Ribozyme-mediated inhibition of HIV 1 suggests nucleolar trafficking of HIV-1 RNA

Abstract

T he expression of HIV type 1 (HIV-1) is controlled by a posttranscriptional mechanism. From a single primary transcript several mRNAs are generated. These RNAs can be divided into three main classes: unspliced 9-kb, singly spliced 4-kb, and the multiply spliced 2-kb RNAs. Each of these RNAs is exported to the cytoplasm for translation and, in the case of the 9-kb RNA, for packaging into virions (1). Normally, pre-mRNAs must undergo a splicing process to remove one or more introns before being exported to the cytoplasm. HIV-1 overcomes this limitation, allowing singly spliced and unspliced RNA to be exported via interaction with its own encoded Rev protein. This regulatory protein binds an RNA stem-loop structure termed the Rev response element located within the env coding region of singly spliced and unspliced HIV RNAs (2-5). Binding of Rev to this element promotes the export, stability, and translation of these HIV-1 RNAs (6-15). The export process is mediated by the nuclear export signal of Rev, which binds the receptor exportin 1͞CRM1. It is believed that CRM1 bridges the interaction of Rev with the nucleoporins required for export to the cytoplasm (16).When Rev and Tat are expressed independently of other HIV transcripts, these proteins localize within the nucleolus of human cells (17)(18)(19)(20)(21)(22). The simultaneous presence of a nuclear export signal as well as a nuclear import͞localization signal confers upon Rev the ability to shuttle between the nucleus and the cytoplasm (16). It has recently been reported that in HeLa cells, the expression of Rev induces the relocalization of the nucleoporins Nup98 and Nup214, along with a significant fraction of CRM1, into the nucleolus (23). This result has led to the hypothesis that formation of the Rev-CRM1-nucleoporin complex targeted to the nuclear pore complex occurs in the nucleolus. It can be similarly hypothesized that HIV RNAs are also relocalized to the nucleolus before cytoplasmic export. Previous studies, which used in situ hybridization assays to define the subcellular localization of HIV RNAs, failed to detect these RNAs in the nucleoli (24)(25)(26)(27). This failure to detect these RNAs is most likely due to the dynamic process of RNA transport, making it difficult to identify discrete nucleolar localization. Therefore we have investigated the same problem, using an alternative strategy based on the use of nucleolar localized ribozymes.

Nucleolar trafficking is essential for nuclear export of intronless herpesvirus mRNA

“…Although the actual localization of Rev itself to the nucleolus has yet to be shown to be essential for its role in HIV-1 replication, it appears that the nucleolus has a fundamental role to play in HIV-1 replication. Some elegant experiments using a hammerhead ribozyme targeted to the nucleolus and specific for HIV-1 RNA showed that HIV-1 RNA trafficking through the nucleolus is essential for HIV-1 replication (40). Similarly, nucleolar targeting of a RNA-based inhibitor of HIV-1 Tat (a second HIV-1 nucleolar protein) was also able to significantly decrease HIV-1 replication, confirming that nucleolar targeting of Tat is required for HIV-1 replication (41).…”

In Vivo HIV-1 Rev Multimerization in the Nucleolus and Cytoplasm Identified by Fluorescence Resonance Energy Transfer

“…Rev is required for export of viral mRNA to the cytoplasm (1, 5), a process essential for virus replication (5,58). It has been suggested previously that HIV-1 RNA is trafficking through the nucleoli of cells, giving the nucleolus a critical role in HIV-1 RNA export (59). Therefore, it is important to study the mobility and affinity kinetics of Rev within the nucleolus.…”

Resistencia a viroides inducida por ribozimas de cabeza de martillo y RNAs interferentes

“…Dicha colocalización condujo a una eficiencia de corte de cerca del 100% (Samarsky et al, 1999). Esta estrategia ha sido empleada igualmente con éxito en células humanas inhibiendo la replicación del HIV (Michienzi et al, 2000;. Otro factor clave que en este contexto determina la localización es el casete de expresión utilizado (revisado en Castanotto et al, 2000), y más concretamente el tipo de promotor, las modificaciones postranscripcionales in vivo de los dos extremos del transcrito, y las secuencias que lo flanquean.…”

“…Las ribozimas de cabeza de martillo presentan una alta especificidad, pues se diseñan para que sus brazos hibriden con el RNA diana que debe contener un trinucleótido NHH (siendo N cualquier nucleótido y H cualquier nucleótido excepto G), haciendo improbable su acción sobre otros RNAs. Otra ventaja de las ribozimas es que en principio pueden dirigirse a los distintos compartimentos celulares en los que se encuentre el RNA diana (Sullenger y Cech, 1993;Samarski et al, 1999;Michienzi et al, 2000;. Además, las ribozimas tienen capacidad de recambio múltiple, pues una misma molécula de ribozima puede catalizar la degradación de varias moléculas de sustrato.…”