The infectious units in native and alkalidenatured preparations of DNA of herpes simplex virus were characterized with respect to their sensitivity to Neurospora crassa endonuclease, their sedimentation properties in high-salt, neutral sucrose gradients, and their sensitivity to hydrodynamic shearing forces. Infectious molecules in native preparations were resistant to N. crassa endonuclease, sedimented at 56 S, and were highly sensitive to shearing forces. After alkaline denaturation, infectious molecules became sensitive to the N. crassa enzyme, sedimented at 200 S, and were relatively resistant to shear. We conclude that both intact duplex molecules (z100 X 106 daltons) and intact single strands (-50 X 106 daltons) are capable of initiating productive infection.The chromosome of herpes simplex virus (HSV) is a linear, double-stranded DNA molecule of~100 X 106 daltons (1-4). A preliminary report (5) presented evidence that HSV DNA, isolated by mild extraction procedures, could infect primary rabbit-kidney cells or rabbit-skin fibroblasts in the presence of diethylaminoethyl-dextran. The infectious unit was shown to be DNase-sensitive, resistant to anti-HSV serum, and to have a density in CsCl of 1.7 g/cm3. Additional studies (Sheldrick and Laithier, in preparation) have shown that infectivity is protease-resistant, and that progeny virus issuing from DNA-infected cells carry genetic markers present in the infecting DNA. In this communication, we describe experiments that further characterize the infectious unit in native preparations as a molecule of HSV DNA. Moreover, we present evidence that intact single strands (-50 X 106 daltons) of HSV DNA are also infectious.
MATERIALS AND METHODSCulture Medium. Eagle's minimal essential medium (Eurobio, Paris) was supplemented with 3.2 g of glucose, 2.7 g of tryptose phosphate (Difco), and 2.0 g of NaHCO3 per liter. All incubations using this medium were performed in an atmosphere of 5% CO2.Cells. The strain of rabbit-skin fibroblasts, RS537, used in this study was isolated and kindly provided by Dr. G. Orth (6). The cells were propagated at 370 in medium supplemented with 10% calf serum in the absence of antibiotics. Penicillin (100 units/ml) and streptomycin (100 yg/ml) were added to Abbreviations: HSV, herpes simplex virus; PFU, plaque-forming units; PBS, phosphate-buffered saline (pH 7.0). 3621 the medium for the growth of virus and for plaque assays. Cultures were trypsinized twice weekly and reseeded at l/4-1/5 the original cell concentration. The experiments described here were performed with cells between their 40th and 70th passages.Virus. The strain of HSV (subtype 1) used here is designated A44 and has been described (7). It produces a syncytial cytopathic effect in RS537 cultures and, in this sense, is similar to the MP variant described by Hoggan and Roizman (8).Virus Growth Qnd Purification. Confluent cultures of RS537 cells in 1-liter Roux bottles (about 2 to 3 X 107 cells per bottle)were infected with 1 to 2 X 107 plaque-forming units (PFU) of virus i...
