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Herpesvirus of turkeys (HVT) is a potent helper for the defective parvovirus avian adeno-associated virus (AAAV). To study the helper mechanism at the molecular level, we established a complete cosmid library of HVT DNA in a set of seven overlapping clones and transiently cotransfected secondary chicken embryo fibroblast (CEF) cells with AAAV DNA and recombinant cosmids (cBL) (individual as well as in different combinations). Using an AAAV-specific indirect immunofluorescence assay, we identified four regions on the HVT genome, represented by cBL267, cBL27, cBL33, and cBL34, which express helper functions for AAAV. As demonstrated by infection studies with extracts from cotransfected CEF cells, cBL267 promotes productive AAAV growth, while the helper effect induced by cBL27, cBL33, and cBL34 is limited to the synthesis of noninfectious AAAV antigen. In view of the data presented, possible HVT-specific helper mechanisms for AAAV are discussed.

A new feline sarcoma virus designated Theilen-Pedersen (TP1-FeSV) has been isolated from a spontaneous, slowly growing fibrosarcoma of a domestic short-haired 4-year-old castrated cat. The virus codes for a gag-onc fusion protein of 83,000 molecular weight phosphorylated in vivo at serine, threonine, and tyrosine residues. Cells transformed in vitro with TP1-FeSV exhibit five- to 10-fold elevated levels of phosphotyrosine over FeLV-infected cells. The gag-onc polyprotein has associated with it a tyrosyl protein kinase activity which in vitro results in autophosphorylation of the molecule at tyrosine residues. The fusion protein cannot be labeled metabolically with [3H]glucosamine and tunicamycin has no effect on the electrophoretic mobility of the in vivo [32P]orthophosphate-labeled fusion protein. The fusion protein, in common with the gag precursor Pr65gag, can be metabolically labeled with palmitic acid.

The transforming phosphoprotein pp85gag-fes of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV) was purified in a form which exhibits tyrosine-specific kinase activity. Cell lysates of ST-FeSV-transformed mink nonproducer cells were applied to a column of DEAE-Sephacel and eluted with a linear gradient of NaCl in phosphate buffer. Kinase activity was found uncomplexed (pp85gag-fes) in the flow-through and was eluted with 200 mM NaCl in a complex with pp50 and pp90. The flow-through material was further fractionated by chromatography on hydroxylapatite, followed by phosphocellulose and a final gel filtration step using Sephacryl S200. The final preparation was specifically enriched 1400-fold, and tyrosine-specific kinase activity was increased by about 300-fold as determined by autophosphorylation or phosphorylation of casein. Pp85gag-fes kinase activity was inhibited by nicotinamide adenosine dinucleotide (NAD) and slightly inhibited by NADH, while quercetin, a strong inhibitor for pp60src-associated tyrosine kinase activity, had no inhibiting effect.