Influence of the Small Leader Exons 2 and 3 on Human Immunodeficiency Virus Type 1 Gene Expression
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文摘
The human immunodeficiency virus type 1 (HIV-1) uses an elaborate alternative splicing pattern for the generation of both the 1.8-kb as well as the 4-kb classes of mRNA. An additional diversity of transcripts in both classes is created by the optional inclusion of the small exons 2 and 3 in the leader sequence. To analyze a possible influence of these leader exons on HIV-1 gene expression, several series of expression vectors with different leaders were constructed, expressing either Rev and Env or a heterologous coding sequence, i.e., the chloramphenicol acetyl transferase (CAT) ORF. Transfection experiments of HeLa-T4+ cells revealed for all series of constructs that mRNA as well as protein expression was stimulated by the presence of exon 2 and reduced by exon 3. The function of the leader exons 2 and 3 is neither dependent on the regulatory proteins Tat or Rev nor on viral coding sequences. Neither transcription rates nor stability of polyadenylated RNAs were found to be responsible for the different levels of steady-state mRNA. When either exon 2 or 3 was inserted into a heterologous intron, processing of the primary transcripts generated identical mRNA species while maintaining the differences in exon 2/3-dependent mRNA steady-state levels. These results may be explained by exon-specific nuclear RNA degradation rates, as also indicated by results from an in vitro degradation assay using a HeLa nuclear extract.