analysis-rna-processing-reactions-using-cell-free-systems-3-end

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates <em>in vitro</em>

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

The Scripps Research Institute

Cellular proteins are now appreciated as critically involved in all steps of the human immunodeficiency virus type 1 (HIV-1) life cycle, and disrupting host functions essential for virus replication may provide novel antiviral approaches. Selection from a human complementary DNA (cDNA) library for clones able to induce resistance to infection by recombinant HIV-1 genomes resulted in the identification of a gene fragment that potently restricts HIV-1 activity. The active cDNA encodes an N-terminal fragment of the heterogeneous nuclear ribonuclear protein U (hnRNP U). The gene fragment specifically targets the 3' long terminal repeat (3'LTR) in the viral mRNA and blocks the cytoplasmic accumulation of HIV-1 mRNAs. The results suggest that HIV-1 requires machinery for the nuclear export of viral mRNAs that can be specifically blocked by an interfering gene.

Inhibition of HIV-1 gene expression by a fragment of hnRNP U.

Cellular proteins are critically involved in all steps of the human immunodeficiency virus type 1 HIV-1) life cycle. Disruption of host functions essential for virus replication or discovery of new proteins that block viral replication may provide novel antiviral approaches. In recent years, genetic selections for and against genes carried by retroviral vectors have become increasingly powerful, allowing for the isolation of cells with altered susceptibility to virus infection. Screening complementary DNA libraries for clones able to induce resistance to infection by recombinant HIV-1 genomes, has proved to be an excellent tool to identify new interfering factors. The restriction factors TRIM5alpha1, the Zinc Finger Antiviral Protein ZAP) 2 as well as the dominant negative factor N-86-HnRNPU 3 have all been discovered by means of such genetic screens. Here we report the strategy and techniques to prepare a library and isolate HIV antiviral genes, using the identification of N-86-HnRNPU as an example.

Somatic cell genetic analyses to identify HIV-1 host restriction factors.

Viruses often use host machinery in unusual ways to execute different steps during their replication. To identify host factors critical for virus replication, we screened cDNA expression libraries for genes or gene fragments that could interfere with HIV-1 vector transduction. The DNA clone that most potently inhibited HIV-1 expression encoded the N-terminal 91 aa of the eukaryotic initiation factor 3 subunit f (N91-eIF3f). Overexpression of N91-eIF3f or full-length eIF3f drastically restricted HIV-1 replication by reducing nuclear and cytoplasmic viral mRNA levels. N91-eIF3f and eIF3f specifically targeted the 3' long terminal repeat (3'LTR) region in the viral mRNA. We show that the 3' end cleavage of HIV-1 mRNA precursors is specifically reduced in N91-eIF3f expressing cells. Our results suggest a role of eIF3f in mRNA maturation and that it can specifically interfere with the 3' end processing of HIV-1 mRNAs.

Inhibition of HIV-1 replication by eIF3f.

A genetic screen previously identified the N-terminal 91 amino acids of the eukaryotic initiation factor 3 subunit f (N91-eIF3f) as a potent inhibitor of HIV-1 replication. Overexpression of N91-eIF3f or full-length eIF3f reduced the level of HIV-1 mRNAs in the infected cell. Here we show that N91-eIF3f and eIF3f act by specifically blocking the 3' end processing of the HIV-1 pre-mRNA both in vivo and in vitro. Furthermore, the results suggest that eIF3f mediates this restriction of HIV-1 expression through the previously unsuspected involvement of a set of factors that includes eIF3f, the SR protein 9G8, and the cyclin-dependent kinase 11 (CDK11). eIF3f affects HIV-1 3' end processing by modulating the sequence-specific recognition of the HIV-1 pre-mRNA by 9G8.

HIV-1 mRNA 3' end processing is distinctively regulated by eIF3f, CDK11, and splice factor 9G8.

The human immunodeficiency virus type 1 (HIV) Tat protein, a potent activator of HIV gene expression, is essential for integrated viral genome expression and represents a potential antiviral target. Tat binds the 5'-terminal region of HIV mRNA's stem-bulge-loop structure, the transactivation-responsive (TAR) element, to activate transcription. We find that didehydro-Cortistatin A (dCA), an analog of a natural steroidal alkaloid from a marine sponge, inhibits Tat-mediated transactivation of the integrated provirus by binding specifically to the TAR-binding domain of Tat. Working at subnanomolar concentrations, dCA reduces Tat-mediated transcriptional initiation/elongation from the viral promoter to inhibit HIV-1 and HIV-2 replication in acutely and chronically infected cells. Importantly, dCA abrogates spontaneous viral particle release from CD4(+)T cells from virally suppressed subjects on highly active antiretroviral therapy (HAART). Thus, dCA defines a unique class of anti-HIV drugs that may inhibit viral production from stable reservoirs and reduce residual viremia during HAART.

Article	Year
Inhibition of HIV-1 gene expression by a fragment of hnRNP U. Molecular cell\| PubMed ID: 16916646	2006
Somatic cell genetic analyses to identify HIV-1 host restriction factors. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 19020830	2009
Inhibition of HIV-1 replication by eIF3f. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19237569	2009
HIV-1 mRNA 3' end processing is distinctively regulated by eIF3f, CDK11, and splice factor 9G8. Molecular cell\| PubMed ID: 19854136	2009
An analog of the natural steroidal alkaloid cortistatin A potently suppresses Tat-dependent HIV transcription. Cell host & microbe\| PubMed ID: 22817991	2012

Susana T. Valente

Biography

JoVE Journal Publications

Other Publications