Structured RNA elements in divergent retroviruses facilitate various posttranscriptional steps in expression of unspliced viral RNA. The 3′ 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
). The 3′ 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
). The 5′ 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
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) orientation- and position-dependent RNA elements (27
). The RNA structure of the CTE that is predicted by M-Fold software (20
) is a stable stem-loop structure (ΔG = −39.4 kcal/mol) that consists of a degenerate repeat of ~70 nt, a 9-nt terminal loop, and two 16-nt internal loops that are rotated 180° relative to each other (11
). The M-Fold prediction was validated by RNA structure analysis and extensive mutagenesis and functional analysis by a Gag reporter gene assay (11
). Point mutations designed to disrupt the double-stranded stem regions adjacent to the loops eliminated HIV-1 gag
reporter gene activity (11
). Compensatory mutations to restore base pairing of the stem rescued Gag production, confirming that the double-stranded stem structure is essential for activity. The stem has been postulated to orient the single-stranded loop nucleotides into the functionally correct position. Mutagenesis experiments by Tabernero et al. (32
) indicated that the sequence of the internal loops, an AAGA bulge, and the stem structure between the loops are essential for CTE activity. Collectively, the mutagenesis data indicate that the secondary structure of CTE RNA and primary sequence of the unpaired nucleotides in the internal loops are necessary for CTE activity.
The DR is a 135-nt redundant stem-loop structure that also functions in an orientation-dependent manner (33
). Each DR consists of two subelements referred to as DR1 and DR2 (29
). Introduction of substitution mutations into predicted internal and terminal loop sequences in DR1 of the replication-competent Prague Rous sarcoma virus C reduces replication by a factor of 10 to 20 and reduces genomic RNA packaging by a factor of 10 (1
). Two separate point mutations at the predicted stem-loop junction of one of the internal loops of the DR reduced expression of unspliced cat
transcripts to between 10 and 40% of wild-type expression (24
). Similar to the mutational analyses of CTE, these mutagenesis results demonstrated the functional importance of predicted unpaired nucleotides in the internal loops of DR. The role of the predicted double-stranded stem in DR remains to be verified, but it is projected to correctly position the unpaired nucleotides for interaction with functionally relevant cellular proteins.
The posttranscriptional control element in the 5′ SNV LTR was initially identified by its ability to facilitate Rex/Rex-responsive element-independent expression of bovine leukemia virus RNA (2
). Subsequently, the SNV LTR was determined to facilitate Rev/RRE-independent expression of unspliced HIV-1 gag
reporter RNA (6
). Rev/RRE-independent Gag expression is eliminated when the RU5 region of the LTR is positioned in the antisense orientation or the LTR is repositioned to the 3′ UTR (6
). SNV RU5 does not function by derepressing inhibitory sequences located in the HIV-1 gag
). Ribosomal sedimentation and ribosome profile analysis established that SNV RU5 augments polysome loading onto HIV-1 gag
reporter RNA and also nonviral luciferase (luc
) reporter RNA (6
). Experiments with bicistronic luc
reporter plasmids determined that SNV RU5 does not function as an internal ribosome entry sequence (28
). Together these results indicate that the RU5 region of the SNV LTR functions as a unique, orientation-dependent RNA element that enhances translation. Sequence or structural motifs necessary for activity remain to be defined.
Here, results of RNA and protein assays on SNV RU5 deletion and substitution mutants and enzymatic mapping of SNV RU5 RNA identified sequence and structural motifs necessary for activity. The experiments determined that SNV RU5 contains two functionally redundant stem-loops that are necessary for Rev/RRE-independent Gag production. Our genetic and biochemical results indicate that SNV RU5 stem-loop structures present unpaired nucleotides for functional interaction with a cellular posttranscriptional modulator(s). Quantitative RNA analysis by RNase protection assay (RPA) indicates that the mutations do not diminish the steady-state level or cytoplasmic accumulation of gag RNA. Instead, the mutations preclude efficient translational utilization of the cytoplasmic transcripts.