RELATIONSHIP BETWEEN TROPISM, INFECTIVITY, AND NEUTRALIZATION IN HIV

HIV 的趋向性、感染性和中和作用之间的关系

• 批准号: 2568928
• 负责人: K PEDEN
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2568928
• 关键词: CD4 molecule; HIV envelope protein gp120; HIV envelope protein gp41; gene mutation; genetic regulatory element; genetic strain; human immunodeficiency virus 1; human immunodeficiency virus 2; tissue /cell culture; transfection; virus RNA; virus genetics; virus infection mechanism; virus replication

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.

虽然所有决定HIV复制趋向性的因素仍在研究中， 阐明，主要决定因素驻留在病毒的包膜 并且存在于细胞外（gp 120）和跨膜 （gp 41）组分。 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， 位点，PBS和多嘌呤段，PPT）也可以调节复制 在不同的细胞类型。 我们之前的工作包括描述 存在于包膜基因中的向性决定因素。 的方法 包括将复制能力降低的病毒在细胞上传代， 这表明复制能力降低。 因为 艾滋病毒变异的高倾向，变异的出现， 经常看到复制容量增加。 类型和位置 解释观察到的表型的突变已经被 在描述HIV基因组的那些基因或区域方面提供信息 在特定的功能中。 最近，我们应用了 引物结合位点（PBS）和多嘌呤的研究方法 图1示出了ROD株HIV-2的病毒片段（PPT）。 虽然大多数HIV-1和HIV-2病毒株 使用宿主tRNA Lys 3种类作为负链cDNA的引物 合成时，HIV-2的ROD 10克隆在PBS中携带序列变化 导致不同的tRNA被整合。 该tRNA 对应于一种我们称之为tRNA Lys 7的tRNA。 的 tRNA Lys 7是否用于HIV-2 ROD 10复制的问题， 在传代期间是否维持非标准PBS， 通过在PBMC和T细胞系中传代后对PBS进行测序。 那里 即使是单次传代后， PBMC或细胞系。 两次传代后，PBS的比例 对应于Lys 7 tRNA的表达量约为30 - 50%。 其他通道是 正在进行中，以确定非典型的PBS是否完全恢复。 在 此外，由于除了PBS之外还有其他决定因素， 掺入适当的tRNA，我们正在构建杂交病毒， 在ROD 10和ROD 2之间，另一种HIV-2 ROD分离株具有 以tRNALys 3为引物，进行鉴定和定位 这些其他的决定因素。 HIV-2 ROD还有另一个不寻常的特征。 的 PPT并不是大多数人通常的全嘌呤典型元素， 逆转录病毒，但具有被嘧啶残基中断的PPT。 由于该病毒具有感染性，我们对传代病毒的PPT进行了测序 并发现非规范PPT保留在培养物上。 此外，有三个克隆的ROD和所有三个有这个 PPT表明，这种PPT存在于体内，它是完全 功能性的，它被选中。 未来的研究将评估 PPT和RT/RNaseH之间的相互作用，将ROD PPT替换为 一个典型的PPT并测量该PPT的稳定性。