Saurya Shreesh, Lichtenstein Zelman, Karpas Abraham
Department of Haematology, University of Cambridge Clinical School, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
J Clin Virol. 2005 Aug;33(4):324-7. doi: 10.1016/j.jcv.2005.05.003.
Highly Active Antiretroviral Therapy (HAART) has emerged as one the most effective method for treating AIDS patients. However, after variable period of treatment, many AIDS patients on HAART show elevated PCR viral load without a corresponding decline in CD4+ T-cells. Our earlier studies have revealed that the viruses present in the plasma of such patients are not infectious.
The aim of this study is to characterize the changes in the regulatory genes of HIV-1, namely tat, rev and rev response element (RRE) isolated from the plasma of such AIDS patients and to assess their role in role in affecting viral infectivity, hence its contribution, in the 'contradictory phenomenon' of high viral load and high CD4+ T-cell counts.
The viral RNA was isolated from the plasma of HAART patients when they exhibited high plasma viral load and high CD4+ T-cell counts. The target regulatory genes were amplified by RT-PCR and sequenced. Sequences were also obtained from the proviral DNA from the peripheral blood mononuclear cells (PBMCs) of the study subjects. The sequences were compared with the wild type viral sequence to look for the changes induced in them due to HAART regime.
Our data revealed that RRE was missing in the viral particles isolated from the plasma of all study subjects. In two patients, the second exon of the rev gene was missing thereby leading to defective Rev protein. In another patients, Rev synthesis was prematurely stopped due to G135T substitution in the amino terminal domain. No such changes were observed in the corresponding proviral DNA. These changes are likely to result in the assembly of non-infectious virus due to lack of envelope proteins. Absence of RRE and Rev protein also leads to transport and packaging of multiply spliced transcripts into the virions instead of complete genomic RNA.
高效抗逆转录病毒疗法（HAART）已成为治疗艾滋病患者最有效的方法之一。然而，经过不同疗程的治疗后，许多接受HAART治疗的艾滋病患者的聚合酶链反应（PCR）病毒载量升高，而CD4+T细胞却没有相应减少。我们早期的研究表明，这类患者血浆中的病毒没有传染性。
本研究旨在鉴定从这类艾滋病患者血浆中分离出的HIV-1调节基因（即tat、rev和rev反应元件（RRE））的变化，并评估它们在影响病毒感染性方面的作用，从而了解其在病毒载量高和CD4+T细胞计数高这一“矛盾现象”中的作用。
从HAART患者血浆中分离病毒RNA，此时他们表现出高血浆病毒载量和高CD4+T细胞计数。通过逆转录聚合酶链反应（RT-PCR）扩增目标调节基因并进行测序。还从研究对象外周血单个核细胞（PBMC）的前病毒DNA中获取序列。将这些序列与野生型病毒序列进行比较，以寻找HAART治疗方案导致的序列变化。
我们的数据显示，从所有研究对象血浆中分离出的病毒颗粒中缺失RRE。在两名患者中，rev基因的第二个外显子缺失，从而导致Rev蛋白缺陷。在另一名患者中，由于氨基末端结构域的G135T替换，Rev合成提前终止。在相应的前病毒DNA中未观察到此类变化。这些变化可能由于包膜蛋白缺乏而导致非感染性病毒的组装。RRE和Rev蛋白的缺失还导致多重剪接转录本而非完整基因组RNA转运和包装到病毒颗粒中。
