In situ detection of alkaline nuclease activity in cells infected with herpes simplex virus type 1 (HSV-1).
An in situ assay for detection of alkaline nuclease activities has been adapted to the herpes simplex virus type 1 (HSV-1) system. Six major nuclease activities which migrate with molecular weights of 90,000, 85,000, 80,000, 76,000, 71,000 and 65,000, and six minor species of molecular weights 87,000, 81,000, 57,000, 18,500, 17,500 and 16,500 were detected in lysates of HSV-1 infected cells following SDS-polyacrylamide gel electrophoresis and enzyme activation in situ. An ELISA assay and an immunoprecipitation study indicated that the six major HSV-induced nuclease species are virus-specific. Moreover, a reconstruction experiment in which 14C-labelled protein markers were incubated with mock- and HSV-infected cell lysates demonstrates that the nuclease fractions detected in situ were not due to endogenous proteolytic activity. The 80,000, 76,000, 71,000 and 65,000 species were first detected at 4 h post-infection, whereas all others were detectable by 6 h post-infection. The activities of the major cellular nucleases of molecular weights 50,000. 48,000 and 45,000 decreased as a function of time post-infection. The level of expression of each of the virus-induced species was dependent upon the multiplicity of infection, and all virus-induced activities exhibited biochemical properties characteristic of purified HSV-1 alkaline nuclease, including activation and inhibition by specifications. The 76,000 HSV-induced alkaline nuclease species was also demonstrated to possess endonucleolytic activity.
Hafner
J
J
Department of Microbiology, University of New Hampshire, Durham 03824.
Mohammad
F
F
Green
D M
DM
Farber
F E
FE
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D003851:Deoxyribonucleases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000378:metabolism; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D018139:Simplexvirus / Q000201:enzymology*; D014709:Vero Cells
D008345:Manganese; D003851:Deoxyribonucleases; D004720:Endonucleases; D008274:Magnesium; D002118:Calcium