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1.
Virology ; 363(2): 261-71, 2007 Jul 05.
Article in English | MEDLINE | ID: mdl-17331561

ABSTRACT

The HIV-1 Vpu and Env proteins are translated from 16 alternatively spliced bicistronic mRNA isoforms. Translation of HIV-1 mRNAs generally follows the ribosome scanning mechanism. However, by using subgenomic env expression vectors, we found that translation of glycoprotein from polycistronic mRNAs was inconsistent with leaky scanning. Instead a conserved minimal upstream open reading frame (uORF) consisting only of a start and stop codon that overlaps with the vpu start site, appears to augment access to the env start codon downstream. Mutating the translational start and stop codons of this uORF resulted in up to fivefold reduction in Env expression. Removing the vpu uORF and increasing the strength of the authentic vpu initiation sequence abolished Env expression from subgenomic constructs and replication of HIV-1, whereas an identical increase in the strength of the minimal uORF initiation site did not alter Env expression.


Subject(s)
HIV-1/physiology , RNA, Messenger/genetics , Viral Envelope Proteins/genetics , Base Sequence , Gene Expression , HeLa Cells , Humans , Molecular Sequence Data , Open Reading Frames/physiology , Protein Biosynthesis , Sequence Alignment , Up-Regulation
2.
Nucleic Acids Res ; 31(23): 6963-75, 2003 Dec 01.
Article in English | MEDLINE | ID: mdl-14627829

ABSTRACT

RNA duplex formation between U1 snRNA and a splice donor (SD) site can protect pre-mRNA from degradation prior to splicing and initiates formation of the spliceosome. This process was monitored, using sub-genomic HIV-1 expression vectors, by expression analysis of the glycoprotein env, whose formation critically depends on functional SD4. We systematically derived a hydrogen bond model for the complementarity between the free 5' end of U1 snRNA and 5' splice sites and numerous mutations following transient transfection of HeLa-T4+ cells with 5' splice site mutated vectors. The resulting model takes into account number, interdependence and neighborhood relationships of predicted hydrogen bond formation in a region spanning the three most 3' base pairs of the exon (-3 to -1) and the eight most 5' base pairs of the intron (+1 to +8). The model is represented by an algorithm classifying U1 snRNA binding sites which can or cannot functionally substitute SD4 with respect to Rev-mediated env expression. In a data set of 5' splice site mutations of the human ATM gene we found a significant correlation between the algorithmic classification and exon skipping (P = 0.018, chi2-test), showing that the applicability of the proposed model reaches far beyond HIV-1 splicing. However, the algorithmic classification must not be taken as an absolute measure of SD usage as it may be modified by upstream sequence elements. Upstream to SD4 we identified a fragment supporting ASF/SF2 binding. Mutating GAR nucleotide repeats within this site decreased the SD4-dependent Rev-mediated env expression, which could be balanced simply by artificially increasing the complementarity of SD4.


Subject(s)
RNA Splice Sites/genetics , RNA, Small Nuclear/metabolism , Algorithms , Ataxia Telangiectasia Mutated Proteins , Base Sequence , Binding Sites , Cell Cycle Proteins , Cell Extracts , DNA-Binding Proteins , Exons/genetics , Gene Products, env/genetics , HIV-1/genetics , HeLa Cells , Humans , Hydrogen Bonding , Mutation/genetics , Protein Serine-Threonine Kinases/genetics , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/genetics , Ribonucleoprotein, U1 Small Nuclear/metabolism , Ribonucleoprotein, U2 Small Nuclear/metabolism , Tumor Suppressor Proteins
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