Abstract
The HIV Rev protein forms a complex with a 351 nucleotide sequence present in unspliced and incompletely spliced human immunodeficiency virus (HIV) mRNAs, the Rev response element (RRE), to recruit the cellular nuclear export receptor Crm1 and Ran-GTP. This complex facilitates nucleo-cytoplasmic export of these mRNAs. The precise secondary structure of the HIV-1 RRE has been controversial, since studies have reported alternative structures comprising either four or five stem-loops. The published structures differ only in regions that lie outside of the primary Rev binding site. Using in-gel SHAPE, we have now determined that the wt NL4-3 RRE exists as a mixture of both structures. To assess functional differences between these RRE ‘conformers’, we created conformationally locked mutants by site-directed mutagenesis. Using subgenomic reporters, as well as HIV replication assays, we demonstrate that the five stem-loop form of the RRE promotes greater functional Rev/RRE activity compared to the four stem-loop counterpart.
Highlights
In an infected cell, integrated human immunodeficiency virus (HIV) proviral DNA is transcribed to produce a primary transcript that either remains unspliced or is alternatively spliced into multiple mRNA species that retain or lack introns [1,2]
Most complex retroviruses, including HIV [6,7,8], HTLV [9,10], MMTV [11], EIAV[12,13] and Jaagsiekte Sheep virus [14,15], encode a regulatory protein that interacts with a cis-acting element in the viral transcripts to mediate nuclear export and translation of intron-retaining mRNA through the Crm1 pathway
This Rev response element (RRE) is functional, it lacks the lower part of SL I present in the 351 nt RRE structure and it is the form commonly used in gel mobility shift and other studies
Summary
In an infected cell, integrated HIV proviral DNA is transcribed to produce a primary transcript that either remains unspliced or is alternatively spliced into multiple mRNA species that retain or lack introns [1,2]. These mRNAs are all exported to the cytoplasm, where they are packaged as the viral genome or translated into viral proteins, despite the fact that eukaryotic cells have several ‘checkpoints’ that would normally restrict export and expression of the mRNAs that retain introns [3,4,5]. Foamy viruses are unique in that they use a cellular RNAbinding protein, HuR, instead of a viral regulatory protein to facilitate nuclear export of the unspliced viral transcripts through the Crm pathway [20]
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