Involvement of spicules in the formation of vaccinia virus envelopes elucidated by a conditional lethal mutant

The envelope of immature vaccinia virions consists of a lipoprotein bilayer upon which a precise curvature is imposed by acquisition of an external scaffold of spicules. Self-assembly of this tegument was examined employing our ts 6757 mutant, which induces accumulation of immature envelopes at the restrictive temperature. With ts 6757 the envelope bilayers were also assembled into an alternative membrane configuration in the form of flexible cylinders or tubes of uniform width, lacking the spicule coat. Such tubes became extensions of or were continuous with the spherical virion envelopes. The approximately 65 kDa spicule protein, L65, product of gene D13L on the HindIII map, generally designated as a late protein, was expressed as an early function in presence of hydroxyurea, an inhibitor which entirely blocked vaccinia DNA synthesis without stopping assembly of immature envelopes. Labeling of thin sections by immunogold for electron microscopy demonstrated that L65 is present at the surface of immature virions, consistent with the position of spicules on envelopes. Transiency of the spicule scaffold was documented by (a) absence of L65 from intracellular mature virions (IMV) and (b) rapid turnover of L65 during ts 6757 virus replication at the permissive temperature but conservation of this protein at restrictive temperature, as demonstrated in pulse-chase experiments. Time-related decrease in MW of L65 to a smaller polypeptide is interpreted as evidence suggesting that the spicules attached to the envelope are assembled from a higher MW precursor.