Arch Virol (2004) 149: 699–712
Structural characterization of Tobacco etch virus
coat protein mutants
A. E. Voloudakis
, C. A. Malpica
, M.-E. Aleman-Verdaguer
D. M. Stark
, C. M. Fauquet
, and R. N. Beachy
Department of Cell Biology, The Scripps Research Institute,
La Jolla, California, U.S.A.
Donald Danforth Plant Science Center, St. Louis, Missouri, U.S.A.
Received June 4, 2003; accepted October 2, 2003
Published online December 8, 2003
Summary. The assembly of Tobacco etch potyvirus (TEV) coat protein (CP) and
truncated mutants in Escherichia coli was studied. CP from which 28, 63 or 112
amino acids were deleted from the N-terminus polymerized into potyvirus-like
particles (PVLPs). These structures were more rigid and progressively smaller
in diameter than those produced by full length TEV-CP. CP from which 175 N-
terminal amino acids were removed, failed to polymerize. A fragment containing
amino acids 131 to 206 of TEV-CP is sufﬁcient for PVLP assembly in E. coli.
To determine the function of the highly conservedamino acids Ser152,Arg154,
and Asp198 point mutants were generated. The mutant CP63(Asp198Glu) ex-
hibited different spectral properties following circular dichroism analysis showing
a lower amount of α-helix compared to the wild type molecule. No differences
were observed in spectra obtained from ﬂuorescence spectroscopy. The point
mutants bind RNA in vitro to the same degree as the wild type protein. However,
while the wild type and theArg154Gln mutant CP were each able to form PVLPs in
E. coli, the Asp198Glu and the double mutant Ser152Pro/Arg154Gln mutants did
not. These results suggest that the Asp198Glu mutation has an altered secondary
structure which affects the capacity of the protein to polymerize but did not affect
in vitro protein-RNA interactions.
The potyviruses form the largest family of plant pathogenic viruses and infect
a wide variety of host plants. Potyviruses have ﬂexuous, rod-shaped particles,
measuring 12 to 15 nm in diameter and 700 to 900 nm in length. Their genomes
These authors contributed equally to the present work.