Biology subjects

Nouel, J.

Publications and source records attributed to Nouel, J..

3 recordsLinked to original sources

bioRxiv2026

Inactivation of the RB1 and PTPN14 tumor suppressors cooperatively enables the carcinogenic activity of the human papillomavirus E7 oncoprotein

High-risk human papillomavirus (HPV) E7 proteins bind and inactivate host cellular tumor suppressors and are essential for the immortalization of primary human keratinocytes. E7 proteins from high- and low-risk HPV genotypes bind directly to at least two tumor suppressors, RB1 and PTPN14, and inactivate both. We previously characterized mutations in high-risk HPV E7 proteins that selectively abrogate the ability of E7 to bind either RB1 or PTPN14. Here, we established a genetic complementation system using the E7 mutants defective for binding to RB1 or PTPN14. Neither mutant alone could extend the lifespan of primary keratinocytes. When expressed together, the mutants could, like wild-type high-risk HPV E7, extend keratinocyte lifespan. Both high- and low-risk E7 reduced PTPN14 protein levels and reduced expression of keratinocyte differentiation genes, whereas only high-risk E7 reduced steady-state RB1 levels and induced E2F-dependent genes. Depletion of either RB1 or PTPN14 could cooperate with low-risk HPV6 E7 to extend keratinocyte lifespan. Our findings advance the model that inactivation of at least two tumor suppressors is required for the lifespan extension activity of high-risk HPV E7. SignificanceInactivation of the retinoblastoma tumor suppressor (RB1) is necessary but insufficient for HPV E7-mediated immortalization of human cells. In addition to inactivating RB1, HPV E7 proteins also target for degradation PTPN14, a tumor suppressor and inhibitor of the YAP1 oncoprotein. We report genetic complementation experiments demonstrating that RB1 inactivation and PTPN14 inactivation are separate activities of E7. Either depletion of RB1 or PTPN14 can confer lifespan extension activity on a low-risk HPV E7. These findings redefine our understanding of E7 transforming activity. The predominant difference between high- and low-risk E7 is their ability to degrade RB1, but inactivation of both tumor suppressors is required for E7 activity. Targeting either E7/RB1 or E7/PTPN14 could be of therapeutic benefit.

microbiology↗

bioRxiv2023

The vaccinia virus E3L dsRNA binding protein detects distinct production patterns of exogenousand endogenous dsRNA

Double-stranded RNA (dsRNA) is a pathogen associated molecular pattern recognized by multiple pattern recognition receptors and induces innate immune responses. Viral infections can generate dsRNA during virus replication. Genetic mutations can also lead to endogenous dsRNA accumulation. DsRNA is present in multiple conformations such as the A form (A-dsRNA) or Z form (Z-dsRNA). A-dsRNA has been detected from multiple viruses with positive-stranded RNA genomes (+ssRNA) but rarely from viruses with negative RNA genomes (-RNA); Z-dsRNA can be detected from influenza virus and poxvirus infections. Viruses have evolved mechanisms to antagonize cellular antiviral responses triggered by dsRNAs. For example, the vaccinia-virus E3L protein can bind and sequester dsRNA to evade host immune responses. The E3L protein encodes a Z-DNA and a dsRNA binding domains that bind to Z-form nucleic acids or dsRNA, respectively. Here we developed recombinant E3L proteins to detect dsRNA and Z-dsRNA generated from viral infections or endogenous cellular mutations. We demonstrate that the E3L recombinant protein specifically detects A-dsRNA generated from +ssRNA viruses but not-RNA viruses. We observe that among various virus infections assayed, only the influenza A virus generates Z-RNA that can be detected by anti-Z-NA antibody but not by the E3L recombinant protein containing the Z-DNA domain. The E3L recombinant protein can also detect endogenous dsRNA in PNPT1 or SUV3L1 knockout cells. Together we concluded that A-dsRNA can be produced and detected from viruses with +ssRNA genomes but not-RNA genomes, and Z-dsRNA can be produced and detected from influenza A virus. ImportanceThe detection of dsRNAs, which exist in the A-dsRNA or Z-RNA conformation, is important for the induction of innate immune responses. dsRNA are generated during a virus infection due to virus replication, or can accumulate to genetic mutations. We engineered recombinant vaccinia virus E3L protein that can detect A-dsRNA generated during infection with a positive-sense RNA genome virus but not a negative-sense RNA genome virus. Infection with influenza A virus generates Z-RNA that can be detected with an anti-z-antibody but not the E3L recombinant protein. The E3L recombinant protein also detects endogenous dsRNA in PNPT1 or SUV3L knockout cells. These findings highlight important characteristics of dsRNA structure and detection.

microbiology↗

bioRxiv2022

ZER1 contributes to the carcinogenic activity of high-risk HPV E7 proteins

Human Papillomavirus (HPV) E7 proteins bind to host cell proteins to facilitate virus replication. Interactions between HPV E7 and host cell proteins can also drive cancer progression. We hypothesize that HPV E7-host protein interactions specific for high-risk E7 contribute to the carcinogenic activity of high-risk HPV. The cellular protein ZER1 interacts with the E7 protein from HPV16, the genotype most frequently associated with human cancers. The HPV16 E7-ZER1 interaction is unique among HPV E7 tested to date. Other E7 proteins, even from closely related HPV genotypes, do not bind ZER1, which is a substrate specificity factor for a CUL2-RING ubiquitin ligase. In the present study, we investigated the contribution of ZER1 to the carcinogenic activity of HPV16 E7. First, we mapped the ZER1 binding site to specific residues on the C-terminus of HPV16 E7. We showed that the mutant HPV16 E7 that cannot bind ZER1 is impaired in the ability to promote the growth of primary keratinocytes. We found that ZER1 and CUL2 contribute to but are not required for HPV16 E7 to degrade RB1. Cancer dependency data shows that ZER1 is an essential gene in most HPV-positive, but not HPV-negative, cancer cell lines. Depleting ZER1 impaired the growth of primary keratinocytes expressing HPV16 E7 or HPV18 E7 and of HPV16 and HPV18-positive cervical cancer cell lines. Taken together, our work demonstrates that ZER1 contributes to HPV-mediated carcinogenesis and is essential for the growth of HPV-positive cells. IMPORTANCEHPV16 is highly carcinogenic and is the most predominant HPV genotype associated with human cancers. The mechanisms that underlie differences between high-risk HPV genotypes are currently unknown, but many of these differences are likely attributable to the activities of the oncogenic HPV proteins, including E7. The HPV E7 oncoprotein is essential for HPV-mediated carcinogenesis. A large number of HPV E7 targets have been identified. However, it is unclear which of these many interactions contribute to the carcinogenic activity of HPV E7. Here, we characterized the interaction between HPV16 E7 and the host cell protein ZER1, testing whether this genotype-specific interaction could enable the enhanced carcinogenic activity of HPV16 E7. We found that ZER1 binding contributes to the growth promoting activity of HPV16 E7 and to the growth of HPV-positive cervical cancer cells. We propose that ZER1 makes an important contribution to HPV-mediated carcinogenesis.

microbiology↗