New findings in research led by Purdue University biologists provide further evidence that the protein envelope
protecting DNA in viruses evolved billions of years ago from a common ancestor and uses the same basic protein "fold" to
construct the critical outer shell.
The most recent findings, which appear in the current issue of the Proceedings of the National Academy of Sciences, show that
the T4 virus has a similar protein fold in its outer shell, or capsid, as another virus called HK97.
The protein fold of the viral envelope is crucial for the assembly of the capsid, which protects DNA vital to a virus'
ability to infect host organisms and reproduce. The T4 and HK97 viruses are called bacteriophages because they infect
bacteria.
"In the case of the T4 virus, the capsid is made of interconnected six-sided structures called hexamers, which link together
in a honeycomb-like pattern to form a very thin wall that is incredibly stable," said Michael Rossmann, the Hanley
Distinguished Professor of Biological Sciences in Purdue's College of Science. "The capsid surrounds and protects the virus'
DNA, and you can think of it as a balloon that doesn't burst even though you are pumping in more and more air.
"It has now been shown that many viruses have derived their protein envelope from a common ancestor."
The research was performed by Andrei Fokine and Petr Leiman, postdoctoral researchers in Rossmann's laboratory; Mikhail M.
Schneider, in the laboratory of Vadim V. Mesyanzhinov in the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry in
Russia; Bijan Ahvazi and Karen M. Boeshans, in the laboratory of Alasdair C. Steven at the National Institute of Arthritis
and Musculoskeletal and Skin Diseases in Bethesda, Md.; and Lindsay W. Black, from the University of Maryland School of
Medicine in Baltimore.
The findings were posted online May 6.
"This work marks only the second time researchers have shown in atomic detail that different bacteriophages have a similar
fold," Rossmann said. "That's because we and the original discoverers of the special fold in HK97, Jack Johnson and
colleagues from the Scripps Research Institute in La Jolla, Calif., used X-ray crystallography, which leaves no doubt about
the atomic structure."
Capsids contain proteins, which are made of a string of building blocks called amino acids. Proteins fold into specific
shapes, depending mostly on the sequence of amino acids.
"If capsid folds have similar structures and amino acid sequences, then they probably have evolved from a common ancestor,"
Rossmann said.
Purdue structural biologists led by Rossmann wrote a paper published last month announcing they had determined that the phi
29 virus, which attacks the soil bacterium bacillus subtilis, possesses a protein fold in its capsid similar to the fold
found in the HK97 virus, which infects E. coli bacteria.
Researchers used a technique called cryoelectron microscopy to determine the three-dimensional structure of phi 29 down to a
resolution of 7.9 angstroms. An angstrom is one ten-billionth of a meter, or roughly one-millionth as wide as a human hair.
The new paper details how the researchers used X-ray crystallography to analyze the structure of a protein called gp24, which
is a key part of the capsid fold in the T4 virus. X-ray crystallography yields more precise results because it is capable of
higher resolution than cryoelectron microscopy. The researchers used X-ray crystallography to view the gp24 protein at a
resolution of 2.9 angstroms.
The X-ray crystallography data confirmed that gp24 has a fold similar to that of the HK97 capsid protein and also revealed
the structure of an "insertion domain," or a portion of gp24 that may play a role in connecting adjacent subunits to assemble
the capsid. Finding the gp24 structure also enabled the researchers to deduce the structure of another protein, gp23, which
is crucial along with gp24 in forming the hexamer segments of the capsid.
Researchers are studying the T4 virus for many fundamental biological studies, some of which might be used to create new
vaccines to fight viral infections in humans.
"Many people have talked about using T4 or other bacteria viruses as antibiotics," Fokine said. "Although we are conducting
research regarding T4's potential vaccine applications, that is not a focus of the work being reported in this paper."
The T4 capsid is made up of five proteins, which come together like the pieces of a jigsaw puzzle.
One of the proteins in the T4 capsid is called a highly antigenic outer capsid protein, or hoc. Proteins that mimic proteins
from dangerous viruses might be attached to this hoc protein. Then the altered T4 virus could be administered as a vaccine,
prompting an immune response against the harmful viruses.
In collaboration with V. Rao of the Catholic University of America in Washington, D.C., Fokine and Rossmann are conducting
research aimed at using the T4 capsid as a vaccine for anthrax.
To study the structure of gp24, the researchers first turned the protein into a crystal form and then analyzed it with X-ray
crystallography, a technique in which X-rays pass through a crystal, creating a "diffraction pattern" that can be interpreted
with various computational procedures.
"Once we determined the structure of gp24, we were able to figure out the shape of gp23 because we know they have a similar
structure from the similarity of their amino acid sequences," Rossmann said.
The researchers needed to know the structures of both gp23 and gp24 before they could learn the overall organization of the
T4 capsid.
Other scientists, Wah Chiu and his colleagues at Baylor School of Medicine in Houston, had previously found that another
bacteriophage, called P22, contains a similar capsid fold as HK97. The Purdue team has since discovered the same fold in phi
29 and the T4 virus, meaning four bacteriophages have been found to possess the fold, bolstering evidence that the fold is
common among bacteriophages.
"One fact that makes the T4 research especially interesting is that it is a much larger virus than HK97, P22 and phi29, so
the T4 virus is the first large virus to be shown to have a similar fold in its capsid protein, suggesting the fold is likely
a common feature in bacteriophages," Fokine said.
The findings agree with other work in which scientists have discovered that seemingly unrelated viruses that infect mammals
use a similar capsid fold.
The research has been funded by the National Science Foundation and the Human Frontier Science Program.
Writer: Emil Venere, (765) 494-4709, venerepurdue
Sources: Michael Rossmann, (765) 494-4911, mgrindiana.bio.purdue
Andrei Fokine, (765) 494-4925, afokinepurdue
Purdue News Service: (765) 494-2096; purduenewspurdue
Related Web site:
Michael Rossmann: biology.purdue/people/faculty/rossmann/index.htm
A publication-quality graphic is available news.uns.purdue/images/+2005/rossmannT4.jpg.
ABSTRACT
Structural and functional similarities between the capsid proteins of bacteriophages T4 and HK97 point to a common
ancestry
Andrei Fokine†‡, Petr G. Leiman†‡, Mikhail M. Schneider§, Bijan Ahvazi, Karen M. Boeshans, Alasdair C. Steven№№, Lindsay W.
Black‡‡, Vadim V. Mesyanzhinov§, and Michael G. Rossmann.†
†Department of Biological Sciences, Purdue University, West Lafayette, IN
§Laboratory of Molecular Bioengineering, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia
X-Ray Crystallography Facility/National Institute of Arthritis and Musculoskeletal and Skin diseases, Bethesda, MD
№№Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda,
MD
‡‡Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD
‡These authors made equal contributions to this work
Gene produce (gp) 24 of bacteriophage T4 forms the pentameric vertices of the capsid. Using X-ray crystallography, we found
the principal domain of gp24 to have a polypeptide fold similar to that of the HK97 phage capsid protein plus an additional
insertion domain. Fitting gp24 monomers into a cryo0EM density map of the mature T4 capsid suggests that the insertion domain
interacts with a neighboring subunit, affecting a stabilization analogous to the covalent crosslinking in the HK97 capsid.
Sequence alignment and genetic data show that the folds of gp24 and the hexamer-forming capsid protein, gp23*, are similar.
Accordingly, models of gp24* pentamers, gp23* hexamers, and the whole capsid were built, based on a cryo-EM image
reconstruction of the capsid. Mutations in gene 23 that affect capsid shape mape to the capsomer's periphery, whereas
mutations that allow gp23 to substitute for gp24 at the vertices modify the interactins between monomers within capsomers.
Structural data show that capsid proteins of most tailed phages, and some eukaryotic viruses, may have evolved from a common
ancestor.
Contact: Emil Venere
venerepurdue
765-494-4709
Purdue University
purdue
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