Riggs N L, Little S J, Richman D D, Guatelli J C
Department of Medicine, University of California at San Diego.
Virology. 1994 Jul;202(1):264-71. doi: 10.1006/viro.1994.1342.
The replication of HIV-1 mutants containing altered splice acceptor sequences was studied. The splice acceptor sites 5' of the essential tat and rev AUG codons were altered to eliminate specifically spliced species from the viral repertoire of mRNAs. All splice site mutants were attenuated or fully defective. Mutation of the tat splice acceptor (exon 4) caused loss of the mRNA species containing exon 4 and resulted in an attenuated but replication-competent phenotype. Mutation of the rev splice acceptor sites resulted in viral genomes that failed to propagate in vitro. Mutation of the more 5' of the two major rev acceptors (exon 4A) caused loss of the mRNA species containing exon 4A together with a compensatory increase in use of the more 3' of the rev acceptors (exon 4B). Mutation of the splice acceptor for exon 4B caused the unexpected loss of both exon 4A- and 4B-containing mRNAs. In addition to these effects on the rev splice acceptors, mutations at the 4A and 4B sites also resulted in decreased use of the tat splice site (exon 4) located 175 nucleotides upstream. These effects on utilization of the tat splice acceptor site may explain the requirement for tat to efficiently complement these mutants. The 4A mutant was complemented by tat but not by rev. The 4B mutant was complemented by rev but required both tat and rev for maximum complementation. These data suggested a cooperativity among these splice sites necessary for efficient viral replication. They also indicated that while viral replication persisted at low levels in the absence of splicing to the known site 5' of the tat AUG, failure to splice to at least one of the two major sites 5' of the rev AUG resulted in insufficient rev activity for replication competence.
对含有改变的剪接受体序列的HIV-1突变体的复制进行了研究。在必需的tat和rev AUG密码子5'端的剪接受体位点被改变，以从病毒mRNA库中特异性消除剪接的物种。所有剪接位点突变体均减弱或完全缺陷。tat剪接受体（外显子4）的突变导致含有外显子4的mRNA物种丢失，并导致减弱但具有复制能力的表型。rev剪接受体位点的突变导致病毒基因组在体外无法传播。两个主要rev受体中更靠近5'端的（外显子4A）突变导致含有外显子4A的mRNA物种丢失，同时补偿性增加了对更靠近3'端的rev受体（外显子4B）的使用。外显子4B的剪接受体突变导致含有外显子4A和4B的mRNA意外丢失。除了对rev剪接受体的这些影响外，4A和4B位点的突变还导致位于上游175个核苷酸处的tat剪接位点（外显子4）的使用减少。这些对tat剪接受体位点利用的影响可能解释了tat有效补充这些突变体的必要性。4A突变体由tat而不是rev补充。4B突变体由rev补充，但需要tat和rev两者才能实现最大程度的补充。这些数据表明这些剪接位点之间的协同作用对于有效的病毒复制是必要的。它们还表明，虽然在没有剪接到tat AUG 5'端的已知位点的情况下病毒复制仍以低水平持续，但未能剪接到rev AUG 5'端的两个主要位点中的至少一个会导致复制能力的rev活性不足。
