Scientific Report 2008
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
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
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.
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.
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.
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.