Klasens B I, Das A T, Berkhout B
Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands.
Nucleic Acids Res. 1998 Apr 15;26(8):1870-6. doi: 10.1093/nar/26.8.1870.
The presence of a polyadenylation signal in the repeat (R) region of the HIV-1 genome, which is located at both the 5' and 3' ends of the viral transcripts, requires differential regulation of polyadenylation. The HIV-1 poly(A) site can fold in a stable stem-loop structure that is well-conserved among different human and simian immunodeficiency viruses. In this study, we tested the effect of this hairpin on polyadenylation by introducing mutations that either stabilize or destabilize the RNA structure. The HIV-1 sequences were inserted into the pSV2CAT reporter plasmid upstream of the SV40 early poly(A) site. These constructs were transfected into COS cells and transcripts were analyzed for the usage of the HIV-1 versus SV40 poly(A) site. The wild-type HIV-1 poly(A) site was used efficiently in this context and destabilization of the poly(A) hairpin did not affect the polyadenylation efficiency. In contrast, further stabilization of the hairpin severely inhibited HIV-1 polyadenylation. Additional mutations that repair the thermodynamic stability of this mutant hairpin restored the polyadenylation activity. These results indicate that the mechanism of polyadenylation can be repressed by stable RNA structure encompassing the poly(A) signal. Experiments performed at reduced temperatures also suggest an inverse correlation between the stability of the RNA structure and the efficiency of polyadenylation.
HIV-1基因组重复（R）区域存在多聚腺苷酸化信号，该区域位于病毒转录本的5'和3'末端，这需要对多聚腺苷酸化进行差异调节。HIV-1多聚腺苷酸化位点可折叠成稳定的茎环结构，在不同的人类和猿猴免疫缺陷病毒中高度保守。在本研究中，我们通过引入使RNA结构稳定或不稳定的突变，测试了这种发夹结构对多聚腺苷酸化的影响。将HIV-1序列插入到SV40早期多聚腺苷酸化位点上游的pSV2CAT报告质粒中。将这些构建体转染到COS细胞中，并分析转录本对HIV-1与SV40多聚腺苷酸化位点的使用情况。在这种情况下，野生型HIV-1多聚腺苷酸化位点被有效利用，多聚腺苷酸化发夹结构的不稳定并不影响多聚腺苷酸化效率。相反，发夹结构的进一步稳定严重抑制了HIV-1多聚腺苷酸化。修复该突变发夹结构热力学稳定性的其他突变恢复了多聚腺苷酸化活性。这些结果表明，包含多聚腺苷酸化信号的稳定RNA结构可抑制多聚腺苷酸化机制。在较低温度下进行的实验也表明，RNA结构的稳定性与多聚腺苷酸化效率呈负相关。
