Genetic Identification of Adenovirus Type 5 Genes That Influence Viral Spread

Abstract

The mechanisms that control cell-to-cell spread of human adenoviruses (Ad) are not well understood. Two early viral proteins, E1B-19K and E3-ADP, appear to have opposing effects since viral mutants that are individually deficient in E1B-19K produce large plaques (G. Chinnadurai, Cell 33:759-766, 1983), while mutants deficient in E3-ADP produce small plaques (A. E. Tollefson et al., J. Virol. 70:2296-2306, 1996) on infected cell monolayers. We have used a genetic strategy to identify different viral genes that influence adenovirus type 5 (Ad5) spread in an epithelial cancer cell line. An Ad5 mutant (dl327; lacking most of the E3 region) with the restricted-spread (small-plaque) phenotype was randomly mutagenized with UV, and 27 large-plaque (lp) mutants were isolated. A combination of analyses of viral proteins and genomic DNA sequences have indicated that 23 mutants contained lesions in the E1B region affecting either 19K or both 19K and 55K proteins. Four other lp mutants contained lesions in early regions E1A and E4, in the early L1 region that codes for the i-leader protein, and in late regions that code for the viral structural proteins, penton base, and fiber. Our results suggest that the requirement of E3-ADP for Ad spread could be readily compensated for by abrogation of the functions of E1B-19K and provide genetic evidence that these two viral proteins influence viral spread in opposing manners. In addition to E1B and E3 proteins, other early and late proteins that regulate viral replication and infectivity also influence lateral viral spread. Our studies have identified novel mutations that could be exploited in designing efficient oncolytic Ad vectors.The efficiency with which a virus spreads from an infected cell to the neighboring uninfected cells is an important determinant of viral pathogenesis. The mechanisms by which nonenveloped viruses, particularly the adenoviruses, spread laterally are not well understood. It is generally believed that subversion of the cellular macromolecular synthesis machineries and the increased intracellular viral load result in cellular destruction and release of virus particles. The human adenoviruses (Ads) replicate in differentiated epithelial cells. The group C Ads infect these cells in a two-step process. The Ads enter the cells by receptor-mediated endocytosis (12,70). The viral particles first bind to the primary receptor (CAR [coxsackie-adenovirus receptor]) (7, 67) through interaction with the knob structure of the fiber (73, 80). Subsequently, the viral capsid protein penton base interacts with the V type integrins (␣ V ␤ 3 and ␣ V ␤ 5 ) via the RGD motif (78). The interaction of the penton base with the integrins also contributes the characteristic Ad cytopathic effect (CPE) (5). After adsorption and internalization into the endosomes, acidification of the endosomes results in cytosolic penetration of the virus particles which is also aided by the penton base (50, 57). Virus particles are then dismantled in a stepwise manner in the cytosol (24). It is possibl...

Bioselection of a Gain of Function Mutation that Enhances Adenovirus 5 Release and Improves Its Antitumoral Potency

“…A point mutation that induced a COOH terminal truncation in the i-leader protein, resulting in its early accumulation, was shown to be essential for the mutant phenotype (18). Interestingly, the relevance of such mutations in the i-leader protein has been further confirmed by another study (19).…”

“…Bioselection of randomly mutagenized pools of human Ad5 by repeated passaging under a predefined set of conditions is a classic virology strategy that has been recently postulated as a powerful method to develop more potent adenoviruses (18,19). We hypothesized that the passage of Ad5 random mutants in an in vivo murine model of human cancer would provide a selective pressure environment closer to human tumors in the clinical setting, where the presence of a threedimensional tumor stroma, the host immune system, and the proliferating status of tumor cells differ from those encountered in in vitro cell cultures.…”

Combination of oncolytic adenovirus and endostatin inhibits human retinoblastoma in an in vivo mouse model

“…E1A protein extended the half-life of p53 protein, in turn promoting premature apoptosis of the tumor cell and limiting the viral replication ability of Ad-E2F1 p-E1A (18). The E1B19K expressed by Ad-TERT p-E1, a functional Bcl-2 homologue, directly binds Bax, Bak-inhibiting oligomeriza- tion, and mitochondrial pore-formation resulting in apoptosis blockage (29,30). The biological function of E1B19K is to inhibit receptor-induced signaling at the time of cell death by preventing the Bax-Bak association, thus intrinsically inhibiting induced apoptosis through the p53-dependent and p53-independent mechanisms.…”

Anticancer activity of oncolytic adenovirus vector armed with IFN-α and ADP is enhanced by pharmacologically controlled expression of TRAIL

“…37 To assess whether regulatable expression of TRAIL from TetON-TRAIL improves the oncolytic activity of the conditionally replicative Ads KD3 and KD3-IFN, Hep3B and DLD-1 cells were [38][39][40][41] TRAIL-mediated induction of apoptosis, therefore, could potentially decrease oncolytic Ad yields when these two modalities are combined. In contrast, apoptosis induced at late stages of Ad infection can increase the fraction of virus progeny released from infected cells and result in improved spread of virus.…”