Functional similarity between E6 proteins of cutaneous human papillomaviruses and the adenovirus E1A tumor-restraining module.

The adenovirus E1A C-terminal region restrains oncogenic transformation through interaction with three distinct cellular protein complexes that include the DYRK1A/1B/HAN11 complex. The E6 proteins of beta-human papillomaviruses (beta-HPVs) also interact with the DYRK1/HAN11 complex. A variant of HPV5 E6 frequently found in epidermodysplasia verruciformis skin lesions interacted less efficiently with DYRK1A/HAN11. The E6 variant and E7 of HPV5 efficiently coimmortalized primary epithelial cells, suggesting that naturally arising variants may contribute potential oncogenic activities of beta-HPV E6 proteins.

Adenovirus type 5 E1A and E6 proteins of low-risk cutaneous beta-human papillomaviruses suppress cell transformation through interaction with FOXK1/K2 transcription factors.

The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.

Preparation and titration of CsCl-banded adenovirus stocks.

Adenovirus research often requires purified high-titer virus stocks and accurate virus titers for use in experiments. Accurate titers are important for quantitative, interpretable, and reproducible results. This is especially true when there are comparisons of different mutant viruses following infection. This chapter details the large-scale preparation of adenovirus (either replication-competent or replication-defective) in spinner cultures (e.g., KB, HeLa, or 293 cells). Protocols for harvesting cells and isolation of adenovirus by CsCl banding are presented. Methods for titering adenovirus by plaque assay are presented along with a discussion of how plaque assays can be used to determine the kinetics of cell killing and cytolysis by adenoviruses.

Cotton rat tumor model for the evaluation of oncolytic adenoviruses.

Oncolytic human adenovirus (Ad) vectors exert their antitumor effect by replicating in and lysing tumor cells. These vectors are commonly evaluated in immunodeficient mice bearing human tumor xenografts. However, this model suffers because the mice are immunodeficient and are not permissive for human Ads. We have developed a cotton rat model to test the selectivity, immunogenicity, and efficacy of oncolytic Ad vectors. The cotton rat is a rodent species that is semipermissive for human Ads. We show that the cotton cancer rat cell line LCRT supports the replication of human Ad in tissue culture and that the cells are destroyed on virus replication. When injected subcutaneously, LCRT cells formed tumors in immunocompetent cotton rats, and the growth of these tumors was delayed by the injection of an oncolytic Ad vector. Replication of the Ad vector in the tumor was demonstrated by sampling tumor tissue and isolating infectious virus particles at various times after intratumoral injection of the virus. We propose that the cotton rat can be used as an animal model to evaluate oncolytic Ad vectors.

Adenovirus immunoregulatory E3 proteins prolong transplants of human cells in immunocompetent mice.

The majority of proteins encoded in the early 3 (E3) region of human subgroup C adenoviruses function to modulate the host immune response. For example, gp19K, one of these E3 proteins, prevents the major histocompatibility complex type I (MHC-I) from presenting viral antigens on the surface of the infected cell. Other E3 proteins, such as the RID and 14.7K proteins, counteract the effector phase of the cellular immune response. In order to study further the effects of these proteins, we constructed an E1-/E3- adenovirus vector, Ad/E3, that contains all the E3 genes with the exception of the cytolytic adp gene, inserted into the deleted E1 region. The transcription of the E3 genes in this vector is driven by a CMV promoter in place of the native E3 promoter. Ad/E3 expressed close to wild-type adenovirus levels of all E3 proteins, and these proteins appear to function normally in cell culture. For example, in Ad/E3-infected cells, surface expression of MHC-I was down-regulated, as was cell surface display of death receptors Fas and TRAIL Receptor 1. A human cell line of lung origin (A549), which was rapidly rejected after transplantation into C57BL/6 mice, was protected for an extended time from the host immune response after infection with an Ad/E3, and went through a number of divisions in immunocompetent mice. These latter results indicate that the E3 proteins protect cells from destruction by the immune system.

Adenovirus E3-6.7K protein is required in conjunction with the E3-RID protein complex for the internalization and degradation of TRAIL receptor 2.

Adenoviruses (Ads) encode several proteins within the early region 3 (E3) transcription unit that help protect infected cells from elimination by the immune system. Among these immunomodulatory proteins, the receptor internalization and degradation (RID) protein complex, which is composed of the RIDalpha (formerly E3-10.4K) and RIDbeta (formerly E3-14.5K) subunits, stimulates the internalization and degradation of certain members of the tumor necrosis factor (TNF) receptor superfamily, thus blocking apoptosis initiated by Fas and TNF-related apoptosis-inducing ligand (TRAIL). The experiments reported here show that TRAIL receptor 2 (TR2) is cleared from the cell surface in Ad-infected cells. Virus mutants containing deletions that span E3 were used to show that the RID and E3-6.7K proteins are both necessary for the internalization and degradation of TR2, whereas only the RID protein is required for TRAIL receptor 1 downregulation. In addition, replication-defective Ad vectors that express individual E3 proteins were used to establish that the RID and E3-6.7K proteins are sufficient to clear TR2. These data demonstrate that E3-6.7K is an important component of the antiapoptosis arsenal encoded by the E3 transcription unit of subgroup C Ads.

An oncolytic adenovirus vector combining enhanced cell-to-cell spreading, mediated by the ADP cytolytic protein, with selective replication in cancer cells with deregulated wnt signaling.

We have constructed a novel oncolytic adenovirus (Ad) vector named VRX-009 that combines enhanced cell spread with tumor-specific replication. Enhanced spread, which could significantly increase antitumor efficacy, is mediated by overexpression of the Ad cytolytic protein named ADP (also known as E3-11.6K). Replication of VRX-009 is restricted to cells with a deregulated wnt signal transduction pathway by replacement of the wild-type Ad E4 promoter with a synthetic promoter consisting of five consensus binding sites for the T-cell factor transcription factor. Tumor-selective replication is indicated by several lines of evidence. VRX-009 expresses E4ORF3, a representative Ad E4 protein, only in colon cancer cell lines. Furthermore, VRX-009 replicates preferentially in colon cancer cell lines as evidenced by virus productivity 2 orders of magnitude higher in SW480 colon cancer cells than in A549 lung cancer cells. Replication in primary human bronchial epithelial cells and human umbilical vein endothelial cells was also significantly lower than in SW480 cells. When tested in human tumor xenografts in nude mice, VRX-009 effectively suppressed the growth of SW480 colon tumors but not of A549 lung tumors. VRX-009 may provide greater level of antitumor efficacy than standard oncolytic Ad vectors in tumors in which a defect in wnt signaling increases the level of nuclear beta-catenin.

Radiation increases the activity of oncolytic adenovirus cancer gene therapy vectors that overexpress the ADP (E3-11.6K) protein.

We have described three potential adenovirus type 5 (Ad5)-based replication-competent cancer gene therapy vectors named KD1, KD3, and VRX-007. All three vectors overexpress an Ad5 protein named Adenovirus Death Protein (ADP, also named E3-11.6 K protein). ADP is required for efficient lysis of Ad5-infected cells and spread of virus from cell to cell, and thus its overexpression increases the oncolytic activity of the vectors. KD1 and KD3 contain mutations in the Ad5 E1A gene that knock out binding of the E1A proteins to cellular p300/CBP and pRB; these mutations allow KD1 and KD3 to grow well in cancer cells but not in normal cells. VRX-007 has wild-type E1A. Here we report that radiation increases the oncolytic activity of KD1, KD3, and VRX-007. This increased activity was observed in cultured cells, and it was not because of radiation-induced replication of the vectors. The combination of radiation plus KD3 suppressed the growth of A549 lung adenocarcinoma xenografts in nude mice more efficiently than radiation alone or KD3 alone. The combination of ADP-overexpressing vectors and radiation may have potential in treating cancer.

Overexpression of the ADP (E3-11.6K) protein increases cell lysis and spread of adenovirus.

Adenoviruses replicate in the nucleus and induce lytic cell death. We have shown previously that efficient cell lysis and release of adenovirus from infected cells requires an 11.6-kDa protein named Adenovirus Death Protein (ADP). The adp gene is located in the early E3 transcription unit, but the gene is expressed primarily at very late stages of infection. The putative function of ADP was discerned previously from the use of virus mutants that lack functional ADP. Here we describe two adenovirus mutants, named VRX-006 and VRX-007, that overexpress ADP. VRX-006 lacks all other genes in the E3 region, and VRX-007 lacks all other E3 genes except 12.5K. VRX-006 and VRX-007 display the phenotype predicted by the proposed function for ADP: they produce early cytopathic effect, early cell lysis, large plaques, and increased cell-to-cell spread. They grow as well in cultured cells as does adenovirus type 5. These results are consistent with the conclusion that ADP functions in adenovirus infections to promote virus release from cells at the culmination of infection.

Construction and characterization of E1-minus replication-defective adenovirus vectors that express E3 proteins from the E1 region.

Previous research has indicated that the adenovirus protein complex named RID, derived from the E3 transcription unit, functions to remove the receptors named Fas/Apo1/CD95 (Fas) and epidermal growth factor receptor (EGFR) from the surface of cells. (The RID complex is composed of the RIDalpha and RIDbeta polypeptides, previously named 10.4K and 14.5K, respectively.) In response to RID, Fas and EGFR appear to be internalized into endosomes and degraded in lysosomes. Fas is a death receptor in the tumor necrosis factor (TNF) receptor superfamily. RID inhibits apoptosis via the Fas pathway, presumably because RID gets rid of Fas. Earlier work further showed that another adenovirus E3-coded protein, E3-14.7K, inhibits apoptosis induced by TNF. Most of the above studies have been conducted using viable virus mutants that lack one or more of the genes for RID, E3-14.7K, or E1B-19K (this protein, coded by the E1B transcription unit, also inhibits apoptosis via the TNF and Fas pathways). Some studies have also been conducted with the genes for RID or E3-14.7K transiently or stably transfected into cells. We now report a new approach to studying the E3 genes. We have constructed four E1-minus replication-defective vectors that have all the E3 genes deleted from their natural position and then reinserted, in different permutations, into the deleted E1 region under control of the cytomegalovirus immediate early promoter. Vector Ad/RID only has the genes for RIDalpha and RIDbeta. Vector Ad/14.7K only has the gene for E3-14.7K. Vector Ad/RID/14.7K only has the genes for RIDalpha, RIDbeta, and E3-14.7K. Vector Ad/E3 has all E3 genes, but there are two missense mutations in the gene for Adenovirus Death Protein. These vectors expressed RID and/or E3-14.7K, as expected. The RID-expressing vectors forced the internalization and degradation of Fas and EGFR, and they inhibited apoptosis induced through the Fas pathway. These vectors should be useful reagents to study the E3 proteins.

Adenovirus replication-competent vectors (KD1, KD3) complement the cytotoxicity and transgene expression from replication-defective vectors (Ad-GFP, Ad-Luc).

The successful clinical application of adenovirus (Ad) in cancer control has been of limited success because of the current inability to infect the majority of cancer cells with a large amount of vector. In this study, we show that when human lung tumors growing in immunodeficient nude mice were coinfected with a replication-defective (RD) Ad vector expressing green fluorescent protein and a replication-competent (RC) Ad vector named KD3, KD3 enhanced the expression of green fluorescent protein throughout the tumor. Also, KD3 and another RC vector named KD1 complemented the expression of luciferase from a RD vector in a human liver tumor xenotransplant in nude mice. Altogether, these results suggest that the combination of a RD vector with a RC vector might be a more effective treatment for cancer than either vector alone due to more widespread dissemination of the virus.