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Regulation of Cellular and Viral Gene Expression
Summary: Bryan Cullen is interested in using viral systems to study the regulation of gene expression in human cells at the post-transcriptional level.
My laboratory has in the past been highly interested in gene regulation in the pathogenic virus human immunodeficiency virus type 1 (HIV-1), with a particular focus on Tat and Rev, two virus-encoded regulatory proteins that are critical for HIV-1 replication. The Tat protein activates HIV-1 gene expression by mediating recruitment of cellular factors important for effective transcription elongation. In contrast, the Rev protein regulates the export of specific HIV-1 mRNA species from the nucleus, where they are transcribed, to the cytoplasm, where they are used for protein synthesis. Although these two proteins are functionally distinct, they both must bind to viral RNA elements to mediate their biological effect. As a result of our extensive work on these viral regulatory proteins, we have developed a more general interest in the study of human and viral gene regulation at the post-transcriptional level.
Although the functions of HIV-1 Tat and Rev are now well understood, a third important HIV-1 protein, Vif, had remained a mystery. This situation changed with the discovery that Vif blocks the activity of the cellular antiviral factor APOBEC3G. As a result, Vif is essential for the replication of HIV-1 in cells that express APOBEC3G, including primary human lymphocytes and macrophages. Some transformed human T cell lines do not, however, express APOBEC3G, and in these cells HIV-1 lacking Vif grows as well as the wild-type virus. APOBEC3G interferes with HIV-1 replication by specifically interacting with the HIV-1 Gag structural protein and incorporating itself into virion particles. During subsequent viral infection, APOBEC3G interferes with the essential reverse transcription step of the viral life cycle. Vif prevents this effect by binding to APOBEC3G in infected cells and targeting it for degradation.
One of our interests has been how the species tropism of HIV-1 is determined and, more specifically, why HIV-1 is unable to replicate in simian cells. We have now demonstrated that this restriction results from the inability of HIV-1 Vif to bind and degrade simian forms of APOBEC3G. Remarkably, this restriction is due to a single–amino acid difference between the human and simian versions of APOBEC3G. In recent research, we have identified two additional cellular factors, APOBEC3F and APOBEC3B (closely related to, but distinct from, APOBEC3G), that can also restrict the replication of HIV-1 as well as other, unrelated retroviruses. It therefore appears that human cells express a range of antiretroviral factors that HIV-1 has to overcome to replicate.
In the past few years, we have also become interested in areas related to the RNA interference (RNAi) phenomenon. RNAi, which probably initially evolved as an antiviral defense mechanism, allows the cell to specifically degrade mRNAs that bear sequence homology to double-stranded RNA (dsRNA) molecules introduced into the cell. Importantly, dsRNAs are not normally present in healthy cells but are critical intermediates in the replication of many viruses. Once such dsRNAs are detected by the cell, they are cleaved into short, 21- to 23-nt dsRNA fragments, termed small interfering RNAs (siRNAs), which are then incorporated into a large protein complex, the RNA-induced silencing complex (RISC). Once incorporated into RISC, the siRNAs act as guide RNAs to target RISC to mRNAs that are complementary to the siRNA, e.g., viral mRNAs, which are then cleaved by RISC. In this way, RNAi can inhibit virus replication.
Although siRNAs are normally processed from long dsRNAs, there is a second class of ~21-nt noncoding RNAs, termed microRNAs (miRNAs), that are present in all vertebrate cells examined so far and that are of endogenous origin. Specifically, miRNAs are encoded within the genome as RNA stem-loop structures of ~70 nt in length. These short, structured RNAs, which can be viewed as dsRNAs bearing a terminal loop, are processed by the same mechanism as dsRNAs to give ~21-nt noncoding RNAs that are incorporated into a protein complex that we have shown is functionally identical to RISC. Like siRNAs, these miRNAs can also inhibit mRNA expression and, in fact, miRNAs appear to play an important role in metazoan development by blocking the expression of target mRNAs at specific developmental stages. Nearly 300 human miRNAs have been described, and the elucidation of their various roles will be of considerable interest.
A key step in the biogenesis of miRNAs is the export of the hairpin RNA precursor from the nucleus to the cytoplasm, where the precursor is processed into a form that can be incorporated into RISC. Our lab has demonstrated that this nuclear export is mediated by the cellular factor Exportin 5, which plays an essential role in miRNA function. Moreover, Exportin 5 is expressed at low levels in human cells, so that nuclear export has emerged as a key rate-limiting step in miRNA production. We are continuing to examine miRNA biogenesis and function and hope to use our insights to design effective mechanisms for the regulation of human and viral gene expression.
Last updated May 20, 2004
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