720 the Clustal Omega algorithm and colored using the ClustalX color scheme. (B) Cell lysates from

721 N/Tert-1 cells expressing E7 proteins from various HPV genotypes were subjected to

722 SDS/PAGE/Western analysis and probed with antibodies to PTPN14, RB1, HA (E7) and Actin.

723 (C and D) Cell lysates from N/Tert-1 cells expressing WT or variant HPV16, HPV31, HPV18,

724 and HPV6 E7 were subjected to SDS/PAGE/Western analysis and probed with antibodies to

725 PTPN14, RB1, HA (E7) and Actin. Band intensity was quantified using ImageJ. Values

726 represent PTPN14 band intensity relative to Actin band intensity. BG indicates that band

727 intensity was ≤ background intensity.

728

729 Figure 2. PTPN14 binding and degradation by HPV E7 are not required to promote the

730 expression of E2F-regulated genes. Primary HFK were transduced with retroviral vectors

731 encoding various HPV16 and HPV18 E7 proteins. qRT-PCR was used to measure the

732 expression of the E2F-regulated genes (A) CCNE2 and (B) MCM2 relative to GAPDH. Graphs

733 display the mean ± SD of three independent replicates. Statistical significance was determined

734 by ANOVA (**p < 0.01, ****p < 0.0001).

735

736 Figure 3. HPV16 E7 R77S and HPV18 E7 R84S cannot bind to PTPN14. N/Tert-1

737 keratinocytes were transduced with retroviruses encoding various HPV16 E7 (A), HPV18 E7

738 (B), or the empty vector control. HPV E7-FlagHA was immunoprecipitated with anti-HA beads

739 from total cell lysates and coimmunoprecipitation of PTPN14 and RB1 was assessed by

740 SDS/PAGE/Western analysis (upper). Total cell lysates were subjected to SDS/PAGE/Western

741 analysis and probed with antibodies to PTPN14, RB1, HA (E7) and actin (lower).

742

743 Figure 4. HPV16 E7 R77S and HPV18 E7 R84S are impaired in their ability to repress

744 differentiation gene expression induced by calcium treatment. N/Tert-1 keratinocytes

745 expressing HPV16 E7 WT and R77S, HPV18 E7 WT and R84S or the empty vector control

746 were maintained in growth medium (-) or were induced to differentiate with 1.5 mM CaCl2 for

747 72h (+). Cell lysates were subjected to SDS/PAGE/Western analysis and probed with antibodies

748 to PTPN14, IVL, KRT1, KRT10, HA (E7) and actin.

749

750 Figure 5. HPV18 E7 binding to PTPN14 contributes to the repression of differentiation

751 induced by suspension and improves survival of cells grown in suspension. N/Tert-1

752 keratinocytes expressing HPV18 E7 WT, HPV18 E7 R84S, or the empty vector control were

753 induced to differentiate by culturing in ultra-low adherence plates for 12h. (A) qRT-PCR was

754 used to measure the expression of the differentiation marker IVL relative to GAPDH in adherent

755 or suspended cells. Graph displays the mean ± SD of three independent replicates. (B) Survival

756 after suspension was assessed by replating 1,000 cells from suspension, allowing cells to grow

757 for 5 days, and measuring total viable cell area by crystal violet staining. Graph displays the

758 mean ± SD of three independent replicates. Representative images of crystal violet staining are

759 displayed. Statistical significance was determined by ANOVA (*p < 0.05, ***p < 0.001).

760

761 Figure 6. Repression of differentiation is the predominant effect of PTPN14 binding by

762 HPV18 E7 on the transcriptome of primary keratinocytes. Primary HFK were transduced

763 with retroviruses encoding HPV18 E7 WT, HPV18 E7 R84S, or the empty vector control. PolyA-

764 selected RNA collected from early passages was analyzed by RNA-seq. (A) Volcano plot of

765 gene expression changes in HFKs expressing HPV18 WT and HPV18 R84S. Vertical and

766 horizontal dotted lines represent 1.5-fold change and p-adj = 0.05, respectively. Representative

767 genes are highlighted in red. (B) Gene Ontology (GO) enrichment analysis of genes down-

768 regulated in HFKs expressing HPV18 WT compared to HPV18 R84S. GO terms are displayed

769 in rank order of their FDR-adjusted p-value. Circle chart displays the fraction of down-regulated

770 genes that fall into enriched GO Terms. (C) Heat map displaying the expression patterns of

771 selected genes related to differentiation and the cell cycle. Early, early differentiation markers.

772 Des., genes encoding components of the differentiation-associated desmosome complexes.

773 Cornif., genes involved in cornified envelope formation. Sig., genes involved in differentiation-

774 promoting signals. TF., genes encoding differentiation-promoting transcription factors. (D) qRT-

775 PCR was used to measure the expression of the differentiation-related genes IVL, KRT1, and

776 MAF relative to GAPDH. Graphs display the mean ± SD of three independent replicates.

777 Statistical significance was determined by ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p <

778 0.0001).

779

780 Figure 7. PTPN14 degradation is required for repression of differentiation in primary

781 keratinocytes harboring the HPV18 genome. Primary HFK were transfected with pNeo-loxP-

782 HPV18 vectors and NLS-Cre. (A) Schematic of the pNeo-loxP-HPV18 transfection experimental

783 strategy. (B) PCR/agarose gel analysis of DNA isolated from HFK was used to confirm the

784 transfection and excision of the HPV18 genome following selection. (C) Cell lysates were

785 subjected to SDS/PAGE/Western analysis and probed with antibodies to PTPN14, RB1 and

786 actin. (D) Cells were treated with 1.5 mM CaCl2 for 0, 2, and 6 days. qRT-PCR was used to

787 measure the expression of the differentiation markers KRT1 and KRT10 relative to GAPDH.

788 Graphs display the mean ± SD of two or three independent replicates. Statistical significance

789 was determined by repeated measures two-way ANOVA (***p < 0.001, ****p < 0.0001).

790

791 Figure 8. PTPN14 degradation is required for HPV18 E7 to extend the lifespan of primary

792 human keratinocytes. (A) Primary HFK were transduced with retroviruses encoding HPV18 E7

793 WT, HPV18 E7 R84S, or the empty vector control and passaged for up to 49 days. Graph

794 displays the growth of these cell lines over the course of the experiment. Statistical significance

795 was determined from three independent experiments by repeated measures two-way ANOVA

796 (*p < 0.05; **p < 0.01). (B) Primary HFK from two different donors were transduced with the

797 same retroviruses as (A) along with LentiCRISPRv2 lentiviral vectors encoding SpCas9 and

798 nontargeting or PTPN14-directed sgRNAs and cultured for up to 58 days. Graph displays the

799 final population doubling values under each condition along with the mean.

Conserved C-Terminal Arginine A s s e u e yp Gen Speci T 10 20 30 40 50 60 70 80 90 100 110 HPV18 High MHGPKATLQDIVLHLEP-QNEI-PVDLLCHEQLSDSE--EENDEIDGVNHQH LPARRAEPQ-RHTMLCMCC--KCEARIELVVESSADDLRAFQQLFLNT-LSFVCPWCASQQ------7 HPV39 High MRGPKPTLQEIVLDLCP-YNEIQPVDLVCHEQLGESEDEIDEPDHAVNHQHQ LLARRDEPQ-RHTIQCSCC--KCNNTLQLVVEASRDTLRQLQQLFMDS-LGFVCPWCATANQ----- HPV45 High MHGPRETLQEIVLHLEP-QNELDPVDLLCYEQLSESE--EENDEADGVSHAQ LPARRAEPQ-RHKILCVCC--KCDGRIELTVESSAEDLRTLQQLFLST-LSFVCPWCATNQ------HPV59 High MHGPKATLCDIVLDLEP-QNY-EEVDLVCYEQLPDSDSENEKDEPDGVNHPL LLARRAEPQ-RHNIVCVCC--KCNNQLQLVVETSQDGLRALQQLFMDT-LSFVCPLCAANQ------HPV16 High MHGDTPTLHEYMLDLQ---P--ETTDLYCYEQLNDSSE--EEDEIDGPAGQA EPDR-----AHYNIVTFCC--KCDSTLRLCVQSTHVDIRTLEDLLMGT-LGIVCPICSQKP------HPV31 High MRGETPTLQDYVLDLQ---P--EATDLHCYEQLPDSSD--EEDVIDSPAGQA EPDT-----SNYNIVTFCC--QCKSTLRLCVQSTQVDIRILQELLMGS-FGIVCPNCSTRL------9 HPV33 High MRGHKPTLKEYVLDLY---P--EPTDLYCYEQLSDSSDE-D-EGLDRPDGQA QPAT-----ADYYIVTCCH--TCNTTVRLCVNSTASDLRTIQQLLMGT-VNIVCPTCAQQ------α HPV35 High MHGEITTLQDYVLDLE---P--EATDLYCYEQLCDSSEE-EEDTIDGPAGQA KPDT-----SNYNIVTSCC--KCEATLRLCVQSTHIDIRKLEDLLMGT-FGIVCPGCSQRA------HPV52 High MRGDKATIKDYILDLQ---P--ETTDLHCYEQLGDSSDEEDTDGVDRPDGQA EQAT-----SNYYIVTYCH--SCDSTLRLCIHSTATDLRTLQQMLLGT-LQVVCPGCARL------HPV58 High MRGNNPTLREYILDLH---P--EPTDLFCYEQLCDSSDE-DEIGLDGPDGQA QPAT-----ANYYIVTCCY--TCGTTVRLCINSTTTDVRTLQQLLMGT-CTIVCPSCAQQ------6 HPV56 High MHGKVPTLQDVVLELTP-QTEI---DLQCNEQLDSSEDEDE-DEVDHLQERP QQARQAKQHTCYLIHVPCC--ECKFVVQLDIQSTKEDLRVVQQLLMGA-LTVTCPLCASSN------5 HPV51 High MRGNVPQLKDVVLHLTP-QTEI---DLQCYEQFDSSEEEDE---VDNMRDQ- LPERRAGQATCYRIEAPCC--RCSSVVQLAVESSGDTLRVVQQMLMGE-LSLVCPCCANN------HPV6 Low MHGRHVTLKDIVLDLQ--PP--DPVGLHCYEQLVDSSED-EVDEVDGQ--DS QPLK-----QHFQIVTCCC--GCDSNVRLVVQCTETDIREVQQLLLGT-LNIVCPICAPKT------10 HPV11 Low MHGRLVTLKDIVLDLQ--PP--DPVGLHCYEQLEDSSED-EVDKVDKQ--DA QPLT-----QHYQILTCCC--GCDSNVRLVVECTDGDIRQLQDLLLGT-LNIVCPICAPKP------HPV55 Low MHGNYPTLKEIVLELD--PP--DPVGLHCNEQL-DSSED-EVDELATQ--AT QDVT-----QPYQIVTTCG--TCNRNVRLVVQCTGTDICQLHTLLLGS-LEILCPVCAPKN------HPV24 MHGNRPSLKDITLILDE-IP--EIVDLHCDEQFDSSEEE-N----NHQ--LT EPDV-----QAYGVVTTCC--KCGRTVRLVVECGQADLRELEQLFLKT-LTLVCPHCA------1 HPV8 MIGKEVTVQDFVLKLSEIQPEVLPVDLLCEEELPNEQ-ETEEEL------DIERTVFKIVAPCGCSCCQVKLRLFVNATDSGIRTFQELLFRD-LQLLCPECRGNCKHGGS- β HPV25 MIGKEVTLQDFTLELSELQPEVQPVDLFCEEELPAEHQETEEEP------AIDRTPYKVVAPCG--CCEVKLRIFVKATDFGIRTLQNLLIEE-LQLLCPECRGNCKHGGS- 2 HPV17 MIGKEATIPDIVLEL---QQLVQPTDLHCYEELSEEETETEEEP------RRIPYKIVAPCC--FCGSKLRLIVLATHAGIRSQEELLLGE-VQLVCPNCREKLRHD--- γ 1 HPV4 MRGAAPTVADLNLEL---NDLVLPANLLSEEVLQSSDDEYEITE------EESVVPFRIDTCCY--RCEVAVRITLYAAELGLRTLEQLLVEGKLTFCCTACARSLNRNGR- 2 HPV48 MRGDKATIPD--IEL---EELVLPANLISDESLSPD-----ATA------EEEFCPYRIDSKCH--NCGCRIRVTVAATEFGIRCFEQLLLKE-LCLFCPACSRQLPRNGRS μ 1 HPV1 MVGEMPALKDLVLQL---EPSVLDLDLYCYEEVPPDDI--EEEL------VSPQQPYAVVASCA--YCEKLVRLTVLADHSAIRQLEELLLRS-LNIVCPLCTLQRQ-----

B C HPV E7 D HPV E7 HPV E7 186 61 552 Empty PTPN14 150 kDa 16R77S 31R77S 31 18R84S Empty 16 18 16E10K 16R77S 18R84S 6 R77S6 16∆21-24 18D10K RB1 100 Empty 16 18 PTPN14 150 kDa 150 kDa 25 PTPN14 HA (E7) RB1 20 100 RB1 25 100 Actin HA (E7) 25 20 HA (E7) PTPN14/Actin: Actin 20 1.00 0.17 0.02 0.25 0.16 0.15 Actin PTPN14/Actin: BG 0.01 0.57 1.00 0.04 0.48 0.90 PTPN14/Actin: 0.20 0.88 1.27 1.00 0.30 0.99 0.04 0.22 0.45 1.25 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.22.111740; this version posted May 23, 2020. The copyright holder for this preprint (which Awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NDCCNE2 4.0 International license. **** 12 **** **** **** **** 8

Relative mRNA Expression mRNA Relative 0

E7 6E7 10K 1 18 Empty 7 E 7 R77S 16E 6E7 ∆21-2416E 18E7 R84S 1 B MCM2 ** 3 ** ** ** ** 2

Relative mRNA Expression mRNA Relative 0

ty 0K 7S 6E7 7 1 18E7 Emp E7 R84S 8 16E7 E16E7 ∆21-2416E7 R 1 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.22.111740; this version posted May 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

A HPV16 E7 B HPV18 E7 ∆21-24 WT Empty WT R77S R84S Empty 150 kDa PTPN14 150

RB1 100 100 HA (E7) 20 IP: HA (E7) HA IP: 20 150 PTPN14 150 100 RB1 100

Input HA (E7) 20 20 Actin 37 37 1.5 mMCaCl bioRxiv preprint was notcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade PTPN14 HA (E7) KRT10 doi: KRT1 https://doi.org/10.1101/2020.05.22.111740 Actin IVL 2 : available undera + - + - + - + - + - Empty CC-BY-ND 4.0Internationallicense ; this versionpostedMay23,2020. 16 HPV E7 .

16 R77S The copyrightholderforthispreprint(which

18 R84S 37 5 150 kDa 15 20 25 37 50 75 100 150 0 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.22.111740; this version posted May 23, 2020. The copyright holder for this preprint (which Awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 InternationalIVL license. 20 Empty * 18E7 15 18E7 R84S

10 A Expression A N

5 e mR e iv t a l 3

Re 2 1 0 Adherent Suspension

B Growth After Suspension y ) 2 15

*** mpt

*** E ea (mm ea r

d A d 10 ne t Stai t

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y 0 R84 Cr 7 7 E 18 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.22.111740; this version postedEnrichment: May 23, 2020. The genes copyright holderlower for thisin preprint (which Awas not certifiedHPV18 by peer E7 review) WT is the vs author/funder, R84S who hasB granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International18E7 license WT. than R84S Higher in WT in Higher

(24 genes) (24 30 Cornification 200 Keratinocyte Differentiation Skin Development Epidermis Development (247genes) Lower in WT Tissue Development 150 20 Developmental Process IVL Other (FDR) (p-adj) Distribution: 10 10 37 100 5 6 -Log -Log 10 2 247 28 50 Total

36 KRT1 MAF 0 0 -2 -1 0 1 Ranked Biological Process GO Term

Log2(FC)

C Log2(Fold-Change): -101

A Control B C A 18E7 B WT C A 18E7 B R84S C IV L FLG MA F KRT1 DSC1 DSC2 PRR9 DSG1 TGM5 MAFB PCNA HOPX MCM2 KRT10 LCE1A CCNE2 CASP14 CALML5 EarlyDes. Cornif.Sig. TF Cell Differentiation Cycle D IVL KRT1 MAF

on 1.5 2.0 ** 1.5

ssi * * 1.5 ****

xpre * 1.0 1.0 E *** 1.0

mRNA mRNA 0.5 0.5 0.5 ve ve ati l 0.0 0.0 0.0 Relative mRNA Expression mRNA Relative Re Expression mRNA Relative S y 7 4S 7 4 ty 8 ty E R84S 8E R p 8 mp 18E7 mpt 1 7 m 1 7 R8 E E7 E E E 8E 18 18 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.22.111740; this version posted May 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

A HPV18 B HPV18 loxP PCR Genome Primer Sets loxP Neo HPV18 Transfection Vector E7WT E7R84S Resistance loxP + NLS-Cre Neo Excised Resistance loxP HPV18 Total Genome loxP PCR

C D KRT1 KRT10 HPV18 on

30 on 30 E7 WT i E7 WT **** HPV18 s HPV18 **** HPV18E7 R84S HPV18E7 R84S 20 20 Expressi Expres Parental WT E7 E7R84S NA

PTPN14 150 kDa R *** m 10 mRNA 10 *** e e v v i i RB1 t *** a *** 100 l 0 0 Re Relat Actin 0 2 4 6 0 2 4 6 37 Time Post CaCl2 Treatment (days) bioRxiv preprint doi: https://doi.org/10.1101/2020.05.22.111740; this version posted May 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

A B 25 Donor 1 Empty vector 20 Donor 2 20 HPV18 E7 15 HPV18 E7 R84S 15 10

10 * ** 5 ** Post Transduction Post Post Transduction Post 5 Population Doublings Population Population Doublings Population 0

0 -5 0 25 50 Time Post Transduction (Days) sgNT-1 sgNT-2 sgNT-1 sgNT-2 sgNT-1 sgNT-2 sgPTPN14-3 sgPTPN14-4 sgPTPN14-3 sgPTPN14-4 sgPTPN14-3 sgPTPN14-4 Empty HPV18 HPV18 Vector E7 WT E7 R84S