A real-time PCR method to rapidly titer adenovirus stocks.

A critical step in working with adenovirus (Ad) and its vectors is the accurate, reproducible, sensitive, and rapid measurement of the amount of virus present in a stock. Titration methods fall into one of two categories: determination of either the infectious or the particle (infectious plus noninfectious) titer. Determining the infectious titer of a virus stock by plaque assay has important limitations, including cell line-, researcher-, and laboratory-dependent variation in titer, and the length of time required to perform the assay (2-4 wk). A major drawback of particle titration methods is the lack of consistent correlation between the resultant titer and the infectious titer. To overcome these problems, a rapid, sensitive, and reproducible real-time polymerase chain reaction (PCR) assay was developed that detects encapsidated full-length genomes. Importantly, there is a linear correlation between the titer determined by the realtime PCR assay and the infectious titer determined by a plaque assay. This chapter provides step-by-step guidance for preparing viral DNA, conducting the real-time PCR assay, and using the resultant data to calculate a viral titer.

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 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.

Experimental infections of humans with wild-type adenoviruses and with replication-competent adenovirus vectors: replication, safety, and transmission.

Replication-competent (RC) adenoviruses (Ads) are increasingly being developed as oncolytic vectors and as vehicles for delivering vaccine antigens. Although the safety of such vectors in humans is of paramount importance, these vectors pose additional special concerns. Specifically, the prospect of causing Ad-mediated disease in the patient, the amount and sites of Ad replication, the possibility of virus shedding leading to unintended transmission to patient contacts, and the potential for persistence in the inoculated individual must be evaluated. Previous experience with administration of wild-type and RC recombinant Ads to humans may shed light on some of these issues. Experimental infections of humans with natural Ad isolates and RC recombinant vectors show that in adults Ads cause mild or no disease, particularly with Ad serotypes 2 and 5, the serotypes most often used to make recombinant constructs. Other studies show that Ad can replicate in experimentally infected persons, that in some situations Ads can be shed and transmitted to close contacts, and that there is evidence for persistent/latent Ad infection in naturally infected individuals. Overall, these studies indicate that Ads can be safely administered to humans for the treatment of cancer and as antigen delivery vehicles suggesting that the continued development of RC oncolytic and vaccine vectors should be pursued.

Functions and mechanisms of action of the adenovirus E3 proteins.

In the evolutionary battle between viruses and their hosts, viruses have armed themselves with weapons to defeat the host's attacks on infected cells. Various proteins encoded in the adenovirus (Ad) E3 transcription unit protect cells from killing mediated by cytotoxic T cells and death-inducing cytokines such as tumor necrosis factor (TNF), Fas ligand, and TNF-related apoptosis-inducing ligand (TRAIL). The viral protein E3-gp19 K blocks MHC class-I-restricted antigen presentation, which diminishes killing by cytotoxic T cells. The receptor internalization and degradation (RID) complex (formerly E3-10.4 K/14.5 K) stimulates the clearance from the cell surface and subsequent degradation of the receptors for Fas ligand and TRAIL, thereby preventing the action of these important immune mediators. RID also downmodulates the epidermal growth factor receptor (EGFR), although what role, if any, this function has in immune regulation is uncertain. In addition, RID antagonizes TNF-mediated apoptosis and inflammation through a mechanism that does not primarily involve receptor downregulation. E3-6.7 K functions together with RID in downregulating some TRAIL receptors and may block apoptosis independently of other E3 proteins. Furthermore, E3-14.7 K functions as a general inhibitor of TNF-mediated apoptosis and blocks TRAIL-induced apoptosis. Finally, after expending great effort to maintain cell viability during the early part of the virus replication cycle, Ads lyse the cell to allow efficient virus release and dissemination. To perform this task subgroup C Ads synthesize a protein late in infection named ADP (formerly E3-11.6 K) that is required for efficient virus release. This review focuses on recent experiments aimed at discovering the mechanism of action of these critically important viral proteins.

Distinct domains in the adenovirus E3 RIDalpha protein are required for degradation of Fas and the epidermal growth factor receptor.

Adenovirus (Ad) types 2 and 5 encode at least five proteins within the E3 transcription unit that help the virus evade the immune system. Two such proteins, RIDalpha (formerly E3-10.4K) and RIDbeta (formerly E3-14.5K), form the RID (receptor internalization and degradation) complex (formerly E3-10.4K/14.5K). RID mediates clearance from the cell surface and lysosomal degradation of a number of important members in the tumor necrosis factor receptor (TNFR) superfamily and the receptor tyrosine kinase receptor family. Affected receptors include Fas, TRAIL (TNF-related apoptosis-inducing ligand) receptor 1 (TR1), TR2, and epidermal growth factor receptor (EGFR). Degradation of Fas and TRAIL receptors protects Ad-infected cells from apoptosis. To investigate the mechanism of action of RIDalpha, 14 mutant RIDalpha proteins, each containing a three- to five-amino-acid deletion, were constructed and then expressed from the E3 region of a replication-competent recombinant Ad in the same context as wild-type RIDalpha. Each mutant protein was characterized with regard to five physical properties associated with wild-type RIDalpha, namely, protein stability, proteolytic cleavage, insertion into the membrane, complex formation with RIDbeta, and transport to the cell surface. Additionally, the mutant proteins were tested for their ability to mediate internalization and degradation of EGFR and Fas and to protect cells from Fas-mediated apoptosis. The majority of mutant RIDalpha proteins (8 out of 14) were physically similar to wild-type RIDalpha. With regard to functional characteristics, the cytoplasmic domain of RIDalpha is largely unimportant for receptor internalization and degradation and the extracellular domain of RIDalpha is important for down-regulation of EGFR but not Fas.

Adenovirus RIDbeta subunit contains a tyrosine residue that is critical for RID-mediated receptor internalization and inhibition of Fas- and TRAIL-induced apoptosis.

The adenovirus-encoded receptor internalization and degradation (RID) protein (previously named E3-10.4K/14.5K), which is composed of RIDalpha and RIDbeta subunits, down-regulates a number of cell surface receptors in the tumor necrosis factor (TNF) receptor superfamily, namely Fas, TRAIL receptor 1, and TRAIL receptor 2. Down-regulation of these "death" receptors protects adenovirus-infected cells from apoptosis induced by the death receptor ligands Fas ligand and TRAIL. RID also down-regulates certain tyrosine kinase cell surface receptors, especially the epidermal growth factor receptor (EGFR). RID-mediated Fas and EGFR down-regulation occurs via endocytosis of the receptors into endosomes followed by transport to and degradation within lysosomes. However, the molecular interactions underlying this function of RID are unknown. To investigate the molecular determinants of RIDbeta that are involved in receptor down-regulation, mutations within the cytoplasmic tail of RIDbeta were constructed and the mutant proteins were analyzed for their capacity to internalize and degrade Fas and EGFR and to protect cells from death receptor ligand-induced apoptosis. The results demonstrated the critical nature of a tyrosine residue near the RIDbeta C terminus; mutation of this residue to alanine abolished RID function. Mutating the tyrosine to phenylalanine did not abolish the function of RID, arguing that phosphorylation of the tyrosine is not required for function. These data suggest that this tyrosine residue forms part of a tyrosine-based sorting signal (Yxxphi). Additional mutations that target another potential sorting motif and several possible protein-protein interaction motifs had no discernible effect on RID function. It was also demonstrated that mutation of serine 116 to alanine eliminated phosphorylation of RIDbeta but did not affect any of the functions of RID that were examined. These results suggest a model in which the tyrosine-based sorting signal in RID plays a role in RID's ability to down-regulate receptors.

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.