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Persistent human papillomavirus (HPV) infection of stratified squamous epithelial cells causes nearly 5% of cancer cases worldwide. HPV-positive oropharyngeal cancers harbor few mutations in the Hippo signaling pathway compared to HPV-negative cancers at the same anatomical site, prompting the hypothesis that an HPV-encoded protein inactivates the Hippo pathway and activates the Hippo effector yes-associated protein (YAP1). The HPV E7 oncoprotein is required for HPV infection and for HPV-mediated oncogenic transformation. We investigated the effects of HPV oncoproteins on YAP1 and found that E7 activates YAP1, promoting YAP1 nuclear localization in basal epithelial cells. YAP1 activation by HPV E7 required that E7 binds and degrades the tumor suppressor protein tyrosine phosphatase non-receptor type 14 (PTPN14). E7 required YAP1 transcriptional activity to extend the lifespan of primary keratinocytes, indicating that YAP1 activation contributes to E7 carcinogenic activity. Maintaining infection in basal cells is critical for HPV persistence, and here we demonstrate that YAP1 activation causes HPV E7 expressing cells to be retained in the basal compartment of stratified epithelia. We propose that YAP1 activation resulting from PTPN14 inactivation is an essential, targetable activity of the HPV E7 oncoprotein relevant to HPV infection and carcinogenesis. The ‘epithelial’ cells that cover our bodies are in a constant state of turnover. Every few weeks, the outermost layers die and are replaced by new cells from the layers below. For scientists, this raises a difficult question. Cells infected by human papillomaviruses, often known as HPV, can become cancerous over years or even decades. How do infected cells survive while the healthy cells around them mature and get replaced? One clue could lie in PTPN14, a human protein which many papillomaviruses eliminate using their viral E7 protein; this mechanism could be essential for the virus to replicate and cause cancer. To find out the impact of losing PTPN14, Hatterschide et al. used human epithelial cells to make three-dimensional models of infected tissues. These experiments showed that, when papillomaviruses destroy PTPN14, a human protein called YAP1 turns on in the lowest, most long-lived layer of the tissue. Cells in which YAP1 is activated survive while those that carry the inactive version mature and die. This suggests that papillomaviruses turn on YAP1 to remain in tissues for long periods. Papillomaviruses cause about five percent of all human cancers. Finding ways to stop them from activating YAP1 has the potential to prevent disease. Overall, the research by Hatterschide et al. also sheds light on other epithelial cancers which are not caused by viruses.

High-risk human papillomavirus (HPV) E7 proteins enable oncogenic transformation of HPV-infected cells by inactivating host cellular proteins. High-risk but not low-risk HPV E7 target PTPN14 for proteolytic degradation, suggesting that PTPN14 degradation may be related to their oncogenic activity. HPV infects human keratinocytes but the role of PTPN14 in keratinocytes and the consequences of PTPN14 degradation are unknown. Using an HPV16 E7 variant that can inactivate retinoblastoma tumor suppressor (RB1) but cannot degrade PTPN14, we found that high-risk HPV E7-mediated PTPN14 degradation impairs keratinocyte differentiation. Deletion of PTPN14 from primary human keratinocytes decreased keratinocyte differentiation gene expression. Related to oncogenic transformation, both HPV16 E7-mediated PTPN14 degradation and PTPN14 deletion promoted keratinocyte survival following detachment from a substrate. PTPN14 degradation contributed to high-risk HPV E6/E7-mediated immortalization of primary keratinocytes and HPV⁺ but not HPV⁻ cancers exhibit a gene-expression signature consistent with PTPN14 inactivation. We find that PTPN14 degradation impairs keratinocyte differentiation and propose that this contributes to high-risk HPV E7-mediated oncogenic activity independent of RB1 inactivation.

The Hippo kinase cascade inhibits YAP1, an oncoprotein and driver of cell stemness and self-renewal. There is mounting evidence that the Hippo pathway is targeted by tumor viruses including human papillomavirus. The high-risk HPV E7 oncoprotein promotes YAP1 nuclear localization and the carcinogenic activity of high-risk HPV E7 requires YAP1 activity. Blocking HPV E7-dependent YAP1 activation could inhibit HPV-mediated carcinogenesis, but the mechanism by which HPV E7 activates YAP1 has not been elucidated. Here we report that by degrading the tumor suppressor PTPN14, HPV18 E7 inhibits LATS1 kinase, reducing inhibitory phosphorylation on YAP1. These data support that an HPV oncoprotein can inhibit Hippo signaling to activate YAP1 and strengthen the link between PTPN14 and Hippo signaling in human epithelial cells.

Interactions between lysyl–tRNA synthetase (LysRS) and HIV-1 Gag facilitate selective packaging of the HIV-1 reverse transcription primer, tRNALys3. During HIV-1 infection, LysRS is phosphorylated at S207, released from a multi-aminoacyl–tRNA synthetase complex and packaged into progeny virions. LysRS is critical for proper targeting of tRNALys3 to the primer-binding site (PBS) by specifically binding a PBS-adjacent tRNA-like element (TLE), which promotes release of the tRNA proximal to the PBS. However, whether LysRS phosphorylation plays a role in this process remains unknown. Here, we used a combination of binding assays, RNA chemical probing, and small-angle X-ray scattering to show that both wild-type (WT) and a phosphomimetic S207D LysRS mutant bind similarly to the HIV-1 genomic RNA (gRNA) 5′UTR via direct interactions with the TLE and stem loop 1 (SL1) and have a modest preference for binding dimeric gRNA. Unlike WT, S207D LysRS bound in an open conformation and increased the dynamics of both the PBS region and SL1. A new working model is proposed wherein a dimeric phosphorylated LysRS/tRNA complex binds to a gRNA dimer to facilitate tRNA primer release and placement onto the PBS. Future anti-viral strategies that prevent this host factor-gRNA interaction are envisioned.

Persistent human papillomavirus (HPV) infections cause about 4.5% of the human cancer burden. The HPV E7 oncoprotein is one of the primary drivers of HPV-mediated carcinogenesis and facilitates HPV replication. The most well-studied HPV E7 activity is inactivation of the retinoblastoma tumor suppressor (RB1), which dysregulates the cell cycle. However, there is substantial evidence to suggest that HPV E7 must have other activities in addition to RB1 inactivation. HPV E7 proteins from diverse HPV genotypes interact with the host cell tumor suppressor protein tyrosine phosphatase non-receptor 14 (PTPN14). Many HPV E7 proteins also direct PTPN14 for proteosome-mediated degradation. Through my dissertation work, I sought to identify the role of PTPN14 degradation by HPV E7 in the HPV replicative cycle and carcinogenesis. To initially characterize PTPN14 function in keratinocytes, we used mutant HPV E7 proteins that cannot degrade PTPN14, CRISPR knockout of PTPN14, and high throughput RNA sequencing to assess differential gene expression. We found that PTPN14 degradation by HPV E7 broadly represses genes related to keratinocyte differentiation. In a subsequent study, we used three-dimensional organotypic epithelial cultures to study the mechanism downstream of PTPN14 degradation. We found that PTPN14 loss in keratinocytes promotes basal cell-specific activation of the HIPPO pathway transcriptional regulator YAP1. PTPN14 degradation and YAP1 activation by HPV E7 did not prevent terminal differentiation in these cultures. Instead, the repression of differentiation observed in monolayer culture translated to delayed commitment to differentiation and promotion of basal cell identity in the three-dimensional system. This indicated that PTPN14 degradation by HPV E7 may help HPV resist epithelial turnover and facilitate persistent infection. YAP1 and its paralogue TAZ are oncogenes when dysregulated. Accordingly, through these studies we also found that PTPN14 degradation and YAP1/TAZ transcriptional activity contribute to the ability of HPV E7 proteins from oncogenic genotypes to extend the lifespans of primary keratinocytes, a metric of E7 carcinogenic activity. Overall, the experiments described in this dissertation outline the effects of PTPN14 degradation by HPV E7 in keratinocytes and stratified squamous epithelia which have implications for HPV persistence and HPV-mediated carcinogenesis.

Interactions between lysyl-tRNA synthetase (LysRS) and HIV-1 Gag facilitate selective packaging of the HIV-1 reverse transcription primer, tRNA . During HIV-1 infection, LysRS is phosphorylated at S207, released from a multi-aminoacyl-tRNA synthetase complex and packaged into progeny virions. LysRS is critical for proper targeting of tRNA to the primer-binding site (PBS) by specifically binding a PBS-adjacent tRNA-like element (TLE), which promotes release of the tRNA proximal to the PBS. However, whether LysRS phosphorylation plays a role in this process remains unknown. Here, we used a combination of binding assays, RNA chemical probing, and small-angle X-ray scattering to show that both wild-type (WT) and a phosphomimetic S207D LysRS mutant bind similarly to the HIV-1 genomic RNA (gRNA) 5'UTR via direct interactions with the TLE and stem loop 1 (SL1) and have a modest preference for binding dimeric gRNA. Unlike WT, S207D LysRS bound in an open conformation and increased the dynamics of both the PBS region and SL1. A new working model is proposed wherein a dimeric phosphorylated LysRS/tRNA complex binds to a gRNA dimer to facilitate tRNA primer release and placement onto the PBS. Future anti-viral strategies that prevent this host factor-gRNA interaction are envisioned.

Infection with the flavivirus Zika virus (ZIKV) can result in tissue tropism, disease outcome, and route of transmission distinct from those of other flaviviruses; therefore, we aimed to identify host machinery that exclusively promotes the ZIKV replication cycle, which can inform on differences at the organismal level. We previously reported that deletion of the host antiviral ribonuclease L (RNase L) protein decreases ZIKV production. Canonical RNase L catalytic activity typically restricts viral infection, including that of the flavivirus dengue virus (DENV), suggesting an unconventional, proviral RNase L function during ZIKV infection. In this study, we reveal that an inactive form of RNase L supports assembly of ZIKV replication factories (RFs) to enhance infectious virus production. Compared with the densely concentrated ZIKV RFs generated with RNase L present, deletion of RNase L induced broader subcellular distribution of ZIKV replication intermediate double-stranded RNA (dsRNA) and NS3 protease, two constituents of ZIKV RFs. An inactive form of RNase L was sufficient to contain ZIKV genome and dsRNA within a smaller RF area, which subsequently increased infectious ZIKV release from the cell. Inactive RNase L can interact with cytoskeleton, and flaviviruses remodel cytoskeleton to construct RFs. Thus, we used the microtubule-stabilization drug paclitaxel to demonstrate that ZIKV repurposes RNase L to facilitate the cytoskeleton rearrangements required for proper generation of RFs. During infection with flaviviruses DENV or West Nile Kunjin virus, inactive RNase L did not improve virus production, suggesting that a proviral RNase L role is not a general feature of all flavivirus infections.

The placenta combats mother-to-fetus transmission of viruses through the antiviral activities of fetal-derived trophoblasts. Placental trophoblasts employ specialized antiviral strategies to protect against infection while preventing maternal immune rejection of the fetus. However, the full extent of how trophoblasts respond to viral infections is not well understood. To address this, we defined the transcriptional landscape of human trophoblast organoids infected with seven diverse teratogenic viruses. We found that herpesviruses, including HSV-1, HSV-2, and HCMV, did not trigger a typical interferon (IFN) response. Instead, they activated the expression of DUX4 and its downstream target genes, collectively known as DUX4-stimulated genes (DSGs). This program was highly specific for trophoblasts and was associated with cells containing low viral transcripts following HSV-1 infection, suggesting an antiviral activity. Screening of highly expressed DSGs revealed that many of them exhibited anti-herpesvirus activity, indicating the existence of an alternative antiviral pathway similar to the IFN-stimulated gene response. These findings identify DUX4 as a master regulator of a coordinated antiviral program in trophoblasts, specifically targeting a prominent family of teratogenic viruses.

The placenta serves as a barrier against the vertical transmission of viruses, due to the protective functions of fetal-derived trophoblasts. Although some antiviral programs of trophoblasts are well-documented, our understanding of how trophoblasts respond to teratogenic viruses remains incomplete. To address this, we profiled the transcriptional responses of human trophoblast organoids to seven teratogenic viruses. We discovered that herpesviruses including HSV-1, HSV-2, and HCMV did not trigger an interferon (IFN) response but instead induced the expression of DUX4 and downstream target genes, termed DUX4-stimulated genes (DSGs). This program was uniquely, highly expressed in trophoblasts. Through single-cell RNA sequencing, we defined the trophoblast response to DUX4 and demonstrated that this program defines cells with low viral transcripts following HSV-1 infection. Similar to the IFN-stimulated genes, we observed that many DSGs with diverse predicted functions exhibited anti-herpesvirus activity. These findings establish DUX4 and DSGs as a critical antiviral defense mechanism in trophoblasts.Competing Interest StatementThe authors have declared no competing interest.

High-risk human papillomavirus (HPV) oncoproteins inactivate cellular tumor suppressors to reprogram host cell signaling pathways. HPV E7 proteins bind and degrade the tumor suppressor PTPN14, thereby promoting the nuclear localization of the YAP1 oncoprotein and inhibiting keratinocyte differentiation. YAP1 is a transcriptional coactivator that drives epithelial cell stemness and self-renewal. YAP1 activity is inhibited by the highly conserved Hippo pathway, which is frequently inactivated in human cancers. MST1/2 and LATS1/2 kinases form the core of the Hippo kinase cascade. Active LATS1 kinase is phosphorylated on threonine 1079 and inhibits YAP1 by phosphorylating it on amino acids including serine 127. Here, we tested the effect of high-risk (carcinogenic) HPV18 E7 on Hippo pathway activity. We found that either PTPN14 knockout or PTPN14 degradation by HPV18 E7 decreased phosphorylation of LATS1 T1079 and YAP1 S127 in human keratinocytes and inhibited keratinocyte differentiation. Conversely, PTPN14-dependent differentiation required LATS kinases and certain PPxY motifs in PTPN14. Neither MST1/2 kinases nor the putative PTPN14 phosphatase active site were required for PTPN14 to promote differentiation. Taken together, these data support that PTPN14 inactivation or degradation of PTPN14 by HPV18 E7 reduce LATS1 activity, promoting active YAP1 and inhibiting keratinocyte differentiation.