Kim J H, McLinden R J, Mosca J D, Burke D S, Boswell R N, Birx D L, Redfield R R
Division of Retrovirology, Walter Reed Army Institute of Research, Rockville, MD 20850, USA.
J Acquir Immune Defic Syndr Hum Retrovirol. 1996 Aug 1;12(4):329-42. doi: 10.1097/00042560-199608010-00002.
We had previously shown that chronically infected ACH-2 cells (HIVLAI) could be superinfected with HIVRF, that the frequency of superinfection increased with time, and that the transcription of the superinfecting virus exceeded that of the host HIVLAI provirus. In contrast, ACH-2 cells superinfected with a nef-substituted neomycin-resistant (proNEO) provirus were not detectable by DNA polymerase chain reaction (PCR) until geneticin (G418) was added, suggesting that the ability to propagate progressively in culture may be HIV strain specific. Clonal populations of ACH-2 superinfected with proNEO did not demonstrate preferential transcription of the superinfecting virus. However, clones of ACH-2 superinfected with HIVRF (ACH2/RF) showed a preponderance of HIVRF transcripts similar to that seen in bulk populations. Induction of the superinfecting virus by phorbol ester (PMA) occurred more rapidly than the hose provirus and did not equalize transcriptional activity. PCR-derived long terminal repeat (LTR) fragments and Tat cDNAs from A3.01 cells acutely infected with HIVRF or from ACH-2 cells were sequenced and tested for transactivation. The HIVLAI LTR was two to three times more Tat-responsive than the HIVRF LTR. TatRF was two to three times more transcriptionally active on either LTR than TatLAI. Demethylation with 5-azacytidine did not significantly affect HIV expression from the HIVLAI host provirus of superinfected ACH2/RF cell clones. These data suggest that the mechanism of preferential transcription in HIVRF superinfected ACH2/RF may be attributed to the Tat/TAR axis and the effect of the specific locus of host proviral integration.
我们之前已经表明，长期感染的ACH-2细胞（HIVLAI）可被HIVRF超感染，超感染频率随时间增加，且超感染病毒的转录超过宿主HIVLAI前病毒的转录。相比之下，用nef替代的新霉素抗性（proNEO）前病毒超感染的ACH-2细胞，在添加遗传霉素（G418）之前，通过DNA聚合酶链反应（PCR）无法检测到，这表明在培养中逐渐增殖的能力可能具有HIV毒株特异性。用proNEO超感染的ACH-2克隆群体未表现出超感染病毒的优先转录。然而，用HIVRF超感染的ACH-2克隆（ACH2/RF）显示出与大量群体中相似的HIVRF转录本优势。佛波酯（PMA）诱导超感染病毒的速度比宿主前病毒更快，且未使转录活性均衡。对急性感染HIVRF的A3.01细胞或ACH-2细胞的PCR衍生长末端重复序列（LTR）片段和Tat cDNA进行测序并测试其反式激活作用。HIVLAI LTR对Tat的反应性比HIVRF LTR高两到三倍。TatRF对任一LTR的转录活性比TatLAI高两到三倍。用5-氮杂胞苷去甲基化对超感染的ACH2/RF细胞克隆的HIVLAI宿主前病毒的HIV表达没有显著影响。这些数据表明，HIVRF超感染的ACH2/RF中优先转录的机制可能归因于Tat/TAR轴以及宿主前病毒整合特定位点的作用。
