Nuclear interactions are necessary for translational enhancement by spleen necrosis virus RU5

Abstract

The 5' long terminal repeat of spleen necrosis virus (SNV) facilitates Rev/Rev-responsive element (RRE)-independent expression of intron-containing human immunodeficiency virus type 1 (HIV-1) gag. The SNV RU5 region, which corresponds to the 165-nucleotide 5' RNA terminus, functions in a position- and orientation-dependent manner to enhance polysome association of intron-containing HIV-1 gag RNA and also nonviral luc RNA. Evidence is mounting that association with nuclear factors during intron removal licenses mRNAs for nuclear export, efficient translation, and nonsense-mediated decay. This project addressed the relationship between the nuclear export pathway of SNV RU5-reporter RNA and translational enhancement. Results of RNA transfection experiments suggest that cytoplasmic proteins are insufficient for SNV RU5 translational enhancement of gag or luc RNA. Reporter gene assays, leptomycin B (LMB) sensitivity experiments, and RNase protection assays indicate that RU5 gag RNA accesses a nuclear export pathway that is distinct from the LMB-inhibited leucine-rich nuclear export sequence-dependent CRM1 pathway, which is used by the HIV-1 RRE. As a unique tool with which to investigate the relationship between different RNA trafficking routes and translational enhancement, SNV RU5 and Rev/RRE were combined on a single gag RNA. We observed a less-than-synergistic effect on cytoplasmic mRNA utilization. Instead, Rev/RRE diverts RU5 gag RNA to the CRM1-dependent, LMB-inhibited pathway and abrogates translational enhancement by SNV RU5. Our study is the first to show that a nuclear factor(s) directs SNV RU5-containing RNAs to a distinct export pathway that is not inhibited by LMB and programs the intron-containing transcript for translational enhancement.

FIG. 1.

Protein synthesis from capped and polyadenylated reporter transcripts reveals that cytoplasmic proteins are insufficient for translational enhancement by SNV RU5. Shown are representative transcripts used to program three translation systems and their designations, structures, and relative protein-synthetic activities. RNA transfection of 293 cells was performed with 2.5 μg of transcript and Lipofectamine. RRL and 293 cell lysates were programmed with 400 ng of reporter transcript. In each assay system, luc or gag reporter transcripts were used reciprocally to standardize minor differences in RNA transfection efficiency. Reporter proteins were measured by a Gag ELISA or a Luc enzymatic assay. ND, not determined.

FIG. 2.

Effect of LMB on Gag production reveals that SNV RU5 functions independently of CRM1/leucine-rich NES. Results of three to five replicate transfections of 293 cells with the indicated gag reporter plasmids are shown. Reporter proteins were measured 24 h posttreatment with the indicated concentrations of LMB. Gag production is presented relative to that of the mock-treated control.

FIG. 3.

LMB dose-response curves indicate that SNV RU5 confers LMB enhancement in a promoter-independent manner. Results of three replicate transfections of 293 cells with the indicated reporter plasmids are shown. Cell-associated reporter proteins were measured 24 h posttreatment with the indicated concentrations of LMB. Presented is Gag protein production in response to the indicated gag reporter plasmid relative to that of the mock-treated control.

FIG. 4.

RPA of cytoplasmic and nuclear RNAs from transfected 293 cells with or without 24 h of LMB treatment reveals that LMB augments cytoplasmic accumulation of SNV RU5 gag RNA. (A) Relationships among the gag reporter plasmid, an antisense HIV-1 5′ UTR RNA probe, and protected unspliced and spliced transcripts with sizes indicated. ss, splice site; nt, nucleotides. (B) RPA and PhosphorImager analysis of cytoplasmic (30 μg) and nuclear (15 μg) RNAs. Labels indicate the reporter plasmid, LMB treatment, and protected transcript. (C) Northern blot analysis of 10-μg aliquots of the RNA samples hybridized to an actin probe.

FIG. 5.

LMB dose-response curves indicate that LMB enhancement of SNV RU5 gag RNA is mitigated by Rev/RRE. 293 cells were transfected with the indicated gag reporter plasmid, and cell-associated Gag levels are presented relative to that of the mock-treated control.

FIG. 6.

RPA of nuclear and cytoplasmic RNAs from transfected 293 cells to quantify cytoplasmic RNA accumulation in response to Rev/RRE and SNV RU5. Cytoplasmic (30 μg) and nuclear (15 μg) RNAs were protected with a ³²P-labeled antisense HIV-1 5′ UTR RNA probe and a GAPDH RNA probe and were visualized by PhosphorImager analysis. Labels indicate the protected transcripts. (A) RNA expressed from pYW100RRE or pTR155 in the absence or presence of Rev. (B) Translational efficiency is presented relative to that of pTR155. The ratio of Gag protein to cytoplasmic RNA is from Table 4.