Characterization of Human Adenovirus Proteinase Activity in Disrupted Virus Particles

AbstractVirus-coded proteinases are attractive targets for antiviral therapy; however, lack of quick, sensitive, quantitative, and selective assays for enzyme activity makes it difficult to characterize these proteinases and to screen large numbers of potential inhibitors. Here we describe new substrates for the adenovirus proteinase, fluorogenic Rhodamine-based substrates containing tetrapeptides corresponding to sequences cleaved in adenovirus precursor proteins. Proteinase activity in as few as 109disrupted virions could be quantitatively detected in a 30-min assay. With the substrate (Leu-Arg-Gly-Gly-NH)2–Rhodamine, theKmwas 1.4 μM,and theVmaxwas 3.24 pmol substrate hydrolyzed/sec/pmol virus. Enzyme activity was stimulated by dithiothreitol and inhibited by several serine-specific as well as cysteine-specific proteinase inhibitors. In a thiol protection experiment, the virion enzyme was shown to have a cysteine residue with an unusually low pKa, a pKasimilar to that of the active-site nucleophile of the cysteine proteinase papain. The curve ofVmaxas a function of pH is unlike the curve from papain and implied that there are at least three ionizable groups whose protonation state can affect catalysis—one with a pKaof 6.2, another with a pKaof 7.2, and a third with a pKaof 8.3

The Fenice Detector At the E+e- Collider Adone

The FENICE detector, installed at the Frascati e+e- storage ring ADONE, for measuring the neutron e.m. form factors in the timelike region, is described. FENICE is a nonmagnetic detector and consists of a complex array of scintillators, limited streamer tubes and iron converters for detecting mainly the process e+e- --> nnBAR. Antineutrons are identified by the charged prongs produced in their annihilation and the antineutron velocity is measured by the retrieved annihilation time with respect to the beam crossing time. Neutrons are detected in plastic scintillator layers. To reduce the cosmic rays background a concrete shield, covered by an active veto system, is added