TY - JOUR
T1 - A cell line that supports the growth of a defective early region 4 deletion mutant of human adenovirus type 2.
AU - Weinberg, D. H.
AU - Ketner, G.
PY - 1983
Y1 - 1983
N2 - 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, H2dl808, 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 H2dl808. One of the five lines, W162, permits plaque formation by H2dl808 at an efficiency that is greater than 10(6)-fold higher than that of the parental Vero cells and allows the production of high-titer, helper-free H2dl808 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 H2dl808 lysate produced on W162 cells, we have accurately mapped the 808 deletion. It extends from between BclI and SmaI sites at positions 91.4 and 92.0, respectively, to just beyond a HindIII 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.
AB - 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, H2dl808, 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 H2dl808. One of the five lines, W162, permits plaque formation by H2dl808 at an efficiency that is greater than 10(6)-fold higher than that of the parental Vero cells and allows the production of high-titer, helper-free H2dl808 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 H2dl808 lysate produced on W162 cells, we have accurately mapped the 808 deletion. It extends from between BclI and SmaI sites at positions 91.4 and 92.0, respectively, to just beyond a HindIII 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.
UR - http://www.scopus.com/inward/record.url?scp=0020822949&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0020822949&partnerID=8YFLogxK
U2 - 10.1073/pnas.80.17.5383
DO - 10.1073/pnas.80.17.5383
M3 - Article
C2 - 6310575
AN - SCOPUS:0020822949
SN - 0027-8424
VL - 80
SP - 5383
EP - 5386
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 17
ER -