Gf Vande Woude

Purification of Herpesvirus saimiri and properties of the viral DNA

SummaryConditions for growth, concentration, and purification of Herpesvirus saimiri were determined. Optimal yields of infectious Herpesvirus saimiri (HVS) were obtained from infected owl monkey kidney (OMK) cells grown at 32.5° C in medium containing 10 per cent fetal calf serum. Forty-five percent of the initial infectious HVS was recovered after an 18-fold concentration using 8 per cent polyethylene glycol 6000 in the presence of 0.5M NaCl. Polyethylene glycol concentrated HVS was purified in an isopycnic-linear Renografin gradient (1.0–1.3 g/cm3). Ninety-six percent of the infectivity was recovered in a single 1.16 g/cm3 density region. DNA extracted from purified HVS was resolved into two distinct density classes by CsCl equilibrium centrifugation (1.727 and 1.709 g/cm3). DNase treated HVS virions yield four DNA species with densities of 1.727, 1.718, 1.712, and 1.706 g/cm3 in CsCl centrifugation.

The Genetic Basis of Retroviral-Induced Transformation

The mammalian leukemia viruses and the avian leukosis viruses produce tumors in animals after long latent periods. In contrast, the acute transforming retroviruses produce a variety of neoplasias within weeks of infection. These latter viruses differ from the leukemia viruses in that they possess specific sequences (one) that share homology with normal host cellular DNA (for reviews see Tooze l973; Duesberg1919). These one sequences appear to be transduced from the host cell into rescuable viral genomes. Their presence in the virus confers a specific transformed phenotype to the infected host cell or animal. We have been interested in how retroviruses interact with one sequences to effect a transformed phenotype.

TWO REGIONS OF THE MOLONEY LEUKEMIA VIRUS GENOME ARE REQUIRED FOR EFFICIENT TRANSFORMATION BY src/sarc

ABSTRACT Src -containing subgenomic DNA fragments of Moloney sarcoma provirus (MSV) with high transforming activity retain two regions of the parental Moloney leukemia virus (MuLV) genome. One MuLV region is the 600 base pair terminal repeat sequence of the provirus. The presence of this sequence either 5′ or 3′ to an internal MSV src sequence enhances transforming activity 1000-fold. The other MuLV region represents approximately 100 nucleotides preceeding the 5′ end of the src sequence. The requirement of this region for transformation is deduced by i) comparison of the endonuclease restriction maps of three MSV provirus isolates, ii) eliminating specific MuLV sequences in cloned subgenomic fragments without loss of biological activity, and iii) demonstrating that efficient transforming activity of the cell s arc sequence occurs only when MSV sequences have been substituted to replace the normal cell sequences at its 5′ end.