MOLECULAR CLONING AND CHARACTERIZATION OF HUMAN RETROVIRUSES

逆转录病毒基因组之间的异质性是逆转录病毒的一个显著特征。 慢病毒 分子克隆，限制性内切酶分析，Southern杂交 印迹、核苷酸测序和聚合酶链反应技术 用于表征人类免疫缺陷病毒型的分离株 1（HIV-1）。 目的是获得有关 艾滋病毒在其生物化学、结构和多样性方面， 致病性和免疫变异性。 大多数克隆的HIV分离株是嗜T细胞的。 最近，我们成功地 获得完整的和感染性的分子克隆， 嗜巨噬细胞病毒分离株（AD 8）。 众所周知，T细胞是 外周血中HIV复制的靶点。 然而，在这方面， 巨噬细胞代表了大多数人中主要的HIV感染细胞类型。 组织中 巨噬细胞是艾滋病毒的主要宿主， 持续感染多年。 由于其 在感染的小鼠中产生高滴度子代病毒的能力 巨噬细胞，以及巨噬细胞在建立 HIV-1感染，AD 8分子克隆应该是一个有用的试剂 用于生产性感染巨噬细胞的生化分析。 从[SIV-HIV]杂合体获得的肺组织的Southern印迹分析 病毒（SHIV）]感染，CD 4耗竭的食蟹猴显示两个 放射自显影法显示明显条带;一条约为10 Kb（全长 SIV）和一个非常明显的信号约6 Kb（删除的SIV）。 直接从受感染的肺组织中进行分子克隆， 分离出6个克隆。 两个是全长的 而其他4个似乎已被删除（6 Kb）。 这些克隆 目前正在研究中。

Heterogeneity among retroviral genomes is a distinct feature of lentiviruses. Molecular cloning, restriction enzyme analysis, southern blotting, nucleotide sequencing, and polymerase chain reaction techniques were used to characterize isolates of human immunodeficiency virus type 1 (HIV-1). The objective was to obtain information pertaining to the structure and diversity of HIV with respect to its biochemical, pathogenic, and immunological variability. Most HIV isolates cloned are T-cell tropic. Recently, we succeeded in obtaining a complete and infectious molecular clone from a macrophage-tropic viral isolate (AD8). T-cells are known to be the major target for the replication of HIV in peripheral blood. However, macrophages represent the predominant HIV-infected cell type in most tissues. Macrophages are the primary reservoir of HIV and sustain a persistent infection in individuals for many years. Because of its capacity to generate high titers of progeny virus in infected macrophages, and the vital role of macrophages in the establishment of an HIV-1 infection, the AD8 molecular clone should be a useful reagent for the biochemical analyses of productively infected macrophages. Southern blot analysis of lung tissue obtained from a [SIV-HIV hybrid virus (SHIV)] infected, CD4 depleted cynomolgus macaque showed two distinct bands by autoradiography; one approximately 10Kb (full length SHIV) and one very pronounced signal approximately 6Kb (deleted SHIV). Molecularly cloning directly from the infected lung tissue resulted in the isolation of 6 individual clones. Two were full length in size while the other 4 appeared to have been deleted (6Kb). These clones are presently under study.