NUCLEOCYTOPLASMIC EXPORT OF SIMIAN RETROVIRUS RNA GENETIC & PROTEIN FACTORS

以往的报道表明，猿类逆转录病毒(SRV) 血清组3基因组包含一个219个核苷酸的序列，从3‘端延伸而来 基因间非翻译区插入到3‘LTR区，可作为 促进REV非依赖性人类免疫缺陷的顺式作用元件 病毒(HIV)复制。该序列包含一个构成成分 转运元件(CTE)，使未剪接的HIV RNA得以转运 从细胞核到细胞质，在没有rev和 转速响应元件(RRE)。确定潜在的细胞蛋白质 结合相关D2/RHE/OR/V1基因组中CTE区域的因子(S)， 我们使用制备的核提取液进行了RNA迁移率变化分析 来自未感染和D2/RHE/或/V1感染的Raji细胞。vbl.使用 聚合酶链式反应(PCR)，我们产生了代表 D2/RHE/OR/V1间隔区CTE区的三个亚区 间隔区，用于将片段单独亚克隆到pGEM3Zf(+)中。感觉 用体外转录的方法合成RNA。RNA迁移率漂移 使用同源和异源亚区的实验 竞争对手，表示R1、R2和R3 RNA会绑定公共 宿主编码蛋白，与R1和R3 RNA在非重叠位置结合 位于同一蛋白质分子上的位点。此外，异源的 竞争分析还表明，第二家公司的潜在存在 与R2 RNA结合的蛋白质。使用核能也得到了类似的结果 未感染或D2/RHE/或/V1感染的Raji细胞的提取物。至 确定该基因间隔区的功能意义 病毒的产生，我们从一种传染性的 D2/RHE/OR/V1的分子克隆含有转化子的 D2/RHE/或/V1分子克隆将病毒释放到培养基中 在转染后4-5天内；无逆转录酶活性 从含有以下物质的转染体获得的培养基中观察到 即使在转染后5天，突变的重组也是如此。因此， 初步证据表明，基因间隔区的存在 非翻译区可能在猿猴逆转录病毒中起重要作用 粒子形成和/或释放。

Previous reports have indicated that the simian retrovirus (SRV) serogroup 3 genome contains a 219 bp sequence, extending from the 3' intergenic nontranslated region into the 3' LTR, that can serve as a cis-acting element to promote rev-independent human immunodeficiency virus (HIV) replication. This sequence, containing a constitutive transport element (CTE), enables the transport of unspliced HIV RNA from the nucleus to the cytoplasm, in the absence of rev and the rev-responsive element (RRE). To identify potential cellular protein factor(s) that bind the CTE region in the related D2/RHE/OR/V1 genome, we conducted RNA mobility shift assays using nuclear extracts prepared from uninfected and D2/RHE/OR/V1-infected Raji cells. Using polymerase chain reaction (PCR), we generated fragments representing three sub-regions of the CTE region from the D2/RHE/OR/V1 intergenic spacer, for individual subcloning of fragments into pGEM3Zf(+). Sense RNAs were synthesized by in vitro transcription. RNA mobility shift experiments, using both homologous and heterologous sub-region competitors, indicate that R1, R2, and R3 RNAs bind a common host-encoded protein, with R1 and R3 RNAs binding at non-overlapping sites on the same protein molecule. In addition, heterologous competition analyses also indicate the potential existence of a second protein bound to R2 RNA. Similar results were obtained using nuclear extracts from uninfected or D2/RHE/OR/V1-infected Raji cells. To determine the functional significance of this intergenic region to virus production, we deleted the intergenic spacer from an infectious molecular clone of D2/RHE/OR/V1. Transfectants containing the D2/RHE/OR/V1 molecular clone released virus into the culture medium within 4 - 5 days post-transfection; no reverse transcriptase activity was observed in culture medium obtained from transfectants containing the mutant recombinant, even at 5 days post-transfection. Thus, preliminary evidence indicates that the presence of the intergenic nontranslated region may play an important role in simian retrovirus particle formation and/or release.