Ladin B F, Blankenship M L, Ben-Porat T
J Virol. 1980 Mar;33(3):1151-64. doi: 10.1128/JVI.33.3.1151-1164.1980.
The maturation of pseudorabies virus DNA from the replicative concatemeric form to molecules of genome length was examined using nine DNA+ temperature-sensitive mutants of pseudorabies virus, each belonging to a different complementation group. At the nonpermissive temperature, cells infected with each of the mutants synthesized concatemeric DNA. Cleavage of the concatemeric DNA to genome-length viral DNA was defective in all the DNA+ ts mutants tested, indicating that several viral gene products are involved in the DNA maturation process. In none of the ts mutant-infected cells were capsids with electron-dense cores (containing DNA) formed. Empty capsids with electron-translucent cores were, however, formed in cells infected with six of the nine temperature-sensitive mutants; in cells infected with three of the mutants, no capsid assembly occurred. Because these three mutants are deficient both in maturation of DNA and in the assembly of viral capsids, we conclude that maturation of viral DNA is dependent upon the assembly of capsids. In cells infected with two of the mutants (tsN and tsIE13), normal maturation of viral DNA occurred after shiftdown of the cells to the permissive temperature in the presence of cycloheximide, indicating that the temperature-sensitive proteins involved in DNA maturation became functional after shiftdown. Furthermore, because cycloheximide reduces maturation of DNA in wild-type-infected cells but not in cells infected with these two mutants, we conclude that a protein(s) necessary for the maturation of concatemeric DNA, which is present in limiting amounts during the normal course of infection, accumulated in the mutant-infected cells at the nonpermissive temperature. Concomitant with cleavage of concatemeric DNA, full capsids with electron-dense cores appeared after shiftdown of tsN-infected cells to the permissive temperature, indicating that there may be a correlation between maturation of DNA and formation of full capsids. The number of empty and full capsids (containing electron-dense cores) present in tsN-infected cells incubated at the nonpermissive temperature, as well as after shiftdown to the permissive temperature in the presence of cycloheximide, was determined by electron microscopy and by sedimentation analysis in sucrose gradients. After shiftdown to the permissive temperature in the presence of cycloheximide, the number of empty capsids present in tsN-infected cells decreased with a concomitant accumulation of full capsids, indicating that empty capsids are precursors to full capsids.
利用9种伪狂犬病病毒的DNA+温度敏感突变体（每个突变体属于不同的互补群）研究了伪狂犬病病毒DNA从复制性串联体形式成熟为基因组长度分子的过程。在非允许温度下，用每个突变体感染的细胞合成了串联体DNA。在所有测试的DNA+温度敏感突变体中，串联体DNA切割成基因组长度的病毒DNA存在缺陷，这表明几种病毒基因产物参与了DNA成熟过程。在任何温度敏感突变体感染的细胞中都没有形成具有电子致密核心（含有DNA）的衣壳。然而，在9种温度敏感突变体中的6种感染的细胞中形成了具有电子透明核心的空衣壳；在3种突变体感染的细胞中，没有发生衣壳组装。由于这3种突变体在DNA成熟和病毒衣壳组装方面都存在缺陷，我们得出结论，病毒DNA的成熟依赖于衣壳的组装。在感染了两种突变体（tsN和tsIE13）的细胞中，在存在放线菌酮的情况下将细胞转移到允许温度后，病毒DNA发生了正常成熟，这表明参与DNA成熟的温度敏感蛋白在转移后变得有功能。此外，由于放线菌酮会降低野生型感染细胞中DNA的成熟，但不会降低这两种突变体感染细胞中DNA的成熟，我们得出结论，串联体DNA成熟所必需的一种蛋白质（在正常感染过程中含量有限）在非允许温度下在突变体感染的细胞中积累。随着串联体DNA的切割，tsN感染的细胞转移到允许温度后出现了具有电子致密核心的完整衣壳，这表明DNA的成熟与完整衣壳的形成之间可能存在相关性。通过电子显微镜和蔗糖梯度沉降分析确定了在非允许温度下以及在存在放线菌酮的情况下转移到允许温度后，tsN感染的细胞中存在的空衣壳和完整衣壳（含有电子致密核心）的数量。在存在放线菌酮的情况下转移到允许温度后，tsN感染的细胞中存在的空衣壳数量减少，同时完整衣壳积累，这表明空衣壳是完整衣壳的前体。
