Krebs F C, Mehrens D, Pomeroy S, Goodenow M M, Wigdahl B
Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey 17033, USA.
J Biomed Sci. 1998;5(1):31-44. doi: 10.1007/BF02253354.
The generation of genomic diversity during the course of infection has the potential to affect all aspects of HIV-1 replication, including expression of the proviral genome. To gain a better understanding of the impact of long terminal repeat (LTR) sequence diversity on LTR-directed gene expression in cells of the central nervous system (CNS) and immune system, we amplified and cloned LTRs from proviral DNA in HIV-1-infected peripheral blood. Sequence analysis of nineteen LTRs cloned from 2 adult and 3 pediatric patients revealed an average of 33 nucleotide changes (with respect to the sequence of the LAI LTR) within the 455-bp U3 region. Transient expression analyses in cells of neuroglial and lymphocytic origin demonstrated that some of these LTRs had activities which varied significantly from the LAI LTR in U-373 MG cells (an astrocytoma cell line) as well as in Jurkat cells (a CD4-positive lymphocyte cell line). While LTRs which demonstrated the highest activities in U-373 MG cells also yielded high activities in Jurkat cells, the LTRs were generally more active in Jurkat cells when compared to the LAI LTR. Differences in LTR sequence also resulted in differences in transcription factor recruitment to cis-acting sites within the U3 region of the LTR, as demonstrated by electrophoretic mobility shift assays. In particular, naturally occurring sequence variation impacted transcription factor binding to an activating transcription factor/cAMP response element binding (ATF/CREB) binding site (located between the LEF-1 and distal NF-kappaB transcription factor binding sites) that we identified in previous studies of the HIV-1 LTR. These findings suggest that LTR sequence changes can significantly affect basal LTR function and transcription factor recruitment, which may, in turn, alter the course of viral replication in cells of CNS and immune system origin.
在感染过程中基因组多样性的产生有可能影响HIV-1复制的各个方面，包括前病毒基因组的表达。为了更好地理解长末端重复序列（LTR）多样性对中枢神经系统（CNS）和免疫系统细胞中LTR指导的基因表达的影响，我们从HIV-1感染的外周血中的前病毒DNA扩增并克隆了LTR。对从2名成人和3名儿科患者克隆的19个LTR的序列分析显示，在455bp的U3区域内平均有33个核苷酸变化（相对于LAI LTR的序列）。在神经胶质细胞和淋巴细胞来源的细胞中的瞬时表达分析表明，其中一些LTR在U-373 MG细胞（一种星形细胞瘤细胞系）以及Jurkat细胞（一种CD4阳性淋巴细胞系）中的活性与LAI LTR有显著差异。虽然在U-373 MG细胞中表现出最高活性的LTR在Jurkat细胞中也产生高活性，但与LAI LTR相比，这些LTR在Jurkat细胞中通常更活跃。LTR序列的差异也导致转录因子募集到LTR的U3区域内的顺式作用位点的差异，如电泳迁移率变动分析所示。特别是，自然发生的序列变异影响转录因子与我们在先前对HIV-1 LTR的研究中鉴定的激活转录因子/cAMP反应元件结合（ATF/CREB）结合位点（位于LEF-1和远端NF-κB转录因子结合位点之间）的结合。这些发现表明，LTR序列变化可显著影响基础LTR功能和转录因子募集，进而可能改变CNS和免疫系统来源细胞中的病毒复制进程。
