Identification of a Novel Posttranscriptional Regulatory Element by Using a rev- and RRE-Mutated Human Immunodeficiency Virus Type 1 DNA Proviral Clone as a Molecular Trap

Abstract

Human immunodeficiency virus (HIV) and all other lentiviruses utilize the essential viral protein Rev, which binds to RRE RNA, to export their unspliced and partially spliced mRNAs from the nucleus. We used a revand RRE-defective HIV type 1 (HIV-1) molecular clone in complementation experiments to establish a method for the rapid isolation of posttranscriptional regulatory elements from the mammalian genome by selecting for rescue of virus replication. Viruses rescued by this method contained a novel element with homology to rodent intracisternal A-particle (IAP) retroelements. A functional element was contained within a 247-nucleotide fragment named RNA transport element (RTE), which was able to promote replication of the Rev-and RRE-defective HIV-1 in both human lymphoid cell lines and primary lymphocytes, demonstrating its potent posttranscriptional function. RTE was functional in many cell types, indicating that the cellular factors that recognize RTE are widely expressed and evolutionarily conserved. RTE also promoted RNA export from Xenopus oocyte nuclei. RTE-mediated RNA transport was CRM1 independent, and RTE did not show high affinity for binding to mRNA export factor TAP/NXF1. Since CRM1 and TAP/NXF1 are critical export receptors associated with the two recognized mRNA export pathways, these results suggest that RTE functions via a distinct export mechanism. Taken together, our results identify a novel posttranscriptional control element that uses a conserved cellular export mechanism.The study of retroviral mRNA expression has provided some important insights for the understanding of nucleocytoplasmic export and posttranscriptional regulation in mammalian cells. The process of mRNA splicing and transport is tightly controlled in retroviruses to ensure that both spliced and unspliced mRNAs are produced and transported to polysomes at the appropriate proportions. These pathways are regulated at the posttranscriptional level by cis-acting elements on the viral RNA and by cellular and viral proteins (for reviews see references 12, 25, 33, 46, 55, 60, and 64). The Rev-responsive element (RRE) of HIV-1 was the first to be identified as a unique RNA element within the env coding region of HIV-1. RRE binds the essential protein Rev and promotes the nuclear export, stability, and expression of all viral mRNAs containing RRE. It was subsequently found that all lentiviruses, some oncoretroviruses (for reviews see above), and the type D and the avian leukosis retroviruses have cis-acting RNA elements with analogous function (6, 49, 52, 62, 74). These elements have been shown to mediate RNA export, and some elements can be exchanged among the different viruses (6,47,50,68,74). Functionally related posttranscriptional regulatory elements have also been identified in hepatitis B virus (30), woodchuck hepatitis virus (10), and herpes simplex virus (44). Detailed analyses of some of these elements have shown that they bind different factors and direct RNA to different cellular export pathways, indicating distinct mech...

RNA-binding Motif Protein 15 Binds to the RNA Transport Element RTE and Provides a Direct Link to the NXF1 Export Pathway

“…RNA Export from Xenopus Oocyte Nuclei-The preparation of capped RTE and CTE-containing adenovirus precursor RNA, U1⌬Sm RNA, and U6⌬ss RNA, unlabeled RTE, CTE, and CTEm36 RNAs and oocyte nuclei microinjections were described previously (34,55,56). RNA was extracted from a pool of five oocytes after proteinase K digestion, and equivalents of one-half oocyte were analyzed on 10% polyacrylamide gels containing 7 M urea.…”

“…RNA Transcription and Gel-mobility Shift-The 32 P-labeled RTE and CTE RNAs (34) were prepared as in a previous study (53), and unlabeled RNAs were synthesized using MEGAScript-T7 (Ambion, Austin, TX). 10 nM cold RTE or CTE RNA was added to 10 fmol of radiolabeled RTE or CTE RNA, respectively, to keep the RNA concentration constant for the different probes.…”

“…Retroviral transcripts are a notable exception from this rule, because the unspliced transcript encodes the Gag-pol polyprotein and also serves as viral genomic RNAs, and, therefore needs to be exported prior to splicing. To overcome the general requirement of splicing before export, simian Type D retroviruses and some retroelements utilize the constitutive transport element (CTE) (29 -33) that serves as a high affinity NXF1 ligand (3), thereby providing constitutive, splicing-independent export signals.We had discovered and characterized a novel family of cis-acting RNA transport elements (RTEs) that are present abundantly in the mouse genome and are associated with intracisternal A-particle retroelements (IAP) (34,35). RTE is functionally analogous to CTE, yet structurally unrelated, and does not bind to NXF1 with high affinity.…”

“…We had discovered and characterized a novel family of cis-acting RNA transport elements (RTEs) that are present abundantly in the mouse genome and are associated with intracisternal A-particle retroelements (IAP) (34,35). RTE is functionally analogous to CTE, yet structurally unrelated, and does not bind to NXF1 with high affinity.…”

Lentiviral vectors: optimization of packaging, transduction and gene expression

“…These include the constitutive transport elements (CTEs) from the Mason-Pfizer monkey virus (MPMV) [30][31][32] and the simian retrovirus type 1 (SRV-1) [33] or the RNA transport element (RTE) from the intracisternal A particle retroelement [34]. With the exception of the FIV-based system [32], the substitution of RRE/rev with such heterologous export sequences has resulted in some decreased titers in HIV-1-or SIV-based vectors.…”