Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

UNDERSTANDING THE MOLECULAR MACHINERY OF THE L PROTEIN FROM EBOLA VIRUS (#154)

Raphael Caballes 1 , Claire Dickson 1 , Nan Li 1 , David A Jacques 1
  1. University of New South Wales, Kingsford, NSW, Australia

Since the emergence of the Sudan and Zaire Ebola virus (EBOV) strains in the 1970s, it has caused recurring epidemics throughout Africa. EBOV infection leads to fatal haemorrhagic fever with varying mortality rates from 25%-90%, with the Zaire strain proving more fatal. Ebola is a Mononegavirus (negative-sense RNA virus), from the Filoviridae family. The viral genome encodes 7 genes that result to 10 proteins, including the RNA-dependent RNA Polymerase (RdRP) known as the L protein and its P protein cofactor, VP35. The huge L protein (~250 kDa) is crucial in the viral life cycle. Protein L has several domains with distinct functions that allows it to perform transcription and replication of the viral genome. The RdRP domain performs the elongation and transcription of the viral RNA genome, the capping domain attaches a GTP cap to the nascent mRNA transcript, the methyltransferase domain methylates the cap at the 2’-O and N-7 positions of the growing viral mRNA, and the C-terminal domain assists in methylation efficiency. The structures of the RdRP and capping domains of L bound to a tetrameric VP35 of EBOV Zaire had been recently published, but the structures of the other domains of EBOV Zaire L protein remain unknown. Also, the mechanistic switching of L from transcription to replication is still poorly understood. This project aims to structurally characterize the methyltransferase and C-terminal domains (MTCTD) of L to try and understand how these domains undergo conformational change. To view these phenomena, we intend to capture RNA-bound and -unbound states of the MTCTD. AlphaFold was implemented to predict the MTCTD region. The conserved motifs and enzymatic centres for these domains had been placed appropriately. Expression of the MTCTD was done successfully using HEK293T cells. The MTCTD will be subjected for further downstream analyses for methylation activities and attempts on solving its structure.

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