Regulation of gene expression involves sequence elements in nucleic acids. In 
promoters, multiple sequence elements cooperate as functional modules, which in 
combination determine overall promoter activity. We previously developed 
computational tools based on this hierarchical structure for in silico promoter 
analysis. Here we address the functional organization of post-transcriptional 
control elements, using the HIV-1 genome as a model. Numerous mutagenesis 
studies demonstrate that expression of HIV structural proteins is restricted by 
inhibitory sequences (INS) in HIV mRNAs in the absence of the HIV-1 Rev protein. 
However, previous attempts to detect conserved sequence patterns of HIV-1 INS 
have failed. We defined four distinct sequence patterns for inhibitory motifs 
(weight matrices), which identified 22 out of the 25 known INS as well as 
several new candidate INS regions contained in numerous HIV-1 strains. The 
conservation of INS motifs within the HIV genome was not due to overall sequence 
conservation. The functionality of two candidate INS regions was analyzed with a 
new assay that measures the effect of non-coding mRNA sequences on production of 
red fluorescent reporter protein. Both new INS regions showed inhibitory 
activity in sense but not in antisense orientation. Inhibitory activity 
increased by combining both INS regions in the same mRNA. Inhibitory activity of 
known and new INS regions was overcome by co-expression of the HIV-1 Rev 
protein.