The growth of simian virus 40 (SV40) host range/adenovirus helper function mutants in an African green monkey cell line that constitutively expresses the SV40 agnoprotein.

The simian virus 40 T-antigen carboxy-terminal mutants, dlA2459 and dlA2475, are cell line and temperature dependent for growth and plaque formation in monkey kidney cells. Although these mutants did form plaques on BSC-1 cells at 37 degrees C, they were about fivefold less efficient for plaque formation than wild-type simian virus 40. These mutants did not grow in CV-1 cells and did not synthesize agnoprotein in those cells. CV-1 cells which constitutively express the agnoprotein were permissive for mutant plaque formation. However, late mRNAs, virion proteins, and progeny virion yields did not accumulate to wild-type levels during mutant infection of the agnoprotein-producing cells.

Biological properties of simian virus 40 host range mutants lacking the COOH-terminus of large T antigen.

Three mutants of simian virus 40 (SV40), with deletions near the 3' end of the A gene, displayed a host range phenotype for growth and virus production in various African green monkey kidney cell lines. The mutants formed plaques in CV-1P cells at 40.5 degrees, in BSC-1 cells at 37 and 40.5 degrees, and in Vero cells at 32, 37, and 40.5 degrees. Virus yields in these three lines were cold sensitive: the burst size was greatest at 40.5 degrees and least at 32 degrees, but some progeny was produced under all conditions examined. Mutant yields never exceeded 10% of wild-type yields under the most permissive conditions (Vero cells at 37 or 40 degrees) and were less than 1% of wild type under the most restrictive conditions (CV-1P cells at 32 degrees). These mutants can be complemented by any SV40 mutant which produces a large T antigen containing a normal COOH-terminus. Mutants whose T antigens could not be transported to the nucleus were most efficient at complementation. Mutant virus production in a line of rhesus monkey kidney cells and in primary cultures of African green and rhesus monkey kidney cells was also substantially below wild type. These mutants were also completely defective for adenovirus helper function. Our data suggest that the host range property and adenovirus helper function represent the same activities of large T antigen.

Identification of sequences in the herpes simplex virus thymidine kinase gene required for efficient processing and polyadenylation.

The herpes simplex virus (HSV) type 1 thymidine kinase gene (tk) was resected from its 3' end with BAL 31 exonuclease. Two sets of plasmids were isolated that lacked information distal to the two copies of the hexanucleotide 5'-AATAAA-3' located at the 3' end of the HSV tk gene. The presence of a simian virus 40 origin of DNA replication in each plasmid facilitated analysis of patterns of transcription in transfected Cos-1 monkey cells. Transcription analyses were performed with an S1 nuclease protection assay. Efficient processing and polyadenylation at the normal site still occurred when all sequences more than 44 or 46 base pairs (bp) downstream from the first AATAAA were removed (pTK311R/SV010 and pTK209R/SV010). Removal of an additional 7 bp (pTK312R/SV010) decreased the amount of tk mRNA processed at that normal site, and tk mRNA polyadenylated at a cryptic site within pBR322 sequences began to appear. The normal processing and polyadenylation site was not used at all when an additional 12 bp was removed (pTK314R/SV010); the small amount of tk mRNA produced was processed and polyadenylated at the cryptic pBR322 site. The region of the tk gene critical for efficient processing and polyadenylation of tk mRNA is located 20 to 38 bp downstream from the first AATAAA, distal to the polyadenylation site, and as RNA can form a stem-loop structure containing AAUAAA. Similar G + T-rich elements were located in DNA fragments which substitute efficiently for the HSV tk processing and polyadenylation signal and were not found in AATAAA-containing DNA fragments which substitute inefficiently for the HSV tk signal.