Evidence for an "early early" vaccinia virus-induced protein which causes a density change of infected L-M cells.

The density decrease of vaccinia virus-infected L-M cells observed in a Ficoll density gradient by 2 h postinfection was found to be dependent on RNA synthesis and protein synthesis but independent of DNA synthesis. Using low multiplicities of infection, the required RNA and protein species appeared to be synthesized before parental viral DNA became sensitive to DNase, i.e., while the bulk of input virus was still at the core stage of uncoating. To date only thymidine kinase and a vaccinia virus-specific cell surface antigen (as well as the putative uncoating protein) have been shown to be "early early" proteins, i.e., synthesized while parental viral DNA is still enclosed within the core. Both heat- and UV-inactivated virus failed to cause the cell density decrease. The need for a functioning viral genome implies that the required early early RNA and protein species are virus specific and not cell specific. Thus the protein leading to the density decrease of L-M cells, induced very early after infection with vaccinia virus, represents one of the first bits of viral genetic information expressed after infection. Since antibody-neutralized virus is still capable of causing the phenomenon of cell density decrease, the basis of neutralization of vaccinia virus by specific antibody must be other than by inhibiting early early transcription and/or translation.

Sedimentation changes of L cells in a density gradient early after infection with vaccinia virus.

By 2 h postinfection, LM cells infected with vaccinia virus show a shift in their distribution when separated on a Ficoll density gradient. This shift is dependent on both time and the multiplicity of infection and is due, at least in part, to an increase in cell size. Those cells which do shift in position in the gradient represent infected members of the population. Physical changes induced in virus-infected cells can be utilized for studying early events in virus replication.

Direct measurement of acetylesterase in living protist cells.

The fluorogenic acetylesterase (acetic ester hydrolase EC 3.1.1.6.) substrate, fluorescein diacetate, was used to measure enzyme activity in living protist cells. The visual enzyme assay was done by monitoring fluorochromasia by fluorescent microscopy. Quantitative fluorogenic assays were done by measuring the evolved fluorescein in a fluorometer. Of 59 strains of bacteria, 35 were fluorochromatically positive. Eight of the fluorochromatically negative strains were fluorogenically positive. Of 22 strains of slime molds and fungi, all were fluorochromatically positive. Three out of 12 different algae were fluorochromatically positive. Several unidentified protozoa were also fluorochromatically positive. Four out of six protozoa were fluorochromatically positive. Structures of special interest showing acetylesterase activity were: the growing hyphal tips of fungi, the vacuolated areas of yeast and protozoa, newly formed bacterial spores or immature fungal spores, "mesosome-like" bodies in Bacillus megaterium, and the cell membrane and nuclear region of green algae. Yeast protoplasts and bacterial protoplasts and spheroplasts were fluorochromatically positive when derived from positive cells and negative when derived from negative cells. There was no correlation between the possession of a capsule and acetylesterase activity. There was no effect on the viability of bacterial cells incubated in the presence of fluorescein diacetate. Paraoxon inhibited bacterial and yeast enzyme at 10(-5)m. Eserine (10(-5)m) and Paraoxon (10(-7)m) inhibited B. megaterium enzyme. Sodium acetate at 10(-2)m did not inhibit bacterial enzyme. The implications of these findings on the location and expression of esterase activity in living cells are discussed.