The RU5 region at the 5 RNA terminus of spleen necrosis virus (SNV) has been shown to facilitate expression of human immunodeficiency virus type 1 (HIV) unspliced RNA independently of the Rev-responsive element (RRE) and Rev. The SNV sequences act as a distinct posttranscriptional control element to stimulate gag RNA nuclear export and association with polyribosomes. Here we sought to determine whether RU5 functions to neutralize the cis-acting inhibitory sequences (INSs) in HIV RNA that confer RRE/Rev dependence or functions as an independent stimulatory sequence. Experiments with HIV gag reporter plasmids that contain inactivated INS-1 indicated that neutralization of INSs does not account for RU5 function. Results with luciferase reporter gene (luc) plasmids further indicated that RU5 stimulates expression of a nonretroviral RNA that lacks INSs. Northern blot and RT-PCR analyses indicated that RU5 does not increase the steady-state levels or nuclear export of the luc transcript but rather that the U5 region facilitates efficient polyribosomal association of the mRNA. RU5 does not function as an internal ribosome entry site in bicistronic reporter plasmids, and it requires the 5-proximal position for efficient function. Our results indicate that RU5 contains stimulatorysequencesthatfunctionina5-proximalpositiontoenhanceinitiationoftranslationofanonretroviralreporter gene RNA. We speculate that RU5 evolved to overcome the translation-inhibitory effect of the highly structured encapsidation signal and other replication motifs in the 5 untranslated region of the retroviral RNA.Highly structured 5Ј untranslated regions (UTRs) are known to inhibit ribosome scanning and prevent efficient translation of a variety of mRNAs (11, 14, 16, 22, 23, 29-31, 35, 42, 43). The 5Ј UTR of the retroviral primary unspliced transcript contains highly structured motifs that direct viral packaging of RNA into infectious virus and other steps in retroviral replication (44). In vitro translation assays with human immunodeficiency virus type 1 (HIV) RNA have verified that the HIV 5Ј UTR inhibits efficient translation (11,30). Efficient posttranscriptional control of HIV gene expression is regulated by the interaction of viral regulatory protein Rev and the Rev-responsive element (RRE) within incompletely spliced HIV transcripts (5,8,24). Rev is essential for the stability and nuclear export of RRE-containing transcripts (9,10,15,27,28) and activates their translational efficiency (1,5,8,24). Some genetically simpler retroviruses utilize an RRE-like constitutive transport element that interacts with cellular Rev-like proteins to modulate cytoplasmic accumulation of the primary unspliced viral transcript (4, 32, 33). However, an analogous RRE/Rev-like RNA-protein interaction that facilitates translation for simple retroviruses has not been characterized.Recent experiments have shown that the 5Ј long terminal repeat (LTR) of spleen necrosis virus (SNV) can facilitate RRE/Rev-independent expression of HIV gag reporter gene RNA over 20,000-fo...

Previous work has shown that spleen necrosis virus (SNV) long terminal repeats (LTRs) are associated with Rex/Rex-responsive element-independent expression of bovine leukemia virus RNA and supports the hypothesis that SNV RNA contains a cis-acting element that interacts with cellular Rex-like proteins. To test this hypothesis, the human immunodeficiency virus type 1 (HIV) Rev/RRE-dependent gag gene was used as a reporter to analyze various SNV sequences. Gag enzyme-linked immunosorbent assay and Western blot analyses reveal that HIV Gag production is enhanced at least 20,000-fold by the 5′ SNV LTR in COS, D17, and 293 cells. Furthermore, SNV RU5 in the sense but not the antisense orientation is sufficient to confer Rev/RRE-independent expression onto a cytomegalovirus-gag plasmid. In contrast, the SNV 3′ LTR and 3′ untranslated sequence between env and the LTR did not support Rev-independent gag expression. Quantitative RNase protection assays indicate that the SNV 5′ RNA terminus enhances cytoplasmic accumulation and polysome association of HIV unspliced and spliced transcripts. However, comparison of the absolute amounts of polysomal RNA indicates that polysome association is not sufficient to account for the significant increase in Gag production by the SNV sequences. Our analysis reveals that the SNV 5′ RNA terminus contains a unique cis-acting posttranscriptional control element that interacts with hypothetical cellular Rev-like proteins to facilitate HIV RNA transport and efficient translation.

The 5 long terminal repeat (LTR) of spleen necrosis virus (SNV) contains a unique posttranscriptional control element that facilitates Rev/Rev-responsive element-independent expression of unspliced human immunodeficiency virus type 1 (HIV-1) gag reporter RNA. HIV-1 Gag expression is eliminated when SNV LTR is repositioned to the 3 untranslated region or when the RU5 region is positioned in the antisense orientation. RU5 corresponds to the 5 RNA terminus, and results presented here indicate that Gag production is sustained upon introduction of transcribed spacers that reposition SNV RU5 35 to 200 nucleotides downstream. Concordant results of deletion and point mutagenesis identified two functionally redundant and synergistic motifs (designated A and C) that are necessary and sufficient for SNV RU5 activity. Enzymatic analysis of SNV RU5 RNA structure determined that A and C correspond to stem-loop structures. Quantitative RNA and protein analysis of A and C mutants revealed that the structural integrity of A and C is necessary for protein production, and loss of function correlates with little change in steady-state level, splicing efficiency, or cytoplasmic accumulation of HIV-1 gag reporter RNA. Instead, the structural mutations eliminate cytoplasmic utilization as an mRNA template for Gag protein production. Point mutations of unpaired loop-and-bulge nucleotides that maintain the structure of A eliminate activity. The results show that the unpaired UUGU loop and U-rich bulges function together and are candidate SNV RU5 binding sites for the host cell protein(s) that directs cytoplasmic utilization of unspliced HIV-1 reporter RNA.Structured RNA elements in divergent retroviruses facilitate various posttranscriptional steps in expression of unspliced viral RNA. The 3Ј untranslated regions (UTRs) of Mason-Pfizer monkey virus (MPMV) and the related simian retrovirus type 1 contain a constitutive transport element (CTE) that directs nuclear export of unspliced RNA in conjunction with host proteins Tap and NXT1/p15 (10,14,26,27). The 3Ј UTRs of avian leukosis virus and the related Rous sarcoma virus contain one and two copies, respectively, of a direct repeat (DR) element that is necessary for cytoplasmic accumulation and stability of unspliced viral RNA and for assembly of progeny virions (1, 22-24, 29, 30, 33). The 5Ј long terminal repeats (LTRs) of spleen necrosis virus (SNV) and MPMV contain a unique posttranscriptional control element that facilitates reporter gene expression from unspliced human immunodeficiency virus type 1 (HIV-1) gag reporter RNA independently of Rev/Rev-responsive element (RRE), and also nonviral luc RNA (6,15,28).Extensive mutagenesis studies of CTE and DR have mapped necessary and redundant structural motifs that present unpaired nucleotides for interaction with cellular posttranscriptional modulators. The MPMV and SRV-1 CTEs share 92% sequence homology and are 154-nucleotide (nt) orientationand position-dependent RNA elements (27, 32). The RNA structure of the CTE that is predicted by ...

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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 orientationdependent 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.Retroviruses contain structured RNA elements that interact with viral and cellular proteins and modulate nuclear export and efficient translation of intron-containing transcripts (reviewed in references 3, 5, and 11). The long terminal repeat (LTR) of spleen necrosis virus (SNV) facilitates Rev/Rev-responsive element (RRE)-independent expression of unspliced human immunodeficiency virus type 1 (HIV-1) gag reporter RNA (7). Quantitative RNA and protein analysis identified a minor 2-fold increase in cytoplasmic accumulation of gag RNA but a greater-than-100-fold increase in Gag protein production in response to the SNV LTR. The 165-nucleotide 5Ј RNA terminus encoded by the RU5 region of the LTR functions in a position-and orientation-dependent manner to direct polysome association and detectable Gag protein synthesis. SNV RU5 functions in a cap-dependent manner (M. Butsch and K. Boris-Lawrie, unpublished data) and is not an internal ribosome entry site (IRES) (35). The SNV U5 region also augments translation of nonviral luciferase (luc) RNA by increasing polysome loading (35). Recently, the R region of human foamy virus (HFV) and RU5 of Mason-Pfizer monkey virus (MPMV) were also shown to program unspliced gag RNA templates for Gag protein synthesis (37; S. Hull and K. BorisL...