NMR assignment of the arenaviral protein Z from Lassa fever virus

Abstract

The arenavirus protein Z from Lassa fever virus was recently found to inhibit mRNA translation through direct interaction with eIF4E. Here, we report the NMR assignment of this RING-containing protein that was determined by triple resonance NMR techniques. Keywords LFV-Z; RING domain; Heteronuclear NMR; translational repressor Biological contextMany arenaviruses cause fatal human hemorrhagic fevers. Infection by Lassa fever virus (LFV) or lymphocytic choriomeningitis virus (LCMV), two members of this arenavirus family, was studied in NIH3T3 cells by indirect immunofluorescence (Borden et al. 1998). The 11kDa arenavirus protein Z was found to directly bind the promyelocytic leukemia (PML) protein, an important regulator of mammalian cell growth. Upon infection, Z causes the relocation of PML nuclear bodies to the cytoplasm. This is likely a means to evade host cell apoptosis, given that PML promotes apoptosis. Also, the purified Z protein inhibits mRNA translation in reticulocyte lysates through direct interaction with the translation initiation factor 4E (eIF4E) (Kentsis et al. 2001). In eukaryotes, eIF4E recognizes the 5'-terminal cap structure of the mRNA, and recruits the large scaffold factor eIF4G to the mRNA. This mRNA-protein complex then binds the 40S preinitiation complex. In contrast to eIF4G and the 4E binding proteins (4E-BPs) that increase eIF4E affinity for the cap, Z was shown to decrease this affinity. Interestingly, all of these regulatory proteins bind the dorsal surface of eIF4E. Also, eIF4G and the 4E-BPs interact with eIF4E through a conserved α-helical recognition motif (YXXXXLϕ, where X is variable and ϕ is a hydrophobic residue), while Z does not contain this motif. Instead, the Z protein contains a RING domain. The RING is a ~60-residue zinc-binding motif that usually uses conserved cysteines and a histidine to bind two zinc atoms. As a first step in providing the structural basis of translation repression by Z, we have undertaken NMR studies and report here the NMR assignments of LFV-Z. Understanding the structure of Z and its interactions with host cell proteins such as PML and eIF4E, may lead to novel therapeutic strategies for Lassa fever infection.Further, Z is used as a model RING protein to study the self-assembly properties of these small domains. For instance, Z assembles into structures visible by electron microscopy (Kentsis et al. 2002a). Such super-molecular assembly of RING domains enhances their biochemical activities e.g. BRCA1/BARD1 and Mdm2 become more efficient E3 ligases when assembled while the Z bodies better inhibit the cap binding activity of eIF4E than Z monomers (Kentsis Correspondence to: Laurent Volpon, laurent.volpon@umontreal.ca. et al. 2002b; Poyunoskvy et al. 2007). Thus, NMR studies could yield molecular insights into these assembly processes. NIH Public Access Methods and experimentsThe full-length cDNA coding for LFV-Z was cloned into pGEX6p-1 (gift from Dr de la Torre and Althea A. Capul, Scripps Research Institute, La Jolla, California) and w...

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“…All constructs were verified by sequencing. (33). Three-dimensional 15 N-edited and 13 C-edited NOESY spectra (100 ms mixing time) were acquired at 600 and 800 MHz.…”

Conserved Residues in Lassa Fever Virus Z Protein Modulate Viral Infectivity at the Level of the Ribonucleoprotein

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