RELATIONSHIP BETWEEN TROPISM, INFECTIVITY, AND NEUTRALIZATION IN HIV

尽管艾滋病毒复制取向的所有决定因素仍在 阐明，主要决定因素存在于病毒的包膜中 并且存在于细胞外(Gp120)和跨膜。 (Gp41)组件。然而，其他基因(gag、nef、vpr、vif)和 顺式作用元件(长末端重复序列、LTR、引物结合 位点、PBS和多聚尿路，PPT)也可以调节复制 在不同的细胞类型中。我们之前的工作涉及刻画 位于包膜基因中的趋向性决定因素。该方法 涉及在细胞上传代复制能力降低的病毒 这表明了复制能力的降低。因为 艾滋病毒变异的高倾向，变异株的出现 复制容量增加是常见的现象。类型和位置 解释观察到的表型的突变的 在描述艾滋病毒基因组的那些基因或区域方面提供了信息 被卷入到特定的功能中。最近，我们已经应用了这个 关于引物结合位点(PBS)和多嘌呤研究的探讨 杆状病毒HIV-2株的路径(PPT)。而大多数HIV-1和HIV-2毒株 用宿主tRNA Lys3种作为负链cDNA3的引物 HIV-2的ROD10克隆在PBS中携带序列变化 这会导致不同的tRNA被掺入。这个tRNA 对应于一种我们称为tRNA Lys7的tRNA物种。这个 TRNA Lys7是否用于HIV-2 ROD10复制和 是否在传代过程中保持非规范的PBS 通过对PBMC和T细胞传代后的PBS进行测序。那里 甚至在通过一段时间后，就迅速转变为规范的PBS 无论是PBMC还是细胞系。在两段时间后，PBS的比例 与Lys7相对应的tRNA约为30%~50%。更多的段落是 正在确定非规范PBS是否完全恢复。在……里面 此外，由于存在PBS以外的其他决定因素 加入适当的tRNA，我们正在构建杂交病毒 在ROD10和ROD2之间，另一株HIV-2 Rod具有 PBS，并以tRNA Lys3为引物，用于鉴定和定位 这些其他决定因素。HIV-2棒还有另一个不同寻常的特征。这个 PPT不是通常的全嘌呤正则元素的大多数 逆转录病毒，但具有被嘧啶残基打断的PPT。 由于这种病毒是传染性的，我们对传代病毒的PPT进行了测序 并发现在培养上保留了非规范的PPT。 此外，有三个Rod的克隆，这三个都有这个 Ppt证明这种PPT在体内存在，它是完全 功能性的，而且它是被选中的。未来的研究将评估 PPT与RT/RNAseH的相互作用 一个规范的PPT，并测量这个PPT的稳定性。

While all the determinants of tropism of HIV replication are still being elucidated, the major determinants reside in the envelope of the virus and are present in both the extracellular (gp120) and the transmembrane (gp41) components. However, other genes (gag, nef, vpr, vif) and cis-acting elements (the long terminal repeat, LTR, the primer binding site, PBS, and the polypurine tract, PPT) may also modulate replication in different cell types. Our previous work has involved characterizing determinants of tropism that reside in the envelope gene. The approach involves passaging a virus with reduced replication capacity on cells in which that reduced replication ability is manifested. Because of the high propensity of HIV to mutate, the emergence of variants with increased replication capacity is frequently seen. The type and location of mutations that account for the observed phenotype have been informative in delineating those genes or regions of the HIV genome invloved in the particular function. Recently, we have applied this approach to the study of the primer binding site (PBS) and the polypurine tract (PPT) of the ROD strain HIV-2. While most HIV-1 and HIV-2 strains use the host tRNA Lys3 species as primer for the minus-strand cDNA synthesis, the ROD10 clone of HIV-2 carries a sequence change in the PBS that results in a different tRNA being incorporated. This tRNA corresponds to a tRNA species that we refer to as tRNA Lys7. The question of whether tRNA Lys7 is used for HIV-2 ROD10 replication and whether the non-canonical PBS is maintained during passage was addressed by sequencing the PBS after passaging in PBMC and T-cell lines. There was rapid conversion to the canonical PBS after even a single passage in either PBMC or cell lines. After two passages, the proportion of the PBS corresponding to the Lys7 tRNA was about 30 to 50%. Further passages are underway to determine if the non-canonical PBS reverts completely. In addition, since there are determinants other than the PBS for incorporation of the appropriate tRNA, we are constructing hybrid viruses between ROD10 and ROD2, another isolate of HIV-2 ROD that has the canonical PBS and uses tRNA Lys3 as primer, in order to identify and map these other determinants. HIV-2 ROD has another unusual feature. The PPT is not the usual all-purine canonical element of the majority of retroviruses but has a PPT that is interrupted with a pyrimidine residue. Since this virus is infectious, we sequenced the PPT from passaged virus and found that the non-canonical PPT was retained on culture. Furthermore, that there are three clones of ROD and all three have this PPT demonstrates that this PPT exists in vivo, that it is fully functional, and that it is selected for. Future studies will assess the interaction between the PPT and RT/RNaseH by replacing the ROD PPT with a canonical PPT and measuring the stability of this PPT.