Edward C. Goodwin

Senescence‐associated β‐galactosidase is lysosomal β‐galactosidase

Replicative senescence limits the proliferation of somatic cells passaged in culture and may reflect cellular aging in vivo. The most widely used biomarker for senescent and aging cells is senescence‐associated β‐galactosidase (SA‐β‐gal), which is defined as β‐galactosidase activity detectable at pH 6.0 in senescent cells, but the origin of SA‐β‐gal and its cellular roles in senescence are not known. We demonstrate here that SA‐β‐gal activity is expressed from GLB1, the gene encoding lysosomal β‐D‐galactosidase, the activity of which is typically measured at acidic pH 4.5. Fibroblasts from patients with autosomal recessive GM1‐gangliosidosis, which have defective lysosomal β‐galactosidase, did not express SA‐β‐gal at late passages even though they underwent replicative senescence. In addition, late passage normal fibroblasts expressing small‐hairpin interfering RNA that depleted GLB1 mRNA underwent senescence but failed to express SA‐β‐gal. GLB1 mRNA depletion also prevented expression of SA‐β‐gal activity in HeLa cervical carcinoma cells induced to enter a senescent state by repression of their endogenous human papillomavirus E7 oncogene. SA‐β‐gal induction during senescence was due at least in part to increased expression of the lysosomal β‐galactosidase protein. These results also indicate that SA‐β‐gal is not required for senescence.

ENDOGENOUS HUMAN PAPILLOMAVIRUS E6 AND E7 PROTEINS DIFFERENTIALLY REGULATE PROLIFERATION, SENESCENCE, AND APOPTOSIS IN HELA CERVICAL CARCINOMA CELLS

ABSTRACT Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins, and repression of HPV gene expression causes the cells to cease proliferation and undergo senescence. However, it is not known whether both HPV proteins are required to maintain the proliferative state of cervical cancer cells, or whether mutations that accumulate during carcinogenesis eliminate the need for one or the other of them. To address these questions, we used the bovine papillomavirus E2 protein to repress the expression of either the E6 protein or the E7 protein encoded by integrated HPV18 DNA in HeLa cervical carcinoma cells. Repression of the E7 protein activated the Rb pathway but not the p53 pathway and triggered senescence, whereas repression of the E6 protein activated the p53 pathway but not the Rb pathway and triggered both senescence and apoptosis. Telomerase activity, cyclin-dependent kinase activity, and expression of c-myc were markedly inhibited by repression of either E6 or E7. These results demonstrate that continuous expression of both the E6 and the E7 protein is required for optimal proliferation of cervical carcinoma cells and that the two viral proteins exert distinct effects on cell survival and proliferation. Therefore, strategies that inhibit the expression or activity of either viral protein are likely to inhibit the growth of HPV-associated cancers.

Human Papillomavirus E7 Repression in Cervical Carcinoma Cells Initiates a Transcriptional Cascade Driven by the Retinoblastoma Family, Resulting in Senescence

ABSTRACT This work demonstrates a central role for the retinoblastoma (Rb) family in driving the transcriptional program of induced and replicative senescence. HeLa cervical carcinoma cells rapidly undergo senescence when the human papillomavirus (HPV) type 18 E7 gene in these cells is repressed by the bovine papillomavirus (BPV) E2 protein. This senescence response requires the endogenous Rb pathway but not the p53 pathway. Microarray analysis 6 days after BPV E2 introduction into HeLa cells identified 224 cellular genes induced by E7 repression and 354 repressed genes. Many repressed genes were involved in cell cycle progression, and numerous induced genes encoded lysosomal proteins. These gene expression changes were blocked by constitutive expression of the wild-type HPV16 E7 or adenovirus E1A gene, but not by E7 or E1A mutants defective for Rb binding. Short hairpin RNAs targeting the Rb family also inhibited these gene expression changes and blocked senescence. Therefore, surprisingly, the transcriptional response to BPV E2 expression was entirely dependent on E7 repression and activation of the Rb family, and the BPV E2 protein did not directly affect the expression of cellular genes. Activation of the Rb family repressed E2F-responsive genes and stimulated transcriptional activators, thereby mobilizing multiple signals, such as repression of B-MYB and DEK, that were independently sufficient to induce senescence. There was extensive overlap between the transcriptional profiles of senescent, late-passage primary human fibroblasts and senescent cervical carcinoma cells, suggesting that this Rb family-mediated transcriptional cascade also plays a central role in replicative senescence.

BiP and Multiple DNAJ Molecular Chaperones in the Endoplasmic Reticulum Are Required for Efficient Simian Virus 40 Infection

ABSTRACT Simian virus 40 (SV40) is a nonenveloped DNA virus that traffics through the endoplasmic reticulum (ER) en route to the nucleus, but the mechanisms of capsid disassembly and ER exit are poorly understood. We conducted an unbiased RNA interference screen to identify cellular genes required for SV40 infection. SV40 infection was specifically inhibited by up to 50-fold by knockdown of four different DNAJ molecular cochaperones or by inhibition of BiP, the Hsp70 partner of DNAJB11. These proteins were not required for the initiation of capsid disassembly, but knockdown markedly inhibited SV40 exit from the ER. In addition, BiP formed a complex with SV40 capsids in the ER in a DNAJB11-dependent fashion. These experiments identify five new cellular proteins required for SV40 infection and suggest that the binding of BiP to the capsid is required for ER exit. Further studies of these proteins will provide insight into the molecular mechanisms of polyomavirus infection and ER function. IMPORTANCE The polyomaviruses, including simian virus 40 (SV40), are important human pathogens and model systems for exploring the general features of virus replication and cell biology. We used a genetic system to interrogate the role of cellular genes in SV40 infection. Based on the results of this unbiased genetic screen and analysis of proteins related to the strongest hit from the screen, we identified five new cellular proteins required for the entry of SV40 into cells. These proteins physically associate with SV40 in the endoplasmic reticulum (ER) during virus entry and are required for exit of the partially disassembled virus from this organelle. These results demonstrate that the polyomaviruses have coopted an ER-localized protein quality control process to initiate disassembly and transit through the cell on their way to the nuclear site of virus replication. The polyomaviruses, including simian virus 40 (SV40), are important human pathogens and model systems for exploring the general features of virus replication and cell biology. We used a genetic system to interrogate the role of cellular genes in SV40 infection. Based on the results of this unbiased genetic screen and analysis of proteins related to the strongest hit from the screen, we identified five new cellular proteins required for the entry of SV40 into cells. These proteins physically associate with SV40 in the endoplasmic reticulum (ER) during virus entry and are required for exit of the partially disassembled virus from this organelle. These results demonstrate that the polyomaviruses have coopted an ER-localized protein quality control process to initiate disassembly and transit through the cell on their way to the nuclear site of virus replication.

Direct binding of retromer to human papillomavirus type 16 minor capsid protein L2 mediates endosome exit during viral infection.

Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.

E2F-Rb complexes assemble and inhibit cdc25A transcription in cervical carcinoma cells following repression of human papillomavirus oncogene expression.

ABSTRACT Expression of the bovine papillomavirus E2 protein in cervical carcinoma cells represses expression of integrated human papillomavirus (HPV) E6/E7 oncogenes, followed by repression of the cdc25A gene and other cellular genes required for cell cycle progression, resulting in dramatic growth arrest. To explore the mechanism of repression of cell cycle genes in cervical carcinoma cells following E6/E7 repression, we analyzed regulation of the cdc25A promoter, which contains two consensus E2F binding sites and a consensus E2 binding site. The wild-type E2 protein inhibited expression of a luciferase gene linked to the cdc25A promoter in HT-3 cervical carcinoma cells. Mutation of the distal E2F binding site in the cdc25A promoter abolished E2-induced repression, whereas mutation of the proximal E2F site or the E2 site had no effect. None of these mutations affected the activity of the promoter in the absence of E2 expression. Expression of the E2 protein also led to posttranscriptional increase in the level of E2F4, p105Rb, and p130 and induced the formation of nuclear E2F4-p130 and E2F4-p105Rb complexes. This resulted in marked rearrangement of the protein complexes that formed at the distal E2F site in the cdc25A promoter, including the replacement of free E2F complexes with E2F4-p105Rb complexes. These experiments indicated that repression of E2F-responsive promoters following HPV E6/E7 repression was mediated by activation of the Rb tumor suppressor pathway and the assembly of repressing E2F4-Rb DNA binding complexes. Importantly, these experiments revealed that HPV-induced alterations in E2F transcription complexes that occur during cervical carcinogenesis are reversed by repression of HPV E6/E7 expression.

Vaccination of Rabbits with an Adenovirus Vector Expressing the Papillomavirus E2 Protein Leads to Clearance of Papillomas and Infection

ABSTRACT Cervical cancer arises from lesions caused by infection with high-risk types of human papillomavirus (HPV). Therefore, vaccination against HPV could prevent carcinogenesis by preventing HPV infection or inducing lesion regression. HPV E2 protein is an attractive candidate for vaccine development because it is required for papilloma formation, is involved in all stages of the virus life cycle, and is expressed in all premalignant lesions as well as some cancers. This study reports vaccination against E2 protein using a rabbit model of papillomavirus infection. A recombinant adenovirus (Ad) vector expressing the E2 protein of cottontail rabbit papillomavirus (CRPV) was tested for therapeutic efficacy in CRPV-infected rabbits. Primary immunization with the Ad-E2 vaccine, compared to immunization with a control Ad vector, reduced the number of papilloma-forming sites from 17 of 45 to 4 of 45. After booster immunization, vaccinated rabbits formed no new papillomas versus an additional 23 papillomas in rabbits that received the control vector. Papillomas in the Ad-E2 vaccinees were significantly smaller than those in the control rabbits, and all four papillomas in the Ad-E2 vaccinated rabbits regressed. No CRPV DNA was detected either in the regression sites or in sites that did not form papillomas, indicating that the vaccination led to clearance of CRPV from all infected sites.

p53 and hTERT determine sensitivity to viral apoptosis

ABSTRACT Apoptosis is a potent host defense against microbes. Most viruses have adapted strategies to counteract this response. Herpes simplex virus (HSV) produces a balance between pro- and antiapoptotic processes during infection. When antiapoptotic signals become limiting, infected cells die through HSV-dependent apoptosis (HDAP). Oncogenic pathways were previously implicated in HDAP susceptibility. Here, we exploited our ability to selectively express all, one, or no oncogenes in the well-defined HeLa cell system to dissect the requirements for HDAP. Human papillomavirus E6 and E7 oncogene expression was inhibited by the E2 viral repressor. Sole expression of E6 mediated HDAP sensitization. Next, two known cellular targets of E6 were independently modulated. This demonstrated that E6 sensitizes HeLa cells to HDAP through hTERT and p53. Given the universality of the apoptotic antiviral response, p53 and telomerase regulation will likely be important for counteracting host defenses in many other viral infections.

High-risk human papillomavirus E6 inhibits monocyte differentiation to Langerhans cells.

High-risk human papillomaviruses (HPVs) cause a variety of malignancies of the mucosal epithelium. However, the local immune evasion strategies used by HPV-transformed cells remain unclear. Here, we examined the effect of HPV-positive cancer cells on human peripheral blood monocytes, which are precursors of Langerhans cells, key antigen-presenting cells in the squamous epithelium. HPV-positive cervical cancer cells and HPV-E6 expressing cells inhibited monocyte differentiation to Langerhans cells in a contact-dependent manner. Unlike Langerhans cells, monocytes that differentiated in the presence of HPV16 E6-expressing cells exhibited high levels of endocytic activity. Our results suggest that cells infected by high-risk HPV evade immune surveillance by blocking the differentiation of monocytes into competent antigen presenting cells.

High-Throughput Cell-Based Screen for Chemicals That Inhibit Infection by Simian Virus 40 and Human Polyomaviruses

ABSTRACT We developed a high-throughput, cell-based screen to identify chemicals that inhibit infection by the primate polyomaviruses. The screen is based on the detection of compounds that inhibit the ability of a replication-defective simian virus 40 (SV40)-based viral vector to cause growth arrest in HeLa cells by repressing the expression of the endogenous human papillomavirus E7 oncogene in these cells. We identified two compounds, ellagic acid and spiperone, that suppressed the ability of the SV40 recombinant virus to inhibit cellular DNA synthesis. These compounds caused a marked reduction of the ability of wild-type SV40 to productively infect permissive monkey cells, even when the compounds were added several hours after infection. The fraction of cells expressing SV40 large T antigen and the levels of T antigen mRNA were reduced in infected human and monkey cells treated with ellagic acid and spiperone, suggesting that these compounds block a step in the virus life cycle prior to SV40 early gene expression. Ellagic acid and spiperone also inhibited large T antigen expression by BK virus and JC virus, two important, pathogenic human polyomaviruses.