Su L, Kaneshima H, Bonyhadi M L, Lee R, Auten J, Wolf A, Du B, Rabin L, Hahn B H, Terwilliger E, Mccune J M
HIV Group, SyStemix, Inc., 1501 California Avenue, Palo Alto, California, 94304, USA.
Virology. 1997 Jan 6;227(1):45-52. doi: 10.1006/viro.1996.8338.
Pathogenic organisms are frequently attenuated after long-term culture in vitro. The mechanisms of the attenuation process are not clear, but probably involve mutations of functions required for replication and pathogenicity in vivo. To identify these functions, a direct comparison must be made between attenuated genomes and those that remain pathogenic in vivo. In this study, we used the heterochimeric SCID-hu Thy/Liv mouse as an in vivo model to define human immunodeficiency virus type 1 (HIV-1) determinants which are uniquely required for replication in vivo. The Lai/IIIB isolate and its associated infectious molecular clones (e.g., HXB2) were found to infect T cell lines but failed to replicate in the SCID-hu Thy/Liv model. When a lab worker was accidentally infected by Lai/IIIB, however, HIV-1 was isolated only from infection of primary PBMC, and not from infection of T cell lines. We hypothesized that the lab worker was exposed to a heterogeneous viral stock which had been attenuated by passage in immortalized T cell lines. Either a rare family member from this stock was selected for in vivo replication or, alternatively, an attenuated genotype dominant in vitro may have reverted to become more infectious in vivo. To address this hypothesis, we have used the SCID-hu Thy/Liv model to study the replication of HXB2 and of HXB2 recombinant viruses with HIV-1 fragments isolated from the infected lab worker. HXB2 showed no or very low levels of replication in the Thy/Liv organ. Replacement of its subgenomic fragment encoding the envelope gene with a corresponding fragment from the lab worker isolate generated a recombinant virus (HXB2/LW) which replicated actively in SCID-hu mice. The NEF mutation in the HXB2 genome is still present in HXB2/LW. Thus, the LW sequences encode HIV-1 determinants which enhance HIV replication in vivo in a NEF-independent mechanism. The specific determinants have been mapped to the V1-V3 regions of the HIV-1 genome. Six unique mutations in the V3 loop region of HXB2/LW have been identified which contribute to the increased replication in vivo.
致病生物在体外长期培养后常出现减毒现象。减毒过程的机制尚不清楚，但可能涉及体内复制和致病性所需功能的突变。为了确定这些功能，必须对减毒基因组与体内仍具致病性的基因组进行直接比较。在本研究中，我们使用异源嵌合的SCID-hu Thy/Liv小鼠作为体内模型，以确定1型人类免疫缺陷病毒（HIV-1）在体内复制所特需的决定因素。发现Lai/IIIB分离株及其相关的感染性分子克隆（如HXB2）能感染T细胞系，但在SCID-hu Thy/Liv模型中无法复制。然而，当一名实验室工作人员意外感染Lai/IIIB时，HIV-1仅从原发性外周血单核细胞感染中分离出来，而不是从T细胞系感染中分离出来。我们推测该实验室工作人员接触到了一种在永生化T细胞系中传代后减毒的异质性病毒株。要么从该病毒株中选择了一个罕见的家族成员进行体内复制，要么体外占主导的减毒基因型可能已回复为在体内更具感染性。为了验证这一假设，我们使用SCID-hu Thy/Liv模型研究了HXB2以及带有从感染的实验室工作人员分离出的HIV-1片段的HXB2重组病毒的复制情况。HXB2在Thy/Liv器官中显示无复制或复制水平极低。用来自实验室工作人员分离株的相应片段替换其编码包膜基因的亚基因组片段，产生了一种重组病毒（HXB2/LW），该病毒在SCID-hu小鼠中能活跃复制。HXB2基因组中的NEF突变在HXB2/LW中仍然存在。因此，LW序列编码以不依赖NEF的机制增强HIV在体内复制的HIV-1决定因素。具体的决定因素已定位到HIV-1基因组的V1-V3区域。已鉴定出HXB2/LW的V3环区域中的六个独特突变，这些突变有助于其在体内复制增加。
