Bordier B, Perala-Heape M, Degols G, Lebleu B, Litvak S, Sarih-Cottin L, Hélène C
Institut de Biochimie Cellulaire et Neurochimie, Centre National de la Recherche Scientifique, Bordeaux, France.
Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9383-7. doi: 10.1073/pnas.92.20.9383.
We have investigated two regions of the viral RNA of human immunodeficiency virus type 1 (HIV-1) as potential targets for antisense oligonucleotides. An oligodeoxynucleotide targeted to the U5 region of the viral genome was shown to block the elongation of cDNA synthesized by HIV-1 reverse transcriptase in vitro. This arrest of reverse transcription was independent of the presence of RNase H activity associated with the reverse transcriptase enzyme. A second oligodeoxynucleotide targeted to a site adjacent to the primer binding site inhibited reverse transcription in an RNase H-dependent manner. These two oligonucleotides were covalently linked to a poly(L-lysine) carrier and tested for their ability to inhibit HIV-1 infection in cell cultures. Both oligonucleotides inhibited virus production in a sequence- and dose-dependent manner. PCR analysis showed that they inhibited proviral DNA synthesis in infected cells. In contrast, an antisense oligonucleotide targeted to the tat sequence did not inhibit proviral DNA synthesis but inhibited viral production at a later step of virus development. These experiments show that antisense oligonucleotides targeted to two regions of HIV-1 viral RNA can inhibit the first step of viral infection--i.e., reverse transcription--and prevent the synthesis of proviral DNA in cell cultures.
我们研究了人类免疫缺陷病毒1型（HIV-1）病毒RNA的两个区域，将其作为反义寡核苷酸的潜在靶点。一种靶向病毒基因组U5区域的寡脱氧核苷酸在体外可阻断HIV-1逆转录酶合成的cDNA的延伸。这种逆转录的阻断与逆转录酶相关的RNase H活性的存在无关。另一种靶向引物结合位点附近位点的寡脱氧核苷酸以RNase H依赖的方式抑制逆转录。这两种寡核苷酸与聚（L-赖氨酸）载体共价连接，并在细胞培养中测试它们抑制HIV-1感染的能力。两种寡核苷酸均以序列和剂量依赖的方式抑制病毒产生。PCR分析表明，它们抑制感染细胞中前病毒DNA的合成。相比之下，靶向tat序列的反义寡核苷酸不抑制前病毒DNA的合成，但在病毒发展的后期步骤抑制病毒产生。这些实验表明，靶向HIV-1病毒RNA两个区域的反义寡核苷酸可抑制病毒感染的第一步，即逆转录，并防止细胞培养中前病毒DNA的合成。
