Baric R S, Fu K, Schaad M C, Stohlman S A
Department of Parasitology and Lab Practice, School of Public Health, University of North Carolina, Chapel Hill 27599-7400.
Virology. 1990 Aug;177(2):646-56. doi: 10.1016/0042-6822(90)90530-5.
MHV-A59 temperature-sensitive mutants, representing one RNA+ and five RNA- complementation groups, were isolated and characterized by genetic recombination techniques. Maximum recombination frequencies occurred under multiplicities of infection greater than 10 each in which 99.99% of the cells were co-infected. Recombination frequencies between different ts mutants increased steadily during infection and peaked late in the virus growth cycle. These data suggest that recombination is a late event in the virus replication cycle. Recombination frequencies were also found to range from 63 to 20,000 times higher than the sum of the spontaneous reversion frequencies of each ts mutant used in the cross. Utilizing standard genetic recombination techniques, the five RNA- complementation groups of MHV-A59 were arranged into an additive, linear, genetic map located at the 5' end of the genome in the 23-kb polymerase region. These data indicate that at least five distinct functions are encoded in the MHV polymerase region which function in virus transcription. Moreover, using well-characterized ts mutants the recombination frequency for the entire 32-kb MHV genome was found to approach 25% or more. This is the highest recombination frequency described for a nonsegmented, linear, plus-polarity RNA virus.
分离出代表一个RNA⁺和五个RNA⁻互补组的MHV - A59温度敏感突变体，并通过基因重组技术对其进行了表征。在感染复数大于10时出现最大重组频率，其中99.99%的细胞被共同感染。不同温度敏感突变体之间的重组频率在感染期间稳步增加，并在病毒生长周期后期达到峰值。这些数据表明重组是病毒复制周期中的晚期事件。还发现重组频率比杂交中使用的每个温度敏感突变体的自发回复频率总和高63至20000倍。利用标准基因重组技术，将MHV - A59的五个RNA⁻互补组排列成一个位于基因组5'端23 kb聚合酶区域的累加线性遗传图谱。这些数据表明，在MHV聚合酶区域中至少编码了五个在病毒转录中起作用的不同功能。此外，使用特征明确的温度敏感突变体，发现整个32 kb MHV基因组的重组频率接近25%或更高。这是针对非节段性、线性、正链RNA病毒描述的最高重组频率。
