An adenovirus ts4 gene function required for DNA encapsidation
The synthesis of Viral DNA by temperature sensitive mutants of adenovirus type 2 was compared with that of WT at the permissive (33C) and non-permissive (39C) temperatures: through centrifugations in alkaline sucrose gradients it was established that none of the 12 mutants tested were defective in DNA synthesis. One of the mutants, ts4 was examined in more detail ; centrifugation experiments in Cesium chloride showed that at 33C, ts4 could produce both the top components (empty capsid) and virus particles as the WT. But at 39C ts4 did not produce infectious, DNA containing particles. This was evident by the absence of the viral band at the density of 1.34 g/cm3. Instead there was an accumulation of top components at 39C. When top components (T.C) from ts4 and WT, or virions synthesized by WT at 39C were negatively stained and examined under the electron microscope, no morphological differences were observed. Double immunodiffusion in agar showed that ts4 made normal fibre and hexon antigens. Attempts were made to "cure" the T.C synthesized by ts4 at 39C by a shift to 33C. These experiments indicated that the T.C made by ts4 at 39C failed to encapsidate DNA even upon transfer to 33C. The shift-up experiments in which the pulse was done at 33C before transferring to 39C, did not reveal any difference between ts4 and WT. SDS-polyacrylamide gel electrophoresis of 35S-methionine labeled infected cells, showed that at 39C ts4 was defective in the processing of precursor proteins: during pulse-chase experiments, polypeptide pVII remained uncleaved. Polypeptide VIII was reduced, whilst polypeptides X to XII were virtually absent. Shift-up and shift-down experiments showed that processing in ts4 was temperature sensitive in the shift-up, but not in the shift-down experiments. Electrophoresis of 35S-methionine labeled, purified virions, and T.C from ts4 and WT (produced at 33C and 39C) indicated the following: (a) polypeptide IIIa of ts4 migrated slower than that of WT whether produced at 39C or 33C, (b) in the T.C of ts4 produced at 39C, polypeptides VIII, XI and XII were present in higher quantities when compared with the WT.