Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors.
We wished to construct cell lines that supply the gene products of gag, pol, and env for the growth of replication-defective reticuloendotheliosis retrovirus vectors without production of the helper virus. To do this, first we located by S1 mapping the donor and acceptor splice sites of reticuloendotheliosis virus strain A. The donor splice site is ca. 850 base pairs from the 5' end of proviral DNA. It is close to or overlaps the encapsidation sequences for viral RNA. The splice acceptor site is ca. 5.6 kilobase pairs from the 5' end of proviral DNA. Therefore, the encapsidation sequences and the donor splice site were removed from viral DNA to give expression of the gag and pol genes without virus production. The promoter in the long terminal repeat was fused to a site near the first ATG codon of the env gene, thereby deleting the encapsidation sequences and the gag and pol genes to give expression of the env gene without virus production. The permissive canine cell line D17 was transfected with the two modified viral DNAs. Two cell clones that contain both modified viral DNAs support the production of replication-defective spleen necrosis virus-thymidine kinase recombinant retrovirus vectors without the production of helper virus. To prevent recombination, the vector contains deletions that overlap with deletions in the integrated helper virus DNAs. This helper cell-vector system will be useful to derive infectious recombinant virus stocks of high titer (over 10(5) thymidine kinase transforming units per ml) which are able to infect avian, rat, and dog cells without the aid of helper virus.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D003001:Cloning, Molecular; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics*; D005814:Genes, Viral; D005822:Genetic Vectors; D006378:Helper Viruses / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D010957:Plasmids; D051381:Rats; D011915:Rats, Inbred BUF; D001356:Reticuloendotheliosis virus / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*; D013937:Thymidine Kinase / Q000172:deficiency; D014162:Transfection; D014779:Virus Replication
D004274:DNA, Recombinant; D004279:DNA, Viral; D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes