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The tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3' splice site.

ABSTRACT: Proportional expression of retroviral genes requires that splicing of the viral primary transcript be an inefficient process. Much of our current knowledge about retroviral suboptimal splicing comes from studies with Rous sarcoma virus. In this report, we describe the use of chimeric introns composed of human beta-globin and human immunodeficiency virus type 1 (HIV-1) splice sites to establish the basis for inefficient splicing of the intron which comprises most of the HIV-1 env coding sequences (referred to as the tat/rev intron). S1 RNA analysis of transfected COS-7 cells revealed that the 3' splice site (3' ss) of this region was significantly less efficient than the 3' ss of the first intron of beta-globin. Deletion of sequences flanking the tat/rev intron 3' ss demonstrated that the requirements for its inefficiency reside within the region that is expected to comprise the essential signals for splicing (i.e., the branchpoint region, the polypyrimidine tract, and the AG dinucleotide). Introduction of an exact copy of the efficient beta-globin branchpoint sequence within a highly conserved region rendered the tat/rev intron 3' ss highly efficient. Improvement of the polypyrimidine tract also increased the splicing efficiency, but to a degree slightly less than that obtained with the branchpoint mutation. Subsequent examination of the tat/rev intron 5' splice site in a heterologous context revealed that it is efficiently utilized. These results indicate that both a poor branchpoint region and a poor polypyrimidine tract are responsible for the low splicing efficiency of the HIV-1 tat/rev intron. It is of fundamental interest to establish the basis for inefficient splicing of the HIV-1 tat/rev intron since it may provide the key to understanding why nuclear export of mRNAs encoding HIV-1 structural proteins is Rev dependent.

SUBMITTER: Staffa A

PROVIDER: S-EPMC236797 | biostudies-other | 1994 May

REPOSITORIES: biostudies-other

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The tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3' splice site.

Staffa A A Cochrane A A

Journal of virology 19940501 5

Proportional expression of retroviral genes requires that splicing of the viral primary transcript be an inefficient process. Much of our current knowledge about retroviral suboptimal splicing comes from studies with Rous sarcoma virus. In this report, we describe the use of chimeric introns composed of human beta-globin and human immunodeficiency virus type 1 (HIV-1) splice sites to establish the basis for inefficient splicing of the intron which comprises most of the HIV-1 env coding sequences ...[more]

PMID: 7512159

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Project description:UnlabelledHerpesviruses have large and complex DNA genomes. The largest among the herpesviruses, those of the cytomegaloviruses, include over 170 genes. Although most herpesvirus gene products are expressed from unspliced transcripts, a substantial number of viral transcripts are spliced. Some viral transcripts are subject to alternative splicing, which leads to the expression of several proteins from a single gene. Functional analysis of individual proteins derived from an alternatively spliced gene is difficult, as deletion and nonsense mutagenesis, both common methods used in the generation of viral gene knockout mutants, affect several or all gene products at the same time. Here, we show that individual gene products of an alternatively spliced herpesvirus gene can be inactivated selectively by mutagenesis of the splice donor or acceptor site and by intron deletion or substitution mutagenesis. We used this strategy to dissect the essential M112/113 gene of murine cytomegalovirus (MCMV), which encodes the MCMV Early 1 (E1) proteins. The expression of each of the four E1 protein isoforms was inactivated individually, and the requirement for each isoform in MCMV replication was analyzed in fibroblasts, endothelial cells, and macrophages. We show that the E1 p87 isoform, but not the p33, p36, and p38 isoforms, is essential for viral replication in cell culture. Moreover, the presence of one of the two medium-size isoforms (p36 or p38) and the presence of intron 1, but not its specific sequence, are required for viral replication. This study demonstrates the usefulness of splice site mutagenesis for the functional analysis of alternatively spliced herpesvirus genes.ImportanceHerpesviruses include up to 170 genes in their DNA genomes. The functions of most viral gene products remain poorly defined. The construction of viral gene knockout mutants has thus been an important tool for functional analysis of viral proteins. However, this strategy is of limited use when viral gene transcripts are alternatively spliced, leading to the expression of several proteins from a single gene. In this study, we showed, as a proof of principle, that each protein product of an alternatively spliced gene can be eliminated individually by splice site mutagenesis. Mutant viruses lacking individual protein products displayed different phenotypes, demonstrating that the products of alternatively spliced genes have nonredundant functions.

Dataset Information

The tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3' splice site.

Publications

The tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3' splice site.

Similar Datasets

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