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Scientific Report 2008


Molecular Biology




RNA-Protein Complexes Mediating Nuclear Transport of HIV mRNAs


J.R. Williamson, F. Agnelli, W. anderson, A. Beck, C. Beuck, A. Bunner, A. Carmel, S. Chen, S., Edgcomb, D. Kerkow, S. Kwan, E. Menichelli, W. Ridgeway, G. Ring, H. Schultheisz, Z. Shajani, E. Sperling, M.T. Sykes, B. Szymczyna, J. Wu

Development of novel therapeutic strategies against HIV infection is a pressing need and requires elucidation of the fundamental mechanisms of HIV replication. Currently prescribed anti-HIV drugs inhibit the viral protease, viral reverse transcriptase, or viral integrase, but these enzymes are only a small fraction of the viral proteins responsible for some key steps in viral replication. To develop new therapeutic strategies, we need to understand additional steps in viral replication and to identify new potential targets.

Rev is an essential HIV protein that is required to mediate transport of HIV viral mRNAs from the nucleus, where they are transcribed, into the cytoplasm, where they are either translated or packaged into new virions. Early in infection, viral mRNAs are fully processed in the nucleus and exported to the cytoplasm, where several small regulatory proteins, including Rev, are synthesized. The Rev protein itself is imported back into the nucleus, where it interacts with newly transcribed viral mRNAs at an RNA structure called the Rev responsive element (RRE; Fig. 1). Binding of Rev to the RRE directs mRNAs from the nucleus to the cytoplasm before full RNA processing takes place. These longer mRNAs code for the structural proteins necessary to assemble a new virus. Thus, binding of Rev to the RRE changes the pattern of gene expression from production of the early regulatory genes to production of the late structural genes, and this binding is therefore a potential point of therapeutic intervention.


Fig. 1. Nuclear transport of HIV Rev. The Rev protein shuttles in and out of the nucleus by sequentially interacting with a series of human factors in 3 key complexes. First, an import complex is formed by association of Rev (yellow) with the nuclear transport factor importin-β (blue). Once in the nucleus, Rev forms an oligomeric RNA complex by binding to the RRE RNA. Multiple copies of Rev bind to the RRE to promote efficient export, but the details of the oligomeric structure are not known. Rev recruits the nuclear transport factor CRM-1 (orange), which facilitates transport of the Rev-RRE complex back to the cytoplasm. Other factors associate with this complex, including the helicases DDX1 and DDX3, to form the export complex. DDX1 (red) interacts directly with Rev, whereas DDX3 (blue) interacts with Rev by indirect binding to CRM-1. The helicases may dissociate proteins from the viral RNA after export to the cytoplasm (lower right) to facilitate translation of the viral mRNA or packaging of the viral genome. Each of these complexes may be a new target for intervention against HIV.

Several human cellular proteins act in concert with Rev to mediate the nuclear transport of viral mRNAs. In collaboration with L.R. Gerace and J.R. Yates, Department of Cell Biology, using a proteomic method to investigate Rev-RRE—associated factors, we identified dozens of such potential cofactor proteins. In particular, we discovered several so-called DEAD-box helicases, named after the conserved sequence signature, that associate with Rev during viral mRNA transport. The helicase DDX1 is thought to associate with Rev while Rev is bound to the RRE RNA in the nucleus; the helicase DDX3 interacts indirectly with Rev through the transport protein CRM-1.

The presence of helicases in the ribonucleoprotein complex for viral mRNA export raises some interesting questions about how Rev functions. Helicases are ATP-dependent motors that can unwind duplex RNAs or displace proteins from RNA-protein complexes. Possibly these helicases play critical roles in either assembling the proper RNA complex for nuclear transport or in disassembling the complex in the cytoplasm to allow translation or packaging of the virus.

We are using biochemical and structural biology approaches to investigate the interactions of Rev with the helicases DDX1 and DDX3. The normal human substrates for these helicases are not known, and we must develop binding and functional assays based on Rev and Rev-RRE complexes. The protein-protein interactions are monitored by using fluorescence assays or isothermal titration calorimetry; formation of RNA-protein complexes is monitored by using polyacrylamide electrophoretic mobility shift assays. In addition, we are developing helicase ATPase assays with a variety of substrates to determine how the helicase activity is modulated in the presence of Rev and RRE. Finally, we are working toward structure determination of protein-protein and protein-RNA complexes to understand the molecular basis for this interaction.

Our results will provide the basis for understanding potentially new targets for antiviral therapy. In addition, although helicases are widespread in human cells, the authentic substrates for these enzymes are known in only a few instances. Studying of Rev as a cargo for nuclear transport and as an authentic substrate for helicases will provide important insights into helicase function. The biochemical and structural work may lead to assays for the discovery of inhibitors of Rev function by a novel mechanism with therapeutic potential.

Publications

Edgcomb, S.P., Aschrafi, A., Kompfner, E., Williamson, J.R., Gerace, L., Hennig, M. Protein structure and oligomerization are important for the formation of export-competent HIV-1 Rev-RRE complexes. Protein Sci. 17:420, 2008.

Hennig, M., Scott, L.G., Sperling, E., Bermel, W., Williamson, J.R. Synthesis of 5-fluoropyrimidine nucleotides as sensitive NMR probes of RNA structure. J. Am. Chem. Soc. 129:14911, 2007.

Naidoo, N., Harrop, S.J, Sobti, M., Haynes, P.A., Szymczyna, B.R., Williamson, J.R., Curmi, P.M., Mabbutt, B.C. Crystal structure of Lsm3 octamer from Saccharomyces cerevisae: implications for Lsm ring organisation and recruitment. J. Mol. Biol. 377:1357, 2008.

Sperling, E., Bunner, A.E., Sykes, M.T., Williamson, J.R. Quantitative analysis of isotope distributions in proteomic mass spectrometry using least-squares Fourier transform convolution. Anal. Chem. 80:4906, 2008.

Vallurupalli, P., Scott, L., Williamson, J.R., Kay, L.E. Strong coupling effects during X-pulse CPMG experiments recorded on heteronuclear ABX spin systems: artifacts and a simple solution. J. Biomol. NMR 38:41, 2007.

 

James R. Williamson, Ph.D.
Professor
Dean, Kellogg School of Science and Technology



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