The 5′ RNA Terminus of Spleen Necrosis Virus Stimulates Translation of Nonviral mRNA

Abstract

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...

Translational Regulation of the JunD Messenger RNA

“…10) (29). Expression from the EMCV IRES control was consistent with previous studies, being up-regulated 10-fold when compared with the no insert construct (24,49). Insertion of a synthetic hairpin sequence into a dicistronic vector was shown previously to inhibit expression of the 5Ј-ORF while simultaneously increasing expression of the 3Ј-ORF in the presence of an IRES (21).…”

“…The insert was then subcloned into the modified pRL-CMV vector after digestion with XhoI and BamHI. The dicistronic construct containing the encephalomyocarditis virus insert was a kind gift from Dr. Kathleen Boris-Lawrie and was described previously (24). A hairpin structure was inserted into the dicistronic constructs by first generating an EcoRI site within the pRL-CMV promoter region upstream of the Renilla luciferase ORF.…”

A novel role of RNA helicase A in regulation of translation of type I collagen mRNAs

“…Recently, it was demonstrated that RHA is a necessary cofactor to promote translation initiation of highly structured mRNAs containing the post-transcriptional control element (PCE) Zhang and Grosse 2004;Hartman et al 2006). PCE is a highly structured, orientationdependent motif present in the 59 UTRs of some retroviral mRNAs (Butsch et al 1999;Roberts and Boris-Lawrie 2000;Hull and Boris-Lawrie 2002). The 59PCE activity is positively regulated by RHA and is essential for the translation efficiency of viral mRNAs (Butsch et al 1999;Hull and Boris-Lawrie 2002).…”

Murine Leukemia Virus Regulates Alternative Splicing through Sequences Upstream of the 5· Splice Site

“…In addition, both deletions affect the translational utilization of the respective RNAs (Fig. 3C), reminiscent of the role of the R/U5 region of spleen necrosis virus in translational regulation (44,45). The main finding of the current study is a novel function for the sequences downstream of the RSL, and especially for the PBS, in retroviral splice regulation.…”