Priano C, Arora R, Butke J, Mills D R
Department of Microbiology and Immunology, State University of New York Health Science Center at Brooklyn 11203, USA.
J Mol Biol. 1995 Jun 2;249(2):283-97. doi: 10.1006/jmbi.1995.0297.
Our laboratory has established a bacteriophage Q beta cDNA-containing plasmid system in which virtually all coding defects present within the 4217 nucleotide Q beta genome can be complemented in trans. In this system, Q beta minus strand RNAs are constitutively transcribed from plasmid cDNA by Escherichia coli RNA polymerase. Replication of these minus strands results in the synthesis of Q beta plus RNA, thereby triggering an infectious cycle in which Q beta phase particles are generated. Genetically engineered Q beta genome mutations that result in defective viral proteins can be complemented in trans by the products of one or more Q beta helper plasmids that express either: (1) Q beta maturation protein, which can complement defects in the Q beta maturation cistron (nucleotides 61 to 1320); (2) Q beta readthrough protein, which can complement defects in the readthrough cistron (nucleotides 1344 to 2330); or (3) Q beta replicase, which can complement defects in the replicase cistron (nucleotides 2352 to 4118). Each plasmid component of this system contains a unique origin of replication and carries a different antibiotic gene, thereby enabling all combinations of these plasmids to coexist in the same host. We have further developed a second series of helper plasmids that generate the corresponding viral proteins of the related group IV RNA phage SP. Each of these SP helper proteins can complement respective defects within the Q beta genome with efficiencies similar to those observed for the Q beta helper proteins. It is now possible to supply functional Q beta or SP proteins in trans to examine Q beta genomes that contain protein coding defects for their ability to synthesize Q beta proteins, replicate Q beta RNA, assemble virions, and/or lyse the host cell.
我们的实验室建立了一个含有噬菌体Qβ cDNA的质粒系统，在该系统中，4217个核苷酸的Qβ基因组中几乎所有的编码缺陷都可以通过反式互补得到弥补。在这个系统中，Qβ负链RNA由大肠杆菌RNA聚合酶从质粒cDNA组成型转录。这些负链的复制导致Qβ正链RNA的合成，从而引发一个感染周期，在此周期中产生Qβ噬菌体颗粒。导致病毒蛋白缺陷的基因工程Qβ基因组突变可以通过一种或多种Qβ辅助质粒的产物进行反式互补，这些辅助质粒表达以下三种蛋白之一：（1）Qβ成熟蛋白，可弥补Qβ成熟顺反子（核苷酸61至1320）中的缺陷；（2）Qβ通读蛋白，可弥补通读顺反子（核苷酸1344至2330）中的缺陷；或（3）Qβ复制酶，可弥补复制酶顺反子（核苷酸2352至4118）中的缺陷。该系统的每个质粒组分都含有一个独特的复制起点，并携带不同的抗生素基因，从而使这些质粒的所有组合都能在同一宿主中共存。我们进一步开发了第二系列的辅助质粒，它们可产生相关的IV组RNA噬菌体SP的相应病毒蛋白。这些SP辅助蛋白中的每一种都能以与Qβ辅助蛋白相似的效率弥补Qβ基因组内各自的缺陷。现在可以通过反式提供功能性Qβ或SP蛋白，来检测含有蛋白质编码缺陷的Qβ基因组合成Qβ蛋白、复制Qβ RNA、组装病毒粒子和/或裂解宿主细胞的能力。
