DIFFERENTIAL GROWTH-KINETICS ARE EXHIBITED BY HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAR MUTANTS
DIFFERENTIAL GROWTH-KINETICS ARE EXHIBITED BY HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAR MUTANTS
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DOI：
10.1128/jvi.68.9.5899-5910.1994
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发表时间：
1994-09-01
影响因子：
5.4
通讯作者：
GAYNOR, RB
中科院分区：
文献类型：
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作者：
HARRICH, D;HSU, C;GAYNOR, RB
人类免疫缺陷病毒1型（HIV-1） TAR元件对反激活蛋白Tat激活基因表达至关重要。诱变研究表明，一种稳定的茎环RNA结构包含从TAR转录的环和凸起结构，是该激活的主要目标。虽然瞬态分析已经确定了对TAR功能至关重要的元素，但由于无法产生含有TAR突变的病毒库，尚未有研究确定TAR在病毒复制中的作用。在目前的研究中，我们开发了一种策略，使我们能够产生稳定的293细胞系，这些细胞系能够产生高滴度的含有TAR突变的不同病毒。从这些细胞系中产生的病毒被用来感染t淋巴细胞细胞系和外周血单核细胞。在所有测试的细胞系中，在上茎、凸起部或环部含有TAR突变的病毒都表现出显著降低HIV-1基因的表达和复制。然而，我们能够从这些TAR突变病毒中分离出稳定表达基因产物的淋巴样细胞系。虽然这些细胞系中的病毒数量大致相当，但与含有野生型病毒的细胞系相比，含有TAR突变病毒的细胞在基因表达方面存在极大缺陷。这种病毒基因表达的下降幅度比之前使用HIV-1长末端重复氯霉素乙酰转移酶基因构建的瞬时表达分析中看到的要大得多。与在t淋巴细胞细胞系中发现的病毒生长缺陷相反，一些含有TAR突变的病毒在活化的外周血单核细胞中基因表达和复制的缺陷要小得多。这些结果表明，尽管被感染的细胞类型也是TAR突变病毒复制特性的主要决定因素，但在所有被测试的细胞系中，维持TAR元件对病毒基因表达和复制至关重要。
The human immunodeficiency virus type 1 (HIV-1) TAR element is critical for the activation of gene expression by the transactivator protein, Tat. Mutagenesis has demonstrated that a stable stem-loop RNA structure containing both loop and bulge structures transcribed from TAR is the major target for tat activation. Though transient assays have defined elements critical for TAR function, no studies have yet determined the role of TAR in viral replication because of the inability to generate viral stocks containing mutations in TAR. In the current study, we developed a strategy which enabled us to generate stable 293 cell lines which were capable of producing high titers of different viruses containing TAR mutations. Viruses generated from these cell lines were used to infect both T-lymphocyte cell lines and peripheral blood mononuclear cells. Viruses containing TAR mutations in either the upper stem, the bulge, or the loop exhibited dramatically decreased HIV-1 gene expression and replication in all tell lines tested. However, we were able to isolate lymphoid cell lines which stably expressed gene products from each of these TAR mutant viruses. Though the amounts of virus in these cell lines were roughly equivalent, cells containing TAR mutant viruses were extremely defective for gene expression compared with cell lines containing wild-type virus. The magnitude of this decrease in viral gene expression was much greater than previously seen in transient expression assays using HIV-1 long terminal repeat chloramphenicol acetyltransferase gene constructs. In contrast to the defects in viral growth found in T-lymphocyte cell lines, several of the viruses containing TAR mutations were much less defective for gene expression and replication in activated peripheral blood mononuclear cells. These results indicate that maintenance of the TAR element is critical for viral gene expression and replication in all cell lines tested, though the cell type which is infected is also a major determinant of the replication properties of TAR mutant viruses.