A novel HPV16 splicing enhancer critical for viral oncogene expression and cell immortalization.

High-risk carcinogenic human papillomaviruses (HPVs), e.g. HPV16, express the E6 and E7 oncogenes from two mRNAs that are generated in a mutually exclusive manner by splicing. The HPV16 E7 mRNA, also known as the E6*I/E7 mRNA, is produced by splicing between splice sites SD226 and SA409, while E6 mRNAs retain the intron between these splice sites. We show that splicing between HPV16 splice sites SD226 and SA409 is controlled by a splicing enhancer consisting of a perfect repeat of an adenosine-rich, 11 nucleotide sequence: AAAAGCAAAGA. Two nucleotide substitutions in both 11 nucleotide sequences specifically inhibited production of the spliced E6*I/E7 mRNA. As a result, production of E7 protein was reduced and the ability of HPV16 to immortalize human primary keratinocytes was abolished. The splicing-enhancing effect was mediated by the cellular TRAP150/THRAP3 protein that also enhanced splicing of other high-risk HPV E6*I/E7 mRNAs, but had no effect on low-risk HPV mRNAs. In summary, we have identified a novel splicing enhancer in the E6 coding region that is specific for high-risk HPVs and that is critically linked to HPV16 carcinogenic properties.

Kaposi's sarcoma-associated herpesvirus glycoprotein K8.1 is critical for infection in a cell-specific manner and functions at the attachment step on keratinocytes.

IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several B cell malignancies and Kaposi's sarcoma. We analyzed the function of K8.1, the major antigenic component of the KSHV virion in the infection of different cells. To do this, we deleted K8.1 from the viral genome. It was found that K8.1 is critical for the infection of certain epithelial cells, e.g., a skin model cell line but not for infection of many other cells. K8.1 was found to mediate attachment of the virus to cells where it plays a role in infection. In contrast, we did not find K8.1 or a related protein from a closely related monkey virus to activate fusion of the viral and cellular membranes, at least not under the conditions tested. These findings suggest that K8.1 functions in a highly cell-specific manner during KSHV entry, playing a crucial role in the attachment of KSHV to, e.g., skin epithelial cells.

Evaluation of the proteomic landscape of HPV E7­induced alterations in human keratinocytes reveal therapeutically relevant pathways for cervical cancer.

The lack of specific and accurate therapeutic targets poses a challenge in the treatment of cervical cancer (CC). Global proteomics has the potential to characterize the underlying and intricate molecular mechanisms that drive the identification of therapeutic candidates for CC in an unbiased manner. The present study assessed human papillomavirus (HPV)­induced proteomic alterations to identify key cancer hallmark pathways and protein­protein interaction (PPI) networks, which offered the opportunity to evaluate the possibility of using these for targeted therapy in CC. Comparative proteomic profiling of HPV­transfected (HPV16/18 E7), HPV­transformed (CaSki and HeLa) and normal human keratinocyte (HaCaT) cells was performed using the liquid chromatography­tandem mass spectrometry (LC­MS/MS) technique. Both label­free quantification and differential expression analysis were performed to assess differentially regulated proteins in HPV­transformed and ­transfected cells. The present study demonstrated that protein expression was upregulated in HPV­transfected cells compared with in HPV­transformed cells. This was probably due to the ectopic expression of E7 protein in the former cell type, in contrast to its constitutive expression in the latter cell type. Subsequent pathway visualization and network construction demonstrated that the upregulated proteins in HPV16/18 E7­transfected cells were predominantly associated with a diverse array of cancer hallmarks, including the mTORC1 signaling pathway, MYC targets V1, hypoxia and glycolysis. Among the various proteins present in the cancer hallmark enrichment pathways, phosphoglycerate kinase 1 (PGK1) was present across all pathways. Therefore, PGK1 may be considered as a potential biomarker. PPI analysis demonstrated a direct interaction between p130 and polyubiquitin B, which may lead to the degradation of p130 via the ubiquitin­proteasome proteolytic pathway. In summary, elucidation of the key signaling pathways in HPV16/18­transfected and ­transformed cells may aid in the design of novel therapeutic strategies for clinical application such as targeted therapy and immunotherapy against cervical cancer.

Transcription Properties of Beta-HPV8 and HPV38 Genomes in Human Keratinocytes.

Persistent infections with high-risk human papillomaviruses (HR-HPV) from the genus alpha are established risk factors for the development of anogenital and oropharyngeal cancers. In contrast, HPV from the genus beta have been implicated in the development of cutaneous squamous cell cancer (cSCC) in epidermodysplasia verruciformis (EV) patients and organ transplant recipients. Keratinocytes are the in vivo target cells for HPV, but keratinocyte models to investigate the replication and oncogenic activities of beta-HPV genomes have not been established. A recent study revealed, that beta-HPV49 immortalizes normal human keratinocytes (NHK) only, when the viral E8^E2 repressor (E8-) is inactivated (T. M. Rehm, E. Straub, T. Iftner, and F. Stubenrauch, Proc Natl Acad Sci U S A 119:e2118930119, 2022, https://doi.org/10.1073/pnas.2118930119). We now demonstrate that beta-HPV8 and HPV38 wild-type or E8- genomes are unable to immortalize NHK. Nevertheless, HPV8 and HPV38 express E6 and E7 oncogenes and other transcripts in transfected NHK. Inactivation of the conserved E1 and E2 replication genes reduces viral transcription, whereas E8- genomes display enhanced viral transcription, suggesting that beta-HPV genomes replicate in NHK. Furthermore, growth of HPV8- or HPV38-transfected NHK in organotypic cultures, which are routinely used to analyze the productive replication cycle of HR-HPV, induces transcripts encoding the L1 capsid gene, suggesting that the productive cycle is initiated. In addition, transcription patterns in HPV8 organotypic cultures and in an HPV8-positive lesion from an EV patient show similarities. Taken together, these data indicate that NHK are a suitable system to analyze beta-HPV8 and HPV38 replication. IMPORTANCE High-risk HPV, from the genus alpha, can cause anogenital or oropharyngeal malignancies. The oncogenic properties of high-risk HPV are important for their differentiation-dependent replication in human keratinocytes, the natural target cell for HPV. HPV from the genus beta have been implicated in the development of cutaneous squamous cell cancer in epidermodysplasia verruciformis (EV) patients and organ transplant recipients. Currently, the replication cycle of beta-HPV has not been studied in human keratinocytes. We now provide evidence that beta-HPV8 and 38 are transcriptionally active in human keratinocytes. Inactivation of the viral E8^E2 repressor protein greatly increases genome replication and transcription of the E6 and E7 oncogenes, but surprisingly, this does not result in immortalization of keratinocytes. Differentiation of HPV8- or HPV38-transfected keratinocytes in organotypic cultures induces transcripts encoding the L1 capsid gene, suggesting that productive replication is initiated. This indicates that human keratinocytes are suited as a model to investigate beta-HPV replication.

Antiviral Effect of hBD-3 and LL-37 during Human Primary Keratinocyte Infection with West Nile Virus.

West Nile virus (WNV) is an emerging flavivirus transmitted through mosquito bites and responsible for a wide range of clinical manifestations. Following their inoculation within the skin, flaviviruses replicate in keratinocytes of the epidermis, inducing an innate immune response including the production of antimicrobial peptides (AMPs). Among them, the cathelicidin LL-37 and the human beta-defensin (hBD)-3 are known for their antimicrobial and immunomodulatory properties. We assessed their role during WNV infection of human primary keratinocytes. LL-37 reduced the viral load in the supernatant of infected keratinocytes and of the titer of a viral inoculum incubated in the presence of the peptide, suggesting a direct antiviral effect of this AMP. Conversely, WNV replication was not inhibited by hBD-3. The two peptides then demonstrated immunomodulatory properties whether in the context of keratinocyte stimulation by poly(I:C) or infection by WNV, but not alone. This study demonstrates the immunostimulatory properties of these two skin AMPs at the initial site of WNV replication and the ability of LL-37 to directly inactivate West Nile viral infectious particles. The results provide new information on the multiple functions of these two peptides and underline the potential of AMPs as new antiviral strategies in the fight against flaviviral infections.

Lyon IARC Polyomavirus Displays Transforming Activities in Primary Human Cells.

Several studies reported the presence of a recently discovered polyomavirus (PyV), Lyon IARC PyV (LIPyV), in human and domestic animal specimens. LIPyV has some structural similarities to well-established animal and human oncogenic PyVs, such as raccoon PyV and Merkel cell PyV (MCPyV), respectively. In this study, we demonstrate that LIPyV early proteins immortalize human foreskin keratinocytes. LIPyV LT binds pRb, accordingly cell cycle checkpoints are altered in primary human fibroblasts and keratinocytes expressing LIPyV early genes. Mutation of the pRb binding site in LT strongly affected the ability of LIPyV ER to induced HFK immortalization. LIPyV LT also binds p53 and alters p53 functions activated by cellular stresses. Finally, LIPyV early proteins activate telomerase reverse transcriptase (hTERT) gene expression, via accumulation of the Sp1 transcription factor. Sp1 recruitment to the hTERT promoter is controlled by its phosphorylation, which is mediated by ERK1 and CDK2. Together, these data highlight the transforming properties of LIPyV in in vitro experimental models, supporting its possible oncogenic nature. IMPORTANCE Lyon IARC PyV is a recently discovered polyomavirus that shows some structural similarities to well-established animal and human oncogenic PyVs, such as raccoon PyV and Merkel cell PyV, respectively. Here, we show the capability of LIPyV to efficiently promote cellular transformation of primary human cells, suggesting a possible oncogenic role of this virus in domestic animals and/or humans. Our study identified a novel virus-mediated mechanism of activation of telomerase reverse transcriptase gene expression, via accumulation of the Sp1 transcription factor. In addition, because the persistence of infection is a key event in virus-mediated carcinogenesis, it will be important to determine whether LIPyV can deregulate immune-related pathways, similarly to the well-established oncogenic viruses.

Restriction of viral gene expression and replication prevents immortalization of human keratinocytes by a beta-human papillomavirus.

SignificanceHigh-risk (HR) human papillomaviruses (HPV) from the genus alpha cause anogenital and oropharyngeal cancers, whereas the contribution of HPV from the genus beta to the development of cutaneous squamous cell cancer is still under debate. HR-HPV genomes display potent immortalizing activity in human keratinocytes, the natural target cell for HPV. This paper shows that immortalization of keratinocytes by the beta-HPV49 genome requires the inactivation of the viral E8^E2 repressor protein and the presence of the E6 and E7 oncoproteins but also of the E1 and E2 replication proteins. This reveals important differences in the carcinogenic properties of HR-HPV and beta-HPV but also warrants further investigations on the distribution and mutation frequencies of beta-HPV in human cancers.

Multiple Salivary Proteins from Aedes aegypti Mosquito Bind to the Zika Virus Envelope Protein.

Aedes aegypti mosquitoes are important vectors of several debilitating and deadly arthropod-borne (arbo) viruses, including Yellow Fever virus, Dengue virus, West Nile virus and Zika virus (ZIKV). Arbovirus transmission occurs when an infected mosquito probes the host's skin in search of a blood meal. Salivary proteins from mosquitoes help to acquire blood and have also been shown to enhance pathogen transmission in vivo and in vitro. Here, we evaluated the interaction of mosquito salivary proteins with ZIKV by surface plasmon resonance and enzyme-linked immunosorbent assay. We found that three salivary proteins AAEL000793, AAEL007420, and AAEL006347 bind to the envelope protein of ZIKV with nanomolar affinities. Similar results were obtained using virus-like particles in binding assays. These interactions have no effect on viral replication in cultured endothelial cells and keratinocytes. Additionally, we found detectable antibody levels in ZIKV and DENV serum samples against the recombinant proteins that interact with ZIKV. These results highlight complex interactions between viruses, salivary proteins and antibodies that could be present during viral transmissions.

Role of E6 in Maintaining the Basal Cell Reservoir during Productive Papillomavirus Infection.

Papillomaviruses exclusively infect stratified epithelial tissues and cause chronic infections. To achieve this, infected cells must remain in the epithelial basal layer alongside their uninfected neighbors for years or even decades. To examine how papillomaviruses achieve this, we used the in vivo MmuPV1 (Mus musculus papillomavirus 1) model of lesion formation and persistence. During early lesion formation, an increased cell density in the basal layer, as well as a delay in the infected cells' commitment to differentiation, was apparent in cells expressing MmuPV1 E6/E7 RNA. Using cell culture models, keratinocytes exogenously expressing MmuPV1 E6, but not E7, recapitulated this delay in differentiation postconfluence and also grew to a significantly higher density. Cell competition assays further showed that MmuPV1 E6 expression led to a preferential persistence of the cell in the first layer, with control cells accumulating almost exclusively in the second layer. Interestingly, the disruption of MmuPV1 E6 binding to MAML1 protein abrogated these phenotypes. This suggests that the interaction between MAML1 and E6 is necessary for the lower (basal)-layer persistence of MmuPV1 E6-expressing cells. Our results indicate a role for E6 in lesion establishment by facilitating the persistence of infected cells in the epithelial basal layer, a mechanism that is most likely shared by other papillomavirus types. Interruption of this interaction is predicted to impede persistent papillomavirus infection and consequently provides a novel treatment target. IMPORTANCE Persistent infection with high-risk HPV types can lead to development of HPV-associated cancers, and persistent low-risk HPV infection causes problematic diseases, such as recurrent respiratory papillomatosis. The management and treatment of these conditions pose a considerable economic burden. Maintaining a reservoir of infected cells in the basal layer of the epithelium is critical for the persistence of infection in the host, and our studies using the mouse papillomavirus model suggest that E6 gene expression leads to the preferential persistence of epithelial cells in the lower layers during stratification. The E6 interaction with MAML1, a component of the Notch pathway, is required for this phenotype and is linked to E6 effects on cell density and differentiation. These observations are likely to reflect a common E6 role that is preserved among papillomaviruses and provide us with a novel therapeutic target for the treatment of recalcitrant lesions.

ΔNp63α promotes Epstein-Barr virus latency in undifferentiated epithelial cells.

Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and contributes to both B-cell and epithelial-cell malignancies. EBV-infected epithelial cell tumors, including nasopharyngeal carcinoma (NPC), are largely composed of latently infected cells, but the mechanism(s) maintaining viral latency are poorly understood. Expression of the EBV BZLF1 (Z) and BRLF1 (R) encoded immediate-early (IE) proteins induces lytic infection, and these IE proteins activate each other's promoters. ΔNp63α (a p53 family member) is required for proliferation and survival of basal epithelial cells and is over-expressed in NPC tumors. Here we show that ΔNp63α promotes EBV latency by inhibiting activation of the BZLF1 IE promoter (Zp). Furthermore, we find that another p63 gene splice variant, TAp63α, which is expressed in some Burkitt and diffuse large B cell lymphomas, also represses EBV lytic reactivation. We demonstrate that ΔNp63α inhibits the Z promoter indirectly by preventing the ability of other transcription factors, including the viral IE R protein and the cellular KLF4 protein, to activate Zp. Mechanistically, we show that ΔNp63α promotes viral latency in undifferentiated epithelial cells both by enhancing expression of a known Zp repressor protein, c-myc, and by decreasing cellular p38 kinase activity. Furthermore, we find that the ability of cis-platinum chemotherapy to degrade ΔNp63α contributes to the lytic-inducing effect of this agent in EBV-infected epithelial cells. Together these findings demonstrate that the loss of ΔNp63α expression, in conjunction with enhanced expression of differentiation-dependent transcription factors such as BLIMP1 and KLF4, induces lytic EBV reactivation during normal epithelial cell differentiation. Conversely, expression of ΔNp63α in undifferentiated nasopharyngeal carcinoma cells and TAp63α in Burkitt lymphoma promotes EBV latency in these malignancies.

The chromatin insulator CTCF regulates HPV18 transcript splicing and differentiation-dependent late gene expression.

The ubiquitous host protein, CCCTC-binding factor (CTCF), is an essential regulator of cellular transcription and functions to maintain epigenetic boundaries, stabilise chromatin loops and regulate splicing of alternative exons. We have previously demonstrated that CTCF binds to the E2 open reading frame (ORF) of human papillomavirus (HPV) 18 and functions to repress viral oncogene expression in undifferentiated keratinocytes by co-ordinating an epigenetically repressed chromatin loop within HPV episomes. Keratinocyte differentiation disrupts CTCF-dependent chromatin looping of HPV18 episomes promoting induction of enhanced viral oncogene expression. To further characterise CTCF function in HPV transcription control we utilised direct, long-read Nanopore RNA-sequencing which provides information on the structure and abundance of full-length transcripts. Nanopore analysis of primary human keratinocytes containing HPV18 episomes before and after synchronous differentiation allowed quantification of viral transcript species, including the identification of low abundance novel transcripts. Comparison of transcripts produced in wild type HPV18 genome-containing cells to those identified in CTCF-binding deficient genome-containing cells identifies CTCF as a key regulator of differentiation-dependent late promoter activation, required for efficient E1^E4 and L1 protein expression. Furthermore, our data show that CTCF binding at the E2 ORF promotes usage of the downstream weak splice donor (SD) sites SD3165 and SD3284, to the dominant E4 splice acceptor site at nucleotide 3434. These findings demonstrate that in the HPV life cycle both early and late virus transcription programmes are facilitated by recruitment of CTCF to the E2 ORF.

Peculiar Histopathologic Feature of an Erythematous/Morbilliform Eruption in a COVID-19-Positive Patient.

ABSTRACT: One of the most common patterns of presentations that have been described in COVID-19 patients includes the erythematous/papular/morbilliform eruptions. However, actually, the diffuse exanthems containing macules and papules were not specific to COVID-19, and even histopathology does not show any specific signs that could help to differentiate COVID-19 skin lesions from non-COVID-19 causes such as drugs or other viral infections. We present the case of a COVID-19-positive woman with a morbilliform rash, whose skin biopsy showed the presence of some peculiar cytopathic epidermal changes that could represent a possible distinctive histopathological feature related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection The presence of viral particles in the keratinocytes with additional positivity of endothelial cells and eccrine glands by immunohistochemistry using an anti-SARS-CoV-2 Spike S1 antibodies supports a causal relation of the lesions with SARS-CoV-2 infection.

Cell-to-cell transmission of HSV1 in human keratinocytes in the absence of the major entry receptor, nectin1.

Herpes simplex virus 1 (HSV1) infects the stratified epithelia of the epidermis, oral or genital mucosa, where the main cell type is the keratinocyte. Here we have used nTERT human keratinocytes to generate a CRISPR-Cas9 knockout (KO) of the primary candidate HSV1 receptor, nectin1, resulting in a cell line that is refractory to HSV1 entry. Nonetheless, a small population of KO cells was able to support infection which was not blocked by a nectin1 antibody and hence was not a consequence of residual nectin1 expression. Strikingly at later times, the population of cells originally resistant to HSV1 infection had also become infected. Appearance of this later population was blocked by inhibition of virus genome replication, or infection with a ΔUL34 virus defective in capsid export to the cytoplasm. Moreover, newly formed GFP-tagged capsids were detected in cells surrounding the initial infected cell, suggesting that virus was spreading following replication in the original susceptible cells. Additional siRNA depletion of the second major HSV1 receptor HVEM, or PTP1B, a cellular factor shown elsewhere to be involved in cell-to-cell transmission, had no effect on virus spread in the absence of nectin1. Neutralizing human serum also failed to block virus transmission in nectin1 KO cells, which was dependent on the receptor binding protein glycoprotein D and the cell-to-cell spread glycoproteins gI and gE, indicating that virus was spreading by direct cell-to-cell transmission. In line with these results, both HSV1 and HSV2 formed plaques on nectin1 KO cells, albeit at a reduced titre, confirming that once the original cell population was infected, the virus could spread into all other cells in the monolayer. We conclude that although nectin1 is required for extracellular entry in to the majority of human keratinocytes, it is dispensable for direct cell-to-cell transmission.

Genomic Instability and DNA Damage Repair Pathways Induced by Human Papillomaviruses.

Human papillomaviruses (HPV) are the causative agents of cervical and other anogenital cancers as well as those of the oropharynx. HPV proteins activate host DNA damage repair factors to promote their viral life cycle in stratified epithelia. Activation of both the ATR pathway and the ATM pathway are essential for viral replication and differentiation-dependent genome amplification. These pathways are also important for maintaining host genomic integrity and their dysregulation or mutation is often seen in human cancers. The APOBEC3 family of cytidine deaminases are innate immune factors that are increased in HPV positive cells leading to the accumulation of TpC mutations in cellular DNAs that contribute to malignant progression. The activation of DNA damage repair factors may corelate with expression of APOBEC3 in HPV positive cells. These pathways may actively drive tumor development implicating/suggesting DNA damage repair factors and APOBEC3 as possible therapeutic targets.

CK2 Phosphorylation of Human Papillomavirus 16 E2 on Serine 23 Promotes Interaction with TopBP1 and Is Critical for E2 Interaction with Mitotic Chromatin and the Viral Life Cycle.

During the human papillomavirus 16 (HPV16) life cycle, the E2 protein interacts with host factors to regulate viral transcription, replication, and genome segregation/retention. Our understanding of host partner proteins and their roles in E2 functions remains incomplete. Here we demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 in vitro and in vivo and that E2 is phosphorylated on this residue during the HPV16 life cycle. We investigated the consequences of mutating serine 23 on E2 functions. E2-S23A (E2 with serine 23 mutated to alanine) activates and represses transcription identically to E2-WT (wild-type E2), and E2-S23A is as efficient as E2-WT in transient replication assays. However, E2-S23A has compromised interaction with mitotic chromatin compared with E2-WT. In E2-WT cells, both E2 and TopBP1 levels increase during mitosis compared with vector control cells. In E2-S23A cells, neither E2 nor TopBP1 levels increase during mitosis. Introduction of the S23A mutation into the HPV16 genome resulted in delayed immortalization of human foreskin keratinocytes (HFK) and higher episomal viral genome copy number in resulting established HFK. Remarkably, S23A cells had a disrupted viral life cycle in organotypic raft cultures, with a loss of E2 expression and a failure of viral replication. Overall, our results demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 and that this interaction is critical for the viral life cycle. IMPORTANCE Human papillomaviruses are causative agents in around 5% of all cancers, with no specific antiviral therapeutics available for treating infections or resultant cancers. In this report, we demonstrate that phosphorylation of HPV16 E2 by CK2 promotes formation of a complex with the cellular protein TopBP1 in vitro and in vivo. This complex results in stabilization of E2 during mitosis. We demonstrate that CK2 phosphorylates E2 on serine 23 in vivo and that CK2 inhibitors disrupt the E2-TopBP1 complex. Mutation of E2 serine 23 to alanine disrupts the HPV16 life cycle, hindering immortalization and disrupting the viral life cycle, demonstrating a critical function for this residue.

The Mus musculus Papillomavirus Type 1 E7 Protein Binds to the Retinoblastoma Tumor Suppressor: Implications for Viral Pathogenesis.

The species specificity of papillomaviruses has been a significant roadblock for performing in vivo pathogenesis studies in common model organisms. The Mus musculus papillomavirus type 1 (MmuPV1) causes cutaneous papillomas that can progress to squamous cell carcinomas in laboratory mice. The papillomavirus E6 and E7 genes encode proteins that establish and maintain a cellular milieu that allows for viral genome synthesis and viral progeny synthesis in growth-arrested, terminally differentiated keratinocytes. The E6 and E7 proteins provide this activity by binding to and functionally reprogramming key cellular regulatory proteins. The MmuPV1 E7 protein lacks the canonical LXCXE motif that mediates the binding of multiple viral oncoproteins to the cellular retinoblastoma tumor suppressor protein, RB1. Our proteomic experiments, however, revealed that MmuPV1 E7 still interacts with RB1. We show that MmuPV1 E7 interacts through its C terminus with the C-terminal domain of RB1. Binding of MmuPV1 E7 to RB1 did not cause significant activation of E2F-regulated cellular genes. MmuPV1 E7 expression was shown to be essential for papilloma formation. Experimental infection of mice with MmuPV1 expressing an E7 mutant that is defective for binding to RB1 caused delayed onset, lower incidence, and smaller sizes of papillomas. Our results demonstrate that the MmuPV1 E7 gene is essential and that targeting noncanonical activities of RB1, which are independent of RB1's ability to modulate the expression of E2F-regulated genes, contribute to papillomavirus-mediated pathogenesis. IMPORTANCE Papillomavirus infections cause a variety of epithelial hyperplastic lesions, or warts. While most warts are benign, some papillomaviruses cause lesions that can progress to squamous cell carcinomas, and approximately 5% of all human cancers are caused by human papillomavirus (HPV) infections. The papillomavirus E6 and E7 proteins are thought to function to reprogram host epithelial cells to enable viral genome replication in terminally differentiated, normally growth-arrested cells. E6 and E7 lack enzymatic activities and function by interacting and functionally altering host cell regulatory proteins. Many cellular proteins that can interact with E6 and E7 have been identified, but the biological relevance of these interactions for viral pathogenesis has not been determined. This is because papillomaviruses are species specific and do not infect heterologous hosts. Here, we use a recently established mouse papillomavirus (MmuPV1) model to investigate the role of the E7 protein in viral pathogenesis. We show that MmuPV1 E7 is necessary for papilloma formation. The retinoblastoma tumor suppressor protein (RB1) is targeted by many papillomaviral E7 proteins, including cancer-associated HPVs. We show that MmuPV1 E7 can bind RB1 and that infection with a mutant MmuPV1 virus that expresses an RB1 binding-defective E7 mutant caused smaller and fewer papillomas that arise with delayed kinetics.

Phosphorylation of RCC1 on Serine 11 Facilitates G1/S Transition in HPV E7-Expressing Cells.

Persistent infection of high-risk human papillomavirus (HR-HPV) plays a causal role in cervical cancer. Regulator of chromosome condensation 1 (RCC1) is a critical cell cycle regulator, which undergoes a few post-translational modifications including phosphorylation. Here, we showed that serine 11 (S11) of RCC1 was phosphorylated in HPV E7-expressing cells. However, S11 phosphorylation was not up-regulated by CDK1 in E7-expressing cells; instead, the PI3K/AKT/mTOR pathway promoted S11 phosphorylation. Knockdown of AKT or inhibition of the PI3K/AKT/mTOR pathway down-regulated phosphorylation of RCC1 S11. Furthermore, S11 phosphorylation occurred throughout the cell cycle, and reached its peak during the mitosis phase. Our previous data proved that RCC1 was necessary for the G1/S cell cycle progression, and in the present study we showed that the RCC1 mutant, in which S11 was mutated to alanine (S11A) to mimic non-phosphorylation status, lost the ability to facilitate G1/S transition in E7-expressing cells. Moreover, RCC1 S11 was phosphorylated by the PI3K/AKT/mTOR pathway in HPV-positive cervical cancer SiHa and HeLa cells. We conclude that S11 of RCC1 is phosphorylated by the PI3K/AKT/mTOR pathway and phosphorylation of RCC1 S11 facilitates the abrogation of G1 checkpoint in HPV E7-expressing cells. In short, our study explores a new role of RCC1 S11 phosphorylation in cell cycle regulation.

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry.

Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced cell binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.

Herpes simplex virus 2 (HSV-2) evolves faster in cell culture than HSV-1 by generating greater genetic diversity.

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) are prevalent human pathogens of clinical relevance that establish long-life latency in the nervous system. They have been considered, along with the Herpesviridae family, to exhibit a low level of genetic diversity during viral replication. However, the high ability shown by these viruses to rapidly evolve under different selective pressures does not correlates with that presumed genetic stability. High-throughput sequencing has revealed that heterogeneous or plaque-purified populations of both serotypes contain a broad range of genetic diversity, in terms of number and frequency of minor genetic variants, both in vivo and in vitro. This is reminiscent of the quasispecies phenomenon traditionally associated with RNA viruses. Here, by plaque-purification of two selected viral clones of each viral subtype, we reduced the high level of genetic variability found in the original viral stocks, to more genetically homogeneous populations. After having deeply characterized the genetic diversity present in the purified viral clones as a high confidence baseline, we examined the generation of de novo genetic diversity under culture conditions. We found that both serotypes gradually increased the number of de novo minor variants, as well as their frequency, in two different cell types after just five and ten passages. Remarkably, HSV-2 populations displayed a much higher raise of nonconservative de novo minor variants than the HSV-1 counterparts. Most of these minor variants exhibited a very low frequency in the population, increasing their frequency over sequential passages. These new appeared minor variants largely impacted the coding diversity of HSV-2, and we found some genes more prone to harbor higher variability. These data show that herpesviruses generate de novo genetic diversity differentially under equal in vitro culture conditions. This might have contributed to the evolutionary divergence of HSV-1 and HSV-2 adapting to different anatomical niche, boosted by selective pressures found at each epithelial and neuronal tissue.

Hippo signaling effectors YAP and TAZ induce Epstein-Barr Virus (EBV) lytic reactivation through TEADs in epithelial cells.

The Epstein-Barr virus (EBV) human herpesvirus is associated with B-cell and epithelial-cell malignancies, and both the latent and lytic forms of viral infection contribute to the development of EBV-associated tumors. Here we show that the Hippo signaling effectors, YAP and TAZ, promote lytic EBV reactivation in epithelial cells. The transcriptional co-activators YAP/TAZ (which are inhibited by Hippo signaling) interact with DNA-binding proteins, particularly TEADs, to induce transcription. We demonstrate that depletion of either YAP or TAZ inhibits the ability of phorbol ester (TPA) treatment, cellular differentiation or the EBV BRLF1 immediate-early (IE) protein to induce lytic EBV reactivation in oral keratinocytes, and show that over-expression of constitutively active forms of YAP and TAZ reactivate lytic EBV infection in conjunction with TEAD family members. Mechanistically, we find that YAP and TAZ interact with, and activate, the EBV BZLF1 immediate-early promoter. Furthermore, we demonstrate that YAP, TAZ, and TEAD family members are expressed at much higher levels in epithelial cell lines in comparison to B-cell lines, and find that EBV infection of oral keratinocytes increases the level of activated (dephosphorylated) YAP and TAZ. Finally, we have discovered that lysophosphatidic acid (LPA), a known YAP/TAZ activator that plays an important role in inflammation, induces EBV lytic reactivation in epithelial cells through a YAP/TAZ dependent mechanism. Together these results establish that YAP/TAZ are powerful inducers of the lytic form of EBV infection and suggest that the ability of EBV to enter latency in B cells at least partially reflects the extremely low levels of YAP/TAZ and TEADs in this cell type.