Research summary:

RNA is unique: it can encode genetic information and also perform a myriad of other biological tasks. This multi-functionality is conferred, in part, by the ability of RNA to fold into specific and dynamic three-dimensional structures that often interact with other components of the cell. The diversity of RNA function is particularly useful to viruses, which tend to have genomes that are much smaller than cell-based life and have evolved elegant, subtle, and yet often complex ways to use RNA as part of their infection strategy.

My research focuses primarily on the structure and function of RNA molecules from viruses. Many of these viral RNAs manipulate the host cell’s biological machinery through direct intermolecular interactions that are poorly understood. By studying these RNAs and the processes they drive, we learn several things: 1) How the virus operates; 2) How the basic cellular machinery operates; 3) How we might block or inhibit viruses; and 4) The basic rules of RNA and RNA-protein complex structure and function. We explore these systems with a variety of methods, to include X-ray crystallography (Figure 1), functional assays, cell-based approaches, biochemistry, and biophysics.



Specific projects that my lab is pursuing include:

Viral IRES RNAs: molecular hijackers. Certain viruses recruit, position, and activate a host cell’s ribosomes by a process that does not require the mRNA to be capped, but rather is driven by a structured RNA called an internal ribosome entry site (IRES). IRESs often operate using far fewer protein factors than are needed by the canonical cap-dependent mechanism, and this raises an interesting question: How can RNA structure functionally replace the cap and many protein factors, and in so doing “hijack” the host cell’s ribosomes? Using a combination of structural, biochemical, biophysical, and cell-based approaches we are trying to understand the mechanism of IRESs from viruses as diverse as HIV-1, the hepatitis C virus (HCV), and the intergenic region (IGR) of the Dicistroviridae.

In one project, we are using in vitro and cell-based assays to understand how the 5’ leader of the full HIV-1 RNA in involved in translation of viral proteins. Previous reports have identified this leader as a cell-cycle dependent IRES RNA; we now seek to describe the detailed molecular mechanism that lies behind this observed regulation.

In addition, we are exploring the mechanism by which the HCV IRES is able to manipulate the host cell’s translation machinery, with a particular interest in the steps involved in forming 80S ribosomes on the IRES RNA, activating those ribosomes for protein synthesis, and the structural changes in the IRES RNA that accompany these steps. Our strategy is to use functional, biochemical, and structural studies to build a detailed model of HCV IRES function.

In another project, we solved the complete structure of an IGR IRES (Figure 2) and then using published cryo-EM reconstructions we have proposed a comprehensive structure-based model for the mechanism of this IRES that includes the idea of tRNA P/E hybrid state mimicry. Now, we are testing this model using methods such as single-molecule FRET and continued biochemical and structural approaches.



Structure and function of the ciRNA, an antiviral countermeasure. Our collaborator, Dr. David Barton, recently discovered a sequence in the protein-coding portion of the poliovirus (and other group C enteroviruses) RNA genome that is a competitive inhibitor RNA (ciRNA) of RNase L. RNase L is part of the cell’s normal interferon-induced antiviral pathway and hence the ciRNA is an “antiviral countermeasure.” We are exploring the three-dimensional structure of the ciRNA,its interactions with the endonuclease domain of RNase L, and also trying to locate ciRNAs in other viruses (and perhaps cellular organisms). These studies give us insight into how structured viral RNAs can manipulate a host cell’s machinery, and also how a single RNA sequence evolved under two independent pressures: to encode genetic information to inhibit an RNase.

Multifunctional tRNA-like structures. Certain economically important plant-infecting RNA virus genomes become aminoacylated at their 3’ ends by the host cell’s aminoacyl tRNA- synthetases (AARSs) (Figure 3). This reaction depends on RNA sequences called “tRNA-like structures” (TLSs) that reside at the 3’ end of the genome and that can have sequences and secondary/tertiary structures that differ substantially from authentic tRNAs. TLSs interact with AARSs and other proteins to include the viral RNA-dependent RNA polymerases (RDRP). It has been proposed that TLSs regulate several steps in the viral replication cycle, raising the question: What are the three-dimensional structures and structural dynamics that allow this multifunctionality? How are structurally divergent TLSs able to recruit and manipulate the host cells AARSs? Using biophysical methods such as small-angle X-ray scattering combined with biochemical assays and X-ray crystallography, we are seeking a deeper understanding of these virally-encoded molecular mimics with the long-term goal of understanding how viral RNAs manipulate the cellular machinery (Figure 3).



RNA-peptide complexes. A long-term goal of RNA structural biologists is to inform the design of small-molecule drugs that target RNA. Attempts to rationally design new drugs that target RNA has not yet come to fruition. To inform structure-based drug design efforts, my lab is studying the interactions between small amphiphilic peptides with modified side-chains (designed by Prof. Jaehoon Yu) and RNA targets from HIV-1 and HCV. By solving the structures of these peptide-RNA complexes by X-ray crystallography, our goal is to better understand how different chemical moieties can interact specifically with RNA, and then use this information to guide future rational design of small molecules.

Discovery of new RNA-protein complexes in viral infections. During infection, viral proteins often bind to cellular RNAs, and viral RNAs can bind to cellular proteins. These interactions are critical to the virus, but in even the best-studied viruses we have identified probably only a subset of these interactions. To address this gap, we are identifying new RNA-protein complexes that occur during certain viral infections. We are interested in interactions involving cellular proteins and those involving virally-encoded proteins. Once identified, our long-term task is to biochemically, functionally, and biophysically characterize these RNA-protein complexes and to use this information to answer specific questions of how each virus operates in the cell.

Selected recent publications from the Kieft Lab

Hammond. J.A.,  Rambo, R.P., Filbin. M.E.,& Kieft, J.S. (2008). Comparison and functional implications of the 3-D architectures of viral tRNA-like structures. RNA (in press).

Costantino, D.A., Pfingsten, J.S., Rambo, R.P., & Kieft. J.S. (2008) tRNA-mRNA mimicry drives translation initiation from a viral IRES. Nat. Struct. Mol. Biol.15, 57-64.

Keel, A.Y., Rambo. R.P., Batey, R.T., & Kieft, J.S. (2007) A general method to solve the phase problem in RNA crystallography. Structure 15, 761-772.

*Batey, R.T. & *Kieft, J.S. Improved native affinity purification of RNA (2007) RNA, 13, 1384-1389.
*These authors contributed equally to this work

Pfingsten, J.S., Costantino, D.A & Kieft, J.S. (2007) Conservation and diversity among the three-dimensional folds of the Dicistroviridae intergenic region IRESes. J. Mol. Biol., 370, 356-369.

Pfingsten, J.S., Costantino, D.A., & Kieft, J.S. (2006) Structural basis for ribosome recruitment and manipulation by a viral IRES. Science, 314, 1450-1454.

Costantino, D. & Kieft, J.S. (2005). A preformed compact ribosome-binding domain in the cricket paralysis-like virus IRES RNAs. RNA 11, 332-343.

*Kieft, J.S. & *Batey, R.T. (2004). A general method for rapid and nondenaturing purification of RNAs. RNA 10, 988-995.  
*These authors contributed equally to this work

 Recent reviews articles and book chapters from the Kieft Lab

Filbin, M.E. & Kieft, J.S. (2009) Towards a structural understanding of IRES RNA function. Curr Opin Struct Biol 19, 267-276

Kieft, J.S. (2009) Comparing the three-dimensional structures of Dicistroviridae IGR IRES RNAs with other viral RNA structures. Virus Res. 139, 148-156. (Coeditor of this edition).

Kieft, J.S. Biophysical analysis of IRES RNAs from the Dicistroviridae: linking architecture to function. Springer Series in Biophysics, 13: 317-334.

Kieft, J.S. (2008) Comparing the three-dimensional structures of Dicistroviridae IGR IRES RNAs with other viral RNA structures. Virus Res. Epub ahead of print, PMID: 18672012 (Coeditor of this edition)

Kieft, J.S.  (2008) Viral IRES RNA structures and ribosome interactions. TiBS, 33, 274-283.

Pfingsten, J.S. & Kieft, J.S. (2008) RNA structure-based translation initiation: Lessons from the Dicistroviridae intergenic region IRESs.  RNA, 14, 1255-63.

Kieft, J.S., Costantino, D.A., Filbin, M.E., Hammond J., and Pfingsten, J.S. (2007) Structural methods for studying IRES function. Methods in Enzymology,  430, 333-371.

Kieft, J.S. & Pfingsten, J. (2005) Weapons in the molecular arms race (News and Views). Nat. Struct. Mol. Biol. 12, 938-939.