Kitamura Y, Ishikawa T, Okui N, Kobayashi N, Kanda T, Shimada T, Miyake K, Yoshiike K
Division of Molecular Genetics, National Institute of Infectious Diseases, Musashimurayama, Tokyo, Japan.
J Acquir Immune Defic Syndr Hum Retrovirol. 1999 Feb 1;20(2):105-14. doi: 10.1097/00042560-199902010-00001.
Retroviruses including HIV-1 integrates a DNA copy of their RNA genome into cellular DNA of the infected cell. This reaction, essential and unique to replication of retroviruses, is mediated by the viral enzyme, integrase (IN). We constructed a recombinant gene encoding a single-chain, antigen-binding peptide (scAb2-19), which interacted with a carboxyl terminal part of HIV-1 IN. HeLa CD4 cells expressing scAb2-19 localized in either cytoplasmic or nuclear compartment were resistant to HIV-1 infection at an multiplicity of infection (MOI) of 0.25 or 0.063, but the resistance was overcome when MOI was increased to 1. To determine whether this resistance was due to inhibition of the early events, transduction experiments were performed with a replication-incompetent HIV-1 vector carrying bacterial lacZ driven by an internal Tat-independent cytomegalovirus immediate early promoter. Both cytoplasmic and nuclear expressions of scAb2-19 resulted in decrease in the transduction efficiency on HeLa CD4 cells. This implies that an early step of replication--before or during integration--was affected by the scAb2-19. Furthermore, cytoplasmic expression of scAb2-19 did not affect the viral amount released from the cells transfected with HIV-1 infectious clone DNA (pLAI). However, infectivity relative to reverse transcriptase activity was lower for virions released from the 293T cells cotransfected with pLAI and the cytoplasmic scAb2-19 expression plasmid than for those released from the 293T cells transfected with pLAI alone. This implies that scAb2-19 reduced infectivity of released virions by interfering a late step of the viral replication. The single-chain, antigen-binding peptide molecule may prove useful not only for studies of the functions of IN and its role in the viral life cycle but also for developing a gene therapy strategy against AIDS.
包括HIV-1在内的逆转录病毒会将其RNA基因组的DNA拷贝整合到被感染细胞的细胞DNA中。这种反应是逆转录病毒复制所必需且独特的，由病毒酶整合酶（IN）介导。我们构建了一个编码单链抗原结合肽（scAb2-19）的重组基因，该肽与HIV-1 IN的羧基末端部分相互作用。表达scAb2-19的HeLa CD4细胞定位于细胞质或细胞核区室，在感染复数（MOI）为0.25或0.063时对HIV-1感染具有抗性，但当MOI增加到1时，这种抗性就会被克服。为了确定这种抗性是否是由于早期事件的抑制，我们用携带由内部Tat非依赖性巨细胞病毒立即早期启动子驱动的细菌lacZ的无复制能力的HIV-1载体进行了转导实验。scAb2-19在细胞质和细胞核中的表达均导致HeLa CD4细胞的转导效率降低。这意味着复制的早期步骤——在整合之前或期间——受到scAb2-19的影响。此外，scAb2-19的细胞质表达不影响从用HIV-1感染性克隆DNA（pLAI）转染的细胞中释放的病毒量。然而，与单独用pLAI转染的293T细胞相比，与pLAI和细胞质scAb2-19表达质粒共转染的293T细胞释放的病毒粒子相对于逆转录酶活性的感染性较低。这意味着scAb2-19通过干扰病毒复制的后期步骤降低了释放的病毒粒子的感染性。单链抗原结合肽分子不仅可能对研究IN的功能及其在病毒生命周期中的作用有用，而且对开发抗艾滋病的基因治疗策略也可能有用。
