Molecular genetics of herpes simplex virus. VI. Characterization of a temperature-sensitive mutant defective in the expression of all early viral gene products.

The herpes simplex virus 1 (HFEM) mutant tsB7 failed to express any detectable viral polypeptides and did not significantly inhibit host cell protein synthesis in infected cells maintained at the nonpermissive temperature. The mutant could complement the growth of a coinfecting temperature-sensitive mutant virus differing in plaque phenotype and thus appeared capable of penetrating doubly infected cells. The yield of tsB7 was enhanced by the coinfecting virus but not to the extent that the coinfecting virus was enhanced. Coinfection studies suggested that the tsB7 defect was complemented in trans, but poorly, by the wild-type parent and other viruses. Marker rescue of tsB7 by transfection with herpes simplex virus 2 XbaI DNA fragments mapped the mutation between 0.45 and 0.70 map units. Analysis of the DNA structure of the ts(+) intertypic recombinants generated by this rescue showed that the herpes simplex virus 2 DNA substitutions all contained the region between 0.46 and 0.52 map units, thus further defining the map position of the mutation. Analyses of the polypeptides expressed by these intertypic recombinants defined the genome location of the genes specifying polypeptides 2, 6, 10, 32, 43, and 44 and indicated that the mutation maps in or near genes coding for virion structural polypeptides. This region of the genome is represented as stable transcripts and cytoplasmic mRNA only after viral DNA replication (P. C. Jones and B. Roizman, J. Virol. 31:299-314, 1979), and thus this gene appears to be a late function. These results are consistent with the ts mutation in tsB7 being in a gene coding for a virion component which functions before expression of the alpha genes early in infection. The most likely explanation is that the mutant is blocked at a stage of uncoating and the defect is complemented, although poorly, by a coinfecting virus gene product.

Fast axonal transport in the presence of high Ca2+: evidence that microtubules are not required.

Microtubules have long been associated with the mechanism of fast axoplasmic transport, although experimental evidence to support an involvement has been equivocal. Electron microscopic studies demonstrated that incubation of the axons of excised rat sciatic nerves in media containing 75 mM Ca2+ caused complete loss of microtubules within 6 hr. To evaluate the role of microtubules in fast anterograde transport, studies of transport in nerves exposed to these conditions were undertaken. Prior to measurement of axoplasmic transport, nerves ligated distal to the dorsal root ganglia were preincubated in vitro in 75 mM Ca2+ for 0-6 hr. Fast axonal transport was subsequently monitored by measuring the amount of trichloroacetic acid-insoluble radioactivity that accumulated at the ligature after incubation for 12-18 hr with L-[3H]proline. Nerves in which microtubules had been depolymerized by preincubation in high Ca2+ maintained control levels of transport. We conclude that intact microtubules are not required for fast anterograde axoplasmic transport.