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Enteroviruses such as poliovirus and coxsackievirus shut off cap-dependent protein synthesis (translation) within 2 hours of infection while allowing cap-independent translation of mRNAs containing IRES elements to continue. Cap-dependent translation is used to produce 96% of all cellular proteins, and the loss of protein homeostasis in the cell is a major cause of cell death.

We have shown that enteroviral 2A protease (2Apro) and 3C protease (3Cpro) are viral proteins required for translation shutoff. 2Apro cleaves the translation initiation factor eIF4G. eIF4G is the central protein involved in initiation of cap-dependent translation since it binds to the 5´ end of capped mRNA and provides a molecular bridge that binds mRNA to 40S ribosomal subunits. Cleavage destroys its ability to function in cap-dependent translation initiation. We have also discovered that enterovirus 2A pro and 3C pro both cleave poly(A)-binding protein (PABP) in infected cells. The cleavage site for both proteases lies in the C-terminal domain of PABP in a region whose function has not been determined. This region somehow regulates ribosome termination, re-initiation and mRNA stability, but these important mechanisms are only beginning to emerge. Thus, the virus attacks proteins which binds to both ends of mRNA, supporting the importance of 5´-3´ interactions that circularize mRNA in translation regulation. Work is continuing to determine the precise function of 5´-3´ interactions and the role of PABP and eIF4G in regulation.

We have also recently learned that poliovirus 3Cpro causes cleavage of a protein called G3BP. G3BP is responsible for nucleating the formation of large mRNP structures in cells called Stress Granules. Stress Granules are thought to be a structure where translationally silenced mRNAs assemble and reside until either signals place the mRNA back into polysomes, or conversely, the mRNAs are transferred to adjacent particles called P-bodies, where they are degraded. Translational silencing by microRNAs is thought to regulate expression of about 50% of human genes, and may proceed by this stress granule pathway. We are performing experiments to discern why enteroviruses need to disrupt stress granule formation and how G3BP functions.

During the initiation of apoptosis or transformation leading to cancer in human cells there are large changes in the types of mRNA species which can bind ribosomes and be translated into proteins. We have elucidated changes in translation factor function and composition that occur during these processes that in turn control gene expression in cells.  This work is now expanding into investigation of eIF3e, which dually helps regulate translation of many proteins and also regulates proteasome function and activity.

IRES (internal ribosome entry site) elements are found on mRNAs of many important regulatory proteins including c-myc and VEGF. IRES elements confer altered regulation of translation on mRNA, allowing specific up or down regulation. There are many classes of IRES elements emerging which function quite differently. We have discovered several key apoptosis regulatory proteins whose mRNAs contain IRES elements. Several of these elements (cIAP-1, Bcl-2) have been cloned and are being studied to characterize their mechanisms of action and how they regulate expression of apoptotic factors. The complex changes in translation regulation involving these factors in early phases of apoptosis may control the outcome of the decision to carry out cell death. In addition, we are characterizing control of expression of eIF4GI itself. We have found that eIF4GI exists as five different isoforms. Our data indicates that alternate splicing, use of different promoters, use of at least 2 types of IRES elements, alternate AUG selection and other translation control mechanism all are used to control the abundance and type of eIF4GI isoform which is expressed in cells. We are investigating this complex regulation scheme and how individual eIF4GI isoforms may differentially regulate translation of other proteins.

Recent Publications (PubMed)

Kuyumcu-Martinez NM, Van Eden ME, Younan P, Lloyd RE. (2004) Cleavage of Poly(A)-Binding Protein By Poliovirus 3C Protease Inhibits Host Cell Translation: A Novel Mechanism for Host Translation Shutoff. Mol. Cell Biol. 24: 1779-90.

Kuyumcu-Martinez M, Belliot G, Sosnovtsev SV, Chang KO, Green KY, Lloyd RE. (2004) The calicivirus 3CL proteinase inhibits cellular translation by cleavage of poly(A)-binding protein. J.Virol. 78: 8172-8182

.Sherrill K, Van Eden ME, Lloyd RE. (2004) Bcl-2 translation is mediated via internal ribosome entry during cell stress. J. Biol. Chem. 279: 29066-29074.

VanEden ME, Byrd MP, Sherrill K,Lloyd RE. (2004) Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress. RNA 10: 469-481.

Byrd, M.P., M. Zamora and Lloyd RE. (2005) Translation of eukaryotic translation initiation factor 4GI (eIF4GI) proceeds from multiple mRNAs containing a novel cap-dependent IRES that is active during poliovirus infection. J. Biol. Chem. 280: 18610-22.

Lloyd RE. (2006) Translational control by viral proteinases. Virus Res. 119:76-88.

White, J., A.M. Cardenas, W.E. Marissen and R.E. Lloyd.  (2007) Inhibition of cytoplasmic mRNA stress granule formation by a viral proteinase.   Cell Host & Microbe 2: 295-305.

Bonderoff, J.M. and R.E. Lloyd. 2008.  CVB Translation: Lessons from the polioviruses. p123-147.In Current Topics in Microbiology and Immunology: The Group B Coxsackieviruses.  S. Tracy, S. Oberste and K. Drescher eds., Springer-Verlag.

Sherrill, K.W., M.P. Byrd and R.E. Lloyd.  2008. Translation of the transcript encoding cIAP2 is mediated exclusively by a stress-modulated ribosome shunt. Mol. Cell. Biol. 28:2011-2022.

Rivera, C. and R.E. Lloyd.  2008.  Modulation of enteroviral proteinase cleavage of poly(A)-binding protein (PABP) by PABP-associated factors. Virology 375: 59-72.

de Breyne, S.,  J.M. Bonderoff, K.M. Chumakov, R.E. Lloyd, and C.U.T. Hellen. 2008.  Cleavage of eukaryotic initation factor eIF5B by enterovirus 3C proteases. Virology, in press.