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Studies of Severe Acute Respiratory Syndrome Virus and Other Coronaviruses
B. Neuman, R. Burrer, A. Kim, J. Abma, J. Meisner, A. Paulino, M.J. Buchmeier
We used a novel method of delivery to determine the sequence-specific antiviral efficacy of peptide-conjugated
morpholino antisense oligomers against mouse hepatitis virus and severe acute respiratory syndrome–associated
coronavirus (SARSCoV). Specific antisense activity designed to block translation of the viral
replicase polyprotein was first confirmed by reduction of luciferase expression from a target
sequence containing a reporter construct in both cell-free and transfected cell culture assays.
Peptide-conjugated morpholino oligomers had low toxicity in DBT astrocytoma and Vero-E6 cells.
Micromolar concentrations of oligomers were delivered to more than 80% of cells and inhibited
viral titers by 10- to 1000-fold in a sequence-specific and dose-response manner.
We tested 7 mechanistically different approaches to inhibiting SARSCoV with antisense morpholino oligomers, in addition to chemical
and coiled-coil fusion inhibiting treatments. Synthesis of viral protein and viral growth, spread,
and cytopathic effects were significantly reduced by several compounds. Conjugation of a peptide
related to the HIV type 1 Tat was a novel method for delivery of antisense morpholino oligomers.
Inhibition of virus infectivity by peptide-conjugated morpholino oligomer was comparable to
the antiviral activity of the aminoglycoside hygromycin B used at a concentration 5- to 10-fold
higher than that of the oligomer. These results suggest that this composition of antisense compound
may be therapeutic for control of coronavirus infection.
We proposed a model based on electron cyromicroscopy (Fig. 1) for the structure of SARSCoV.
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| Fig. 1.
Electron cryomicrographs of SARSCoV. Purified virus was frozen in vitreous ice and
imaged in an electron microscope by R. Milligan, Department of Cell Biology. The
images reveal an enveloped virion approximately 83 nm in diameter surrounded by
a fringe of glycoprotein spikes projecting approximately 17 nm into the medium.
On the basis of these images, a reconstruction of the virion structure and organization
is in progress. |
Coronaviruses are pleomorphic, with a gaussian distribution
of particle diameters around a mean of 82.0 ±
1.0 nm, and package a single copy of the 30-kb genome per virion. Virions contain the surface attachment
and fusion transmembrane glycoprotein spike, a triple-pass integral membrane glycoprotein,
the viral nucleoprotein, and trace amounts of a small hydrophobic budding factor. Electron cryomicroscopy
and image analysis of SARSCoV and a murine coronavirus revealed concentric paracrystalline shells
of density near the virion surface. The internal scaffold layers were arranged in 3 types of orthorhombic
units. The main structural proteins were identified in refined images on the basis of position,
predicted size, and copy number. We characterized the structure of the 300-Å transmembrane
protein complex surrounding the viral spike protein oligomer. Coronaviruses have a novel architecture
that resembles the organization of retrovirus and arenavirus particles.
Arenavirus Structure, Function, and Immunology
J. Botten, B. Neuman, A. Saunders, E. Burke, B. Adair, A. Kim, J. Abma, A. Paulino, J. Meisner, M. Yeager, M.J. Buchmeier
Arenaviruses are rodent-borne agents of various diseases, including potentially lethal human hemorrhagic
fevers. Arenavirus particles are pleomorphic, ranging from 44 to about 500 nm in diameter, and
contain variable numbers of a bisegmented ambisense single-stranded RNA genome. Electron cryomicroscopy
and image analysis of New World Pichinde and Tacaribe arenaviruses and Old World lymphocytic choriomeningitis
virus revealed that the distribution of arenavirus diameters was consistent with conserved periodic
classes of virion size that may be related by modular inclusion or exclusion of 4-ribonucleoprotein
units. Analysis of en face images revealed paracrystalline arrays with a center-to-center spacing
of approximately 57, 62, and 74 Å, respectively. Analysis of edge-on views showed that the
surface glycoproteins interact with the packaged viral RNA via a multilayer scaffold on the inner
surface of the envelope (Fig. 1). These structural features resemble the supramolecular design
of retroviruses in which surface glycoproteins interact with the matrix domain of gag and the nucleocapsid
domain of GAG binds the genomic RNA.
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| Fig. 1. Electron cryomicroscopy and image analysis of arenaviruses.
A, The arenavirus glycoprotein complexes are present in 2-dimensional, orthorhombic
arrays on the surface of the virion. Images of New World Pichinde (top row) and
Tacaribe (middle row) arenaviruses and Old World lymphocytic choriomeningitis virus
(bottom row) are shown. Scale bar = 50 Å. B, Organization of the glycoprotein
complex is revealed in 2 views of a 3-dimensional reconstruction of the Pichinde
virus glycoprotein complex. |
Recovery from Lassa virus infection usually occurs before the appearance of neutralizing antibodies, indicating that cellular immunity
plays a primary role in viral clearance. To date, the role of Lassa virus–specific CD8+
T cells has not been evaluated in humans. To facilitate studies of T-cell responses to infection
with Lassa virus, we sought to identify human cytotoxic T lymphocyte epitopes, peptide sequences
that in association with proteins on antigen-presenting cells are required for antigen recognition
by specific cytotoxic T cells. We searched for peptides encoded by the genes for the glycoprotein
precursor and the nucleoprotein of 2 genetically distinct strains of Lassa virus.
We identified 130 potential HLA-A2, HLA-A3, or HLA-B7 supertype-restricted cytotoxic T lymphocyte epitopes. Each peptide was assayed for
MHC binding by using a panel of 5 purified human MHC class I molecules per supertype. We found 48 peptides
that bound 2 or more alleles with affinities of 500 nM or greater, values indicative of immunogenicity.
To determine whether these peptides were immunogenic in vivo, we immunized HLA transgenic mice
with pools of 3–5 nonoverlapping peptides. CD8+ T cells were isolated from spleens
and assayed for the ability to produce IFN-γ
in response to each peptide.
We identified 19 HLA-A2 peptides and 4 HLA-A3 peptides that are immunogenic in the transgenic mouse system. We did not detect any HLA-B7 peptides
that were immunogenic. Four of the immunogenic Lassa virus peptide sequences are conserved (100%
homology) in other members of the arenavirus family (range, 1–5 viruses). We also found that
effector cells immunized against Lassa virus peptides can recognize variant peptides from other
arenaviruses. The immunogenic epitopes identified in this study will aid in the characterization
of T-cell responses to Lassa virus and in the design of arenavirus vaccines.
PUBLICATIONS
Neuman, B.W., Stein, D.A., Kroeker,
A.D., Paulino, A.D., Moulton, H.M., Iversen, P.L., Buchmeier, M.J.
Antisense morpholino oligomers directed against the 5¢-end
of the genome inhibit coronavirus proliferation and growth. J. Virol. 78:5891, 2004.
Rempel, J.D., Murray, S.J., Meisner,
J., Buchmeier, M.J. Differential regulation of innate and
adaptive immune responses in viral encephalitis. Virology 318:381, 2004.
Rempel, J.D., Murray, S.J., Meisner,
J., Buchmeier, M.J. Mouse hepatitis virus neurovirulence:
evidence of a linkage between S glycoprotein expression and immunopathology. Virology 318:45,
2004.
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