A cell line that supports the growth of a defective early region 4 deletion mutant of human adenovirus type 2.

Abstract

Cell lines that produce viral gene products and that can support the growth of viral mutants lacking those products have been valuable in the genetic analysis of the transforming regions of several animal viruses. To extend the advantages of such complementing cell systems to regions of the adenoviral genome not directly involved in transformation, we have constructed a cell line that will support the growth of a defective adenoviral deletion mutant, H2dU808, that lacks most of early region 4 (E4). The right-hand terminal adenovirus 5 EcoRI restriction fragment, which contains all of E4, was first inserted into the vector pSV2gpt, and the recombinant plasmid was introduced into Vero cells by calcium phosphate precipitation. Clones containing the hybrid plasmid were selected by their resistance to mycophenolic acid. Five mycophenolic acid-resistant clones were then tested for the ability to support the growth of H2di808. One of the five lines, W162, permits plaque formation by H2i808 at an efficiency that is >106-fold higher than that of the parental Vero cells and allows the production of high-titer, helper-free H2di808 stocks. Thus, W162 cells are permissive for at least one defective E4 mutant. The line carries, as expected, an intact E4, detected by hybridization. Using an H2d08 lysate produced on W162 cells, we have accurately mapped the 808 deletion. It extends from between Bcl I and Sma I sites at positions 91.4 and 92.0, respectively, to just beyond a HindIl site at position 97.2 and, therefore, falls entirely within E4. H2dl808 and W162 should be of value in determining the physiological role of E4 in adenoviral infection.Studies that probe the functions of viral genes are frequently dependent upon the availability of viral mutants. Most of the mutants that have proved useful in studying viral gene function are conditionally defective; in animal virus systems, the majority of these are temperature sensitive (1, 2). An alternative approach to the isolation of conditionally lethal mutations exploits the fact that some virally transformed cells will support the growth of mutants with defects in transforming regions. For example, polyoma virus-transformed mouse cells support the growth of hr-t mutants which carry lesions in the polyoma early region (3); COS cells, a line of simian virus 40 (SV40)-transformed monkey cells, support the growth of SV40 early region mutants (4); and 293 cells, a line of human cells transformed by sheared adenoviral DNA (5), support the growth of mutants of adenoviral early region 1 (6-8). In each of these cases, mutant viruses can be propagated efficiently on the transformed cell line, and their phenotypes subsequently can be analyzed in normal, nonpermissive cell types. The value of a complementing cell line in the isolation and propagation of viral mutants is probably best illustrated by the last example; the examination of a wide variety of mutants of adenoviral early region 1 (El) has provided a detailed picture of the functions of that region (6-13).In the examples ...

Transgene amplification and persistence after delivery of retroviral vector and packaging functions with E1/E4-deleted adenoviruses

“…E1/E4-deleted adenoviruses do not replicate in these two cell lines at the MOIs used (E. Vigne, unpublished observations). 13 Their use herein therefore validates our data on transgene persistence (see below). NIH-3T3 and W162 cells coinfected with AdTK/ENV and AdGAG/POL (MOI of 200 IU/cell for each virus) were mixed at a 1:3 ratio with noninfected cells before being passaged twice weekly for 59 and 94 days, respectively.…”

“…IGRP2 cells, an E1/E4-transcomplementing cell line derived from 293 12 and W162 cells 13 were maintained in minimum Eagle's medium supplemented with 10% fetal bovine serum (FBS). Human-derived NCI-H460 non-small cell carcinoma cells (HTB177; American Type Culture Collection, Manassas, Va) and NIH-3T3 fibroblasts were cultivated in Dulbecco's modified Eagle's medium and RPMI 1640, respectively, supplemented with 10% FBS.…”

Persistent transgene expression in mouse liver following in vivo gene transfer with a ΔE1/ΔE4 adenovirus vector

“…12 The E4 for comparison. Both ⌬E1 and ⌬E1/⌬E4 vectors used in gene region encodes proteins that regulate viral early-toin vivo gene transfer studies were screened using PCRlate phase transition, and cause host protein synthesis based, 11 as well as cell based assays 11,17 and found to shutoff. 13 Removal of the E1 and E4 gene regions from be free of E4-containing (⌬E1/E4+), E1-containing the recombinant adenovirus vectors should dramatically (E1+/⌬E4), and replication-competent adenovirus (RCA, reduce viral late gene expression due to failure to E1+/E4+).…”

Development and application of adenoviral vectors for gene therapy of cancer

“…191 The E4 fragment carried by a plasmid vector (pSV2gpt) was transfected into Vero cells by calcium phosphate precipitation. The functional cell line that was cloned as a result (W162) permitted plaque formation by H2 dl808 at an efficiency that is Ͼ106-fold higher than that of the parental Vero cells and allowed the production of high-titer, helper-free H2 dl808 stocks.…”

“…207 In the cell line W162, 191 constitutive expression of E4 In the presence of the mini-Ad vector genome, the helper Ad also supports DNA replication of the mini-Ad vector genome, which is preferentially packaged because it contains a wt (unmodified) packaging signal that has a high affinity (relative to the modified signal of the helper virus) for the limiting amount of the packaging proteins. 35,36 Purification of the mini-Ad vector can be achieved by biochemical or physical methods such as banding the virus on CsCl gradients using an ultracentrifuge.…”