The 5′ RNA Terminus of Spleen Necrosis Virus Contains a Novel Posttranscriptional Control Element That Facilitates Human Immunodeficiency Virus Rev/RRE-Independent Gag Production

Abstract

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.

Translational control of retroviruses

“…In these viruses, the region of genomic RNA between the 5′-LTR and the R-U5 domain was shown to enhance the translation of SNV genomic RNA and was subsequently termed the post-transcriptional control element (PCE). Interestingly, this effect was also demonstrated when this PCE was inserted upstream of both the main reading frame of the HIV-1 RNA and a Luciferase reporter gene [65][66][67] . Quantitative RNA analysis, together with ribosomal sedimentation profile analysis, has established that this translation enhancement is due to increased ribosome loading of the mRNA.…”

RNA helicases

“…62,70 However downregulation experiments with siRNAs and quantitative RNA analysis determined RHA is not required for retrovirus transcription, RNA splicing or nuclear export, but that RHA is necessary for retrovirus translation. 57,[71][72][73] RHA selectively interacts with structural features of the 5' UTR of many retroviruses and overexpression of RHA increases their translation. 57,[71][72][73] RHA interaction with the 5' UTR increases polysome association and requires the ATP-binding activity of the DEIH helicase domain.…”

“…57,[71][72][73] RHA selectively interacts with structural features of the 5' UTR of many retroviruses and overexpression of RHA increases their translation. 57,[71][72][73] RHA interaction with the 5' UTR increases polysome association and requires the ATP-binding activity of the DEIH helicase domain. 57,73 The molecular basis of RHA translation activity is catalytic rearrangement of the RNP to facilitate ribosome scanning and translation initiation, and possibly and protein-protein interaction that secures a circular polysome for efficient translation reinitiation.…”

Vectors derived from the human immunodeficiency virus HIV-1

“…Butsch et al have shown that the R/U5 region at the 5' RNA terminus of SNV contains a unique cis-acting posttranscriptional control element that interacts with hypothetical cellular Rev-like proteins and contains stimulatory sequences that function in a 5'-proximal position to enhance initiation of translation of a non-retroviral reporter gene RNA. This facilitates Rev/RRE independent transport and efficient translation of HIV-1 RNA (122,123).…”