A subset of human papillomavirus (HPV) infections is causally related to the development of human epithelial tumors and cancers. Like a number of pathogens, HPV entry into target cells is initiated by first binding to heparan sulfonated proteoglycan (HSPG) cell surface attachment factors. The virus must then move to distinct secondary receptors, which are responsible for particle internalization. Despite intensive investigation, the mechanism of HPV movement to and the nature of the secondary receptors have been unclear. We report that HPV16 particles are not liberated from bound HSPG attachment factors by dissociation, but rather are released by a process previously unreported for pathogen-host cell interactions. Virus particles reside in infectious soluble high molecular weight complexes with HSPG, including syndecan-1 and bioactive compounds, like growth factors. Matrix mellatoproteinase inhibitors that block HSPG and virus release from cells interfere with virus infection. Employing a co-culture assay, we demonstrate HPV associated with soluble HSPG-growth factor complexes can infect cells lacking HSPG. Interaction of HPV-HSPG-growth factor complexes with growth factor receptors leads to rapid activation of signaling pathways important for infection, whereas a variety of growth factor receptor inhibitors impede virus-induced signaling and infection. Depletion of syndecan-1 or epidermal growth factor and removal of serum factors reduce infection, while replenishment of growth factors restores infection. Our findings support an infection model whereby HPV usurps normal host mechanisms for presenting growth factors to cells via soluble HSPG complexes as a novel method for interacting with entry receptors independent of direct virus-cell receptor interactions.

Human papillomaviruses (HPVs) cause benign and malignant tumors of the mucosal and cutaneous epithelium. The initial events regulating HPV infection impact the establishment of viral persistence, which is requisite for malignant progression of HPV-infected lesions. However, the precise mechanisms involved in HPV entry into host cells, including the cellular factors regulating virus uptake, are not clearly defined. We show that HPV16 exposure to human keratinocytes initiates epidermal growth factor receptor (EGFR)-dependent Src protein kinase activation that results in phosphorylation and extracellular translocation of annexin A2 (AnxA2). HPV16 particles interact with AnxA2 in association with S100A10 as a heterotetramer at the cell surface in a Ca 2؉ -dependent manner, and the interaction appears to involve heparan-sulfonated proteoglycans. We show multiple lines of evidence that this interaction promotes virus uptake into host cells. An antibody to AnxA2 prevents HPV16 internalization, whereas an antibody to S100A10 blocks infection at a late endosomal/lysosomal site. These results suggest that AnxA2 and S100A10 have separate roles during HPV16 binding, entry, and trafficking. Our data additionally imply that AnxA2 and S100A10 may be involved in regulating the intracellular trafficking of virus particles prior to nuclear delivery of the viral genome.

Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-crosslinking analyses that cellular proteins of ϳ60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5 untranslated region (UTR)-and one 3 UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5 UTR with ϳ2.5 M affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.Coronaviruses (CoVs) (59) cause primarily respiratory and gastroenteric diseases in birds and mammals (35,71). In humans, they most commonly cause mild upper respiratory disease, but the recently discovered human CoVs (HCoVs), HCoV-NL63 (65), HCoV-HKU1 (73), and severe acute respiratory syndrome (SARS)-CoV (40) cause serious diseases in the upper and lower respiratory tracts. The SARS-CoV causes pneumonia with an accompanying high (ϳ10%) mortality rate (69). The ϳ30-kb positive-strand CoV genome, the largest known among RNA viruses, is 5Ј capped and 3Ј polyadenylated and replicates in the cytoplasm (41). As with other characterized cytoplasmically replicating positive-strand RNA viruses (3), translation of the CoV genome is an early step in replication, and terminally located cis-acting RNA signals regulate translation and direct genome replication (41). How these happen mechanistically in CoVs is only beginning to be understood.In the highly studied group 2 mouse hepatitis coronavirus model (MHV A59 strain) and its close relative the bovine CoV (BCoV Mebus strain), five higher-order cis-replication signals have been identified in the 5Ј and 3Ј untranslated regions (UTRs). These include two in the 5Ј UTR required for BCoV defective interfering (DI) RNA replication (Fig. 1A) described as stem-loop III (SLIII) (50) and SLIV (51). Recently, the SLI regi...

Vaccines targeting conserved epitopes in the HPV minor capsid protein, L2, can elicit antibodies that can protect against a broad spectrum of HPV types that are associated with cervical cancer and other HPV malignancies. Thus, L2 vaccines have been explored as alternatives to the current HPV vaccines, which are largely type-specific. In this study we assessed the immunogenicity of peptides spanning the N-terminal domain of L2 linked to the surface of a highly immunogenic bacteriophage virus-like particle (VLP) platform. Although all of the HPV16 L2 peptide-displaying VLPs elicited high-titer anti-peptide antibody responses, only a subset of the immunogens elicited antibody responses that were strongly protective from HPV16 pseudovirus (PsV) infection in a mouse genital challenge model. One of these peptides, mapping to HPV16 L2 amino acids 65–85, strongly neutralized HPV16 PsV but showed little ability to cross-neutralize other high-risk HPV types. In an attempt to broaden the protection generated through vaccination with this peptide, we immunized mice with VLPs displaying a peptide that represented a consensus sequence from high-risk and other HPV types. Vaccinated mice produced antibodies with broad, high-titer neutralizing activity against all of the HPV types that we tested. Therefore, immunization with virus-like particles displaying a consensus HPV sequence is an effective method to broaden neutralizing antibody responses against a type-specific epitope.

The induction of mucosal immune responses in the genital tract may be important for increasing the effectiveness of vaccines for sexually transmitted infections (STIs). In this study, we asked whether direct immunization of the mouse genital tract with a non-replicating virus-like particle (VLP)-based vaccine could induce local mucosal as well as systemic antibody responses. Using VLPs derived from two bacteriophages, Qβ and PP7, and from a mammalian virus that normally infects the genital tract, human papillomavirus (HPV), we show that intravaginal aerosol administration of VLPs can induce high titer IgG and IgA antibodies in the female genital tract as well as IgG in the sera. Using a mouse model for HPV infection, we show that intravaginal immunization with either HPV type 16 VLPs or with PP7 bacteriophage VLPs displaying a peptide derived from the HPV minor capsid protein L2 could protect mice from genital infection with an HPV16 pseudovirus. These results provide a general method for inducing genital mucosal and systemic antibody responses using VLP-based immunogens.

The human papillomavirus (HPV) expresses the viral oncogene E7 that inhibits the retinoblastoma protein (RB1). RB1 mediates contradictory cell growth and cell death pathways via E2F family members. Here, we assessed the extent to which HPV oncogenes caused toxicity as measured by mouse survival and tumor growth. Materials/Methods: iHPV mice contained a LoxP-Stop-LoxP (LSL)-iE6E7 transgene in which conditional E6E7 expression is regulated by Cre recombinase. We constitutively expressed HPV oncogenes by breeding iHPV transgenic mice to CMV-Cre transgenic mice expressing Cre recombinase under a CMV promoter (CMV-HPV mice). We induced HPV oncogene expression in adult mice using RosaHPV mice containing the iHPV transgene and a Rosa-CreER tam transgene expressing a tamoxifenregulated (TAM) regulated Cre recombinase in all tissues. We studied primary oral tumors using triple transgenic KHR mice containing a K14-CreER tam transgene, a LSL-iE6E7 transgene and a LSL-Kras G12D transgene that formed HPV-positive oral tumors after TAM treatment. We assessed the role of E2f1 on survival and oral tumor formation using RosaHPV-E2f1-/mice and KHR-E2f1-/mice, respectively, that contained a homozygous E2f1deletion. HPV oncogene expression and E2f target gene expression was assessed by quantitative RT-PCR. Results: Induction of HPV oncogenes caused embryonic lethality as CMV-HPV double transgenic mice were born at significantly lower frequencies compared to mice carrying single transgenes (P<.0001). Tamoxifen treatment of adult RosaHPV (RosaHPV+TAM) mice caused recombination of the LSL-E6E7 transgenes and HPV oncogene expression in all organs tested. Furthermore, RosaHPV+TAM mice had decreased survival compared to vehicle treated RosaHPV mice (median survival: 50d for RosaHPV+TAM vs not reached for RosaHPV-TAM; P<.0001). Decreased survival in RosaHPV+TAM mice was associated with focal necrosis in hepatocytes and pancreatic tissues and the activation of the E2f target genes. Deletion of E2f1 increased survival of RosaHPV+TAM mice indicating that E2f1 mediated HPV oncogene toxicity (median survival: not reached for RosaHPV-E2f1-/mice vs 49 days for RosaHPV-E2f1 +/mice vs 30 days for RosaHPV-E2f1 +/+ ; P<.0001). Compared to tumors with heterozygous loss of E2f1, KHR tumors with homozygous loss of E2f1 grew faster and had more proliferating tumor cells as measured by Pcna immunohistochemistry (tumor volume at d18: 453.7 mm 3 for KHR-E2f1-/vs 139.7 mm 3 for KHR-E2f1 +/-; PZ.0004). Conclusion: Our results indicate that HPV oncogenes activated the E2f1 pathway to cause toxicity in normal mice and to suppress oral tumor growth. These results suggest that selective modulation of the E2f1 pathway, which is activated in HPV tumors, may facilitate tumor regression.

Human papillomaviruses (HPVs) are etiological agents of many anogenital and oropharyngeal cancers. HPV[+] oropharyngeal squamous cell carcinomas (OPSCCs) typically respond more favorably to current treatment regimens (including radiotherapy combined with cisplatin or cetuximab, an EGFR monoclonal antibody), than do HPV-negative OPSCCs. The discrepancy in patient outcomes has been attributed, in part, to substantially fewer genetic mutations present in HPV[+] cancers. Paradoxically, an increasing body of literature notes lower EGFR expression in HPV[+] vs. HPV[-] OPSCC. Yet, HPV oncoproteins E5, E6 and E7 each work to increase EGFR signaling, and MAPK-regulated AP-1 transcription factors direct HPV early gene expression. The goal of this work is to test the hypothesis that upon infection, HPV establishes a feed-forward loop with the EGFR pathway to drive viral gene expression independent of EGFR levels. We modeled early neoplasia with cell lines maintaining episomal HPV16 genomes, and cancers using SCC lines with integrated HPV16 genomes. Cells treated with EGF, EGFR inhibitors (cetuximab, erlotinib), or MEK antagonists (PD98059, trametinib) were evaluated for signaling and viral responses. Consistent with our hypothesis, we found increased EGF-dependent EGFR activation levels in HPV[+] cell lines, as well as heightened MEK1/2 activity independent of EGFR activation in HPV[+] cell lines compared to uninfected cells. RT-qPCR revealed that HPV oncogene transcription was enhanced by EGFR activation, whereas EGFR and MEK inhibitors led to significant declines in HPV transcription. In cells with episomal HPV genomes, lower viral genome levels accompanied inhibitor-reduced viral transcription. These data suggest EGFR/MAPK pathway interference leads to anti-viral affects, which are expected to restore p53 and pRb function and prevent tumorigenesis in vivo. As predicted, cell proliferation and tumor growth in NOD/SCID-gamma mouse HPV[+] SCC xenografts were significantly reduced with EGFR and MEK inhibitors. Experiments are underway to quantify the restored function of p53 and pRb in drug-treated cells and determine if these cells become sensitized to lower doses of cisplatin or radiation. In summary, our results reveal HPV infection creates a feed-forward loop with the EGFR/MAPK proliferation pathway, and EGFR and MEK antagonists have anti-viral effects, including reduction in the E6 and E7 oncoproteins needed to maintain the transformed phenotype in vitro and in vivo. Additionally, the ability of the drugs to reduce episomal viral genome levels suggests a potential curative effect if administered prior to viral genome integration. Lastly, this work reveals an anti-viral molecular mechanism which may account for the favorable outcomes of patients with HPV[+] OPSCC treated with cetuximab-radiotherapy. Citation Format: Anastacia M. Griego, Pamela Barraza, Chelin Hu, Agnieszka Dziduszko, Brianna K. Crowley, Helen J. Hathaway, Julie E. Bauman, Michelle A. Ozbun. The EGFR pathway as the Achilles’ heel for human papillomavirus-induced tumors: EGFR/MAPK pathway inhibitors exhibit antiviral activities and limit tumor growth in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3176. doi:10.1158/1538-7445.AM2014-3176