Berthold E, Maldarelli F
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA.
J Virol. 1996 Jul;70(7):4667-82. doi: 10.1128/JVI.70.7.4667-4682.1996.
Two distinct intranuclear locations were identified for alternatively spliced RNA transcripts expressed from the pNL4-3 infectious molecular clone of human immunodeficiency virus (HIV) type 1. Multiply spliced HIV RNA encoding tat was detected within the nucleus in large clusters; immunostaining and colocalization studies using laser-scanning confocal microscopy revealed that these structures contained the non-small nuclear ribonucleoprotein RNA processing factor, SC35. In contrast, unspliced gag RNA was detected in much smaller granules distributed throughout the nucleus, with little or no association with SC35-containing granules. Analyses of nuclear RNA expressed from recombinant plasmids encoding gag (pCMVgag-2) alone or tat (pCMVtat-2) alone revealed distributions corresponding to those obtained with pNL4-3, indicating that expression within the context of the HIV provirus was not required for the distinct RNA locations detected for these transcripts. The presence of unspliced gag RNA in small granules was confirmed in infections of H9 T-lymphocytic cells, indicating that gag localization was not restricted to transient expression systems. The intranuclear distribution of gag RNA was dependent on specific RNA sequences. Deletion of a portion of the gag gene of pCMVgag-2, containing a cis-repressing inhibitory region, resulted in redirection of unspliced gag RNA from small granules into large SC35-containing clusters. The addition of the Rev-responsive element, RRE, to the deleted pCMVgag-2 construct resulted in RNA transcripts which were no longer associated with SC35. We also identified a cellular intron, rabbit beta-globin-intervening sequence 2 (IVS-2) which, when introduced into pCMVgag-2, redirected unspliced gag RNA into SC35-containing granules and permitted rev-independent Gag expression. These findings suggest that redirecting intranuclear RNA localization may influence gene expression. Color micrographs from this article are available for view at http//128.231.216.2/lmmhome.htm.
在1型人类免疫缺陷病毒（HIV）的pNL4-3感染性分子克隆表达的选择性剪接RNA转录本中，鉴定出了两种不同的核内定位。编码tat的多重剪接HIV RNA在细胞核内以大簇形式被检测到；使用激光扫描共聚焦显微镜进行的免疫染色和共定位研究表明，这些结构包含非小核核糖核蛋白RNA加工因子SC35。相比之下，未剪接的gag RNA在分布于整个细胞核的小得多的颗粒中被检测到，与含SC35的颗粒几乎没有关联。对单独编码gag（pCMVgag-2）或tat（pCMVtat-2）的重组质粒表达的核RNA的分析显示，其分布与用pNL4-3获得的分布相对应，这表明对于这些转录本检测到的不同RNA定位，不需要HIV前病毒环境中的表达。在H9 T淋巴细胞感染中证实了小颗粒中存在未剪接的gag RNA，这表明gag定位不限于瞬时表达系统。gag RNA的核内分布取决于特定的RNA序列。删除pCMVgag-2的gag基因的一部分，该部分包含一个顺式抑制区域，导致未剪接的gag RNA从小颗粒重新定位于含SC35的大簇中。将Rev反应元件RRE添加到缺失的pCMVgag-2构建体中，导致RNA转录本不再与SC35相关联。我们还鉴定出了一种细胞内含子，兔β-珠蛋白间隔序列2（IVS-2），当将其引入pCMVgag-2时，可将未剪接的gag RNA重新定位于含SC35的颗粒中，并允许不依赖Rev的Gag表达。这些发现表明，重新定位核内RNA可能会影响基因表达。本文彩色显微照片可在http//128.231.216.2/lmmhome.htm查看。
