Proc. Natl. Acad. Sci. USA
Vol. 89, pp. 10847-10851, November 1992
Nonreplicating vaccinia vector efficiently expresses
(poxvirus/expression vector/attenuation/host retricin)
GERD SUTrER AND BERNARD MOSS
Laboratory of Viral Diseases, National Institute of Ailergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892
Contributed by Bernard Moss, August 20, 1992
attenuated vaccinia virus strain that has been safety tested in
humans, was evaluated for use as an expression vector. MVA
has multiple genomic deletions and is severely host cell re-
stricted: it grows well in avian cells but is unable to multiply in
human and most other mammalian cells tested. Nevertheless,
we found that replication of viral DNA appeared normal and
that both early and late viral proteins were synthesized in
human cells. Proteolytic processing ofviral structural proteins
was inhibited, however, and only immature virus particles
were detected by electron microscopy. We constructed an
insertion plasmid with theEschenchia coli lacZ gene under the
control of the vaccinia virus late promoter P11, flanked by
sequences ofMVA DNA, to allow homologous recombination
at the site of a naturally occurring 3500-base-pair deletion
within theMVAgenome.MVA recombinants were isolatedand
propagated in permissive avian cells and shown to express the
enzyme 3-galactosidase upon infection of nonpermissive hu-
man cells. The amount of enzyme made was similar to that
produced by a recombinant of vaccinia virus strain Western
Reserve, which also had the lacZ gene under control ofthe P11
promoter, butmultiplied to high titers. Since recombinant gene
expression is unimpaired in nonpermissive human cells, MVA
may serve as a highly efficient and exceptionally safe vector.
Modified vaccinia Ankara (MVA), a highly
The eradication of smallpox was achieved through immuni-
zation with live vaccinia virus (1). Presently, vaccinia virus
is used extensively as a gene expression vector and is under
evaluation as a recombinant vaccine (2). Because vaccinia
virus is infectious for humans, its use in the laboratory has
been affected by safety concerns and regulations (3, 4). For
general vector applications, health risks would be lessened by
the adoption of a highly attenuated vaccinia virus strain.
Several such strains were developed foruse as safersmallpox
vaccines (1). We chose to examine the potential of the
modified vaccinia Ankara (MVA) strain as an expression
vector because ofits extreme attenuation. MVA was derived
from vaccinia virus strain Ankara, referred to here as the
wild-type virus (WT), by over 570 serial passages in chicken
embryo fibroblast cells (CEF) (5). The resulting MVA strain
lost the capacity to productively infect mammalian cells and
suffered six major deletions of DNA totaling 31,000 base
pairs (bp), including at least two host-range genes (refs. 6 and
7; G.S., unpublished data). When tested in a variety ofanimal
species, MVA was proven to be avirulent even in immuno-
suppressed animals. Most importantly, there is clinical expe-
rience using MVA for primary vaccination of over 120,000
humans against smallpox. During extensive field studies,
including high risk patients, no side effects were associated
with the use of the MVA vaccine (5, 8, 9). However, since
MVA cannot replicate in human and most other mammalian
cells, high expression ofrecombinant genes seemed unlikely
in view of reports that other host-range vaccinia virus mu-
tants are inhibited early in infection (10, 11).
Contrary to expectations, we found that the expression of
late, as well as early, viral genes was unimpaired in human
cells despite the inability of MVA to produce infectious
progeny. Moreover, recombinant viruses were able to syn-
thesize high levels of a foreign protein in human cells,
demonstrating the potential of MVA to serve as an excep-
tionally safe and highly efficient expression vector.
MATERIALS AND METHODS
Cells and Viruses. The WT Ankara and MVA strains were
kindly provided by A. Mayr (Veterinary Faculty, University
of Munich). The viruses were routinely propagated and
titered in CEF grown in minimal essential medium (MEM)
supplemented with 10%/ fetal calfserum (FCS). Human HeLa
and 293 cells were also grown in MEM supplemented with
10%1 FCS. Virus multiplication was monitored by infecting
cell monolayers with 0.05 plaque-forming unit (pfu) per cell.
After virus adsorption for 45 min at 37TC, the inoculum was
removed; the cell monolayer was washed once with MEM
and incubated with fresh medium(MEM containing2% FCS)
at 370C in a 5% CO2 atmosphere. At 0, 24, and 48 h
postinfection, virus was harvested by freeze-thawing and
briefsonication ofthe infected cells. The resulting lysate was
titrated on CEF.
Analysis of Viral DNA. Cytoplasmic DNA from infected
293 cells was transferred to a Hybond N+ membrane (Am-
ersham) with a dot blot apparatus and hybridized to a
32P-labeled MVADNA probe. Radioactivity was quantitated
with a Betascope 603 blot analyzer (Betagen, Waltham, MA).
The same DNA preparation from infected 293 cells was also
digested with BstEII and separated by electrophoresis on a
1% agarose gel. The DNA was transferred by Southern blot
to aHybondN+ membrane (Amersham) and hybridized with
a 32P-labeled probe of the terminal segment of the vaccinia
virus genome (12).
Analysis of [35SJMethionine-Labeled Polypeptides. Cell
monolayers grown in 12-well plates were infected with virus
at a multiplicity of 15 pfu per cell. After adsorption of virus
for 45 min at 40C, MEM supplemented with 2% FCS was
added, and the cell cultures were incubated at 370C in a 5%
CO2 atmosphere. At 2, 6, and 12 h after infection, the medium
was removed, and the cultures were washed once with 1 ml
ofmethionine-free MEM. To each well, 0.2 ml ofmethionine-
free MEM supplemented with 50gCi(1 Ci = 37 GBq) of
[35S]methionine was added and incubated for 30 min at 370C.
Cytoplasmic extracts of infected cells were prepared by
incubating each well in 0.2 ml of 0.5% Nonidet P-40 lysis
buffer for 10 min at 370C. For pulse-chase experiments, cells
Abbreviations: CEF, chicken embryo fibroblast cells; FCS, fetal calf
serum; MVA, modified vaccinia Ankara; pfu, plaque-forming unit(s);
WT, wild-type virus.
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Biochemistry: Sutter and Moss
Hours after infectiot
Hours after infection
3 4 5 6
4rr ** **
infection of human cells with
MVA.(A) Multiplicationof MVA
in HeLa (-), 293 (i), and CEF (*)
cells and of WT in HeLa (A) and
293 (A) cells. (B) Viral DNA syn-
thesis was determined by hybrid-
ization of a 32P-labeled viral DNA
probe to DNA isolated from 293
cells at 0, 4, or 8 h after infection
with MVA or WT. Radioactivity
was quantitated using a Betascope
603 blot analyzer. (C) Synthesis of
early and late viral polypeptides in
human cells. HeLa cells were in-
fected with WT or MVA and la-
beled with [35S]methionine at the
indicated hours postinfection
(Pulse Time). Cell lysates were
treated with 2% SDS and 1% di-
thiothreitol and analyzed by elec-
trophoresis on a 10%o polyacryl-
amide gel. Lane U, uninfected cell
extract; lane M, protein standards
(indicated by their molecular
masses in kDa on the left). (D)
Proteolytic processing of viral
proteins. CEF (lanes 1 and 2) or
HeLa (lanes 3-6) cells were in-
fected with MVA or with WT for 6
h and labeled with V35S]methionine
for 30 min. Cells were harvested
afterthe pulse (lanes 1, 3, and 5) or
after a 16-h chase (lanes 2, 4, and
6). Cell lysates were treated with
2% SDS and 1% dithiothreitol and
analyzed by electrophoresis on a
10% polyacrylamide gel. Lanes U
and M are defined as in C, and the
positions of the marker proteins
(in kDa) are indicated on the right.
The uncleaved precursors of the
major late core polypeptides, P4a
and P4b, are marked by arrow-
were infected for 6 h and then incubated in 0.2 ml of
methionine-free MEM containing 50gCiof [35S]methionine
for 30 min. The cells were subsequently washed once with
MEM and incubated in MEM supplemented with 2% FCS for
another 16 h. Cells were lysed as above, and samples were
analyzed by SDS/PAGE.
Electron Microscopic Analysis. Cell monolayers were in-
fected with virus at a multiplicity of 10 pfu per cell. At 16 h
after infection, the medium was removed, and the cells were
fixed by the addition of 2.5% (vol/vol) glutaraldehyde in
Millonig's buffer (13). After 1 h ofincubation at 40C, the cells
were scraped and then pelleted by low-speed centrifugation.
The cell pellet was incubated an additional 30 min in the same
2.5% glutaraldehyde buffer at 40C. Then the buffer was
replaced by Millonig's 0.13 M sodium phosphate buffer (pH
7.4). Transmission electron micrographs were prepared by
Advanced Biotechnology, Silver Spring, MD).
Plasmids. Sequences of MVA DNA flanking the site of a
3500-bp deletion in the HindIII A fragment of the MVA
genome were amplified by PCR and cloned into pGEM 4Z
(Promega). The primers for the left 900-bp DNA flank were
GTGT-3' and 5'-GGGGGGGGTACCTACCAGCCAC-
CGAAAGAG-3' (sites for restriction enzymes EcoRI and
Kpn I are underlined). The primers for the right 600-bp DNA
flank were 5'-GGGGGGCTGCAGTTTGGAAAGTTTTAT-
AGG-3' and 5'-GGGGGGAAGCTTAACTAGTTTCTG-
GTG-3' (sites for the restriction enzymes Pst I and HindIII
are underlined). Between these flanks of MVA DNA, the
Escherichia coli lacZgene under control ofthe vaccinia virus
late promoter P11 and the E. coli gpt gene under control of
the vaccinia virus early/late promoter P7.5 were cloned.
Generation of Recombinant Viruses. CEF infected with
MVA at a multiplicity of 0.05 pfu per cell were transfected
with calcium phosphate-precipitated plasmid as described
(14). Recombinant MVA virus expressing 1-galactosidase
(MVA LZ) was selected by six consecutive rounds ofplaque
purification in CEF stained with 5-bromo-4-chloro-3-indolyl
/3-D-galactoside (300 ,g/ml) (15). After one blind passage on
CEF in the presence of mycophenolic acid, recombinant
MVA virus expressing P-galactosidase and guanine phospho-
ribosyltransferase (MVA LZgpt) was selected by three con-
secutive rounds of plaque purification on CEF in the pres-
ence of mycophenolic acid and 5-bromo-4-chloro-3-indolyl
/3-D-galactoside screening for blue virus plaques (16). Sub-
sequently, viruses were amplified by infection ofCEF mono-
layers, and the DNA was analyzed by Southern blot hybrid-
8-Galactosidase Assay. Confluent HeLa cells and CEF
were infected with 15 pfu per cell ofMVA LZ, MVA LZgpt,
or vaccinia virus Western Reserve recombinant vSC8, which
also expresses theP-galactosidase gene under control of
Proc. Nati. Acad. Sci. USA 89(1992)
Proc. Natl. Acad. Sci. USA 89 (1992)
.09''W' ,, . >X
(D) for 16 h were fixed, sectioned, and examined by electron microscopy. (Bars = 0.1 ,um.)
Electron micrographs ofinfected cells. HeLa cells infected with MVA (A) or WT (B) and CEF cells infected with MVA (C) or WT
vaccinia virus promoter P11 (15). After a 24-h incubation,
cytoplasmic extracts were prepared, and the protein content
(17) and the specific f3-galactosidase activity (18) were de-
Characterization of the Host-Range Defect of MVA in Hu-
man Cells. Previous work had shown that MVA is unable to
multiply in abroad range ofmammalian cell lines (7). In HeLa
and 293 cells, WT increased 10,000-fold in titer, whereas no
increase in MVA was detected (Fig. 1A)- By contrast, MVA
grew well in CEF (Fig. 1A), usually reaching a titer higher
than that ofWT (data not shown). Despite the differences in
the abilities ofWT and MVA to multiply in human cells, viral
DNA replication proceeded similarly (Fig. 1B), and restric-
tion enzyme analysis indicated that the concatemeric forms
of replicative MVA DNA were processed normally to unit
genomes (data not shown).
The replication ofMVADNA implied that the initial stages
of infection comprising viral attachment, entry, early gene
expression, and uncoating occurred in nonpermissive human
cells. SDS/PAGE analysis of extracts of WT- and MVA-
infected HeLa cells thathad been pulse-labeled with [35Slme-
thionine confirmed the synthesis of early viral proteins and
demonstrated that late viral proteins were made at 6 and 12
h after infection (Fig. 1C). The viral protein patterns in WT-
and MVA-infected human cells were very similar. An ex-
ception, the absence ofa 90,000-Da polypeptide from MVA-
infected cells, was attributed to the deletion of the gene
encoding the nonessential A-type inclusion protein homolog
(7). This difference was also noted upon analysis of labeled
proteins from WT- and MVA-infected permissive CEF.
Since pulse-labeling studies showed no significant differ-
ence in early or late viral protein synthesis, we checked to see
if there was a defect in protein processing. When MVA-
infected CEF were labeled with [35S]methionine at 6 h after
infection and then chased with unlabeled amino acids for 16
h, the expected cleavage of at least five polypeptides includ-
ing the major core protein precursors, P4a and P4b, was
observed (Fig. 1D). By contrast, processing ofMVA proteins
in HeLa cells was inhibited, whereas WT proteins were
processed correctly (Fig. 1D).
Virus Morphogenesis in Infected Cells. From studies using
the drug rifampicin, it is known that an interruption of virus
assembly leads to the inhibition ofproteolytic cleavage ofthe
major core precursors P4a and P4b in infected cells (19).
Similarly, when the expression ofan 11,000-DaDNA binding
protein of vaccinia virus is prevented, characteristic imma-
ture virus particles are found in infected cells, and proteolytic
cleavage of P4a and P4b is blocked (20). Since proteolytic
processing of late viral polypeptides was also inhibited in
MVA-infected HeLa cells, we suspected that MVA virion
formation failed to occur in nonpermissive human cells.
Indeed, only immature virus particles appearing as circular
spicule-coated membranes encircling granular material and
Biochemistry:Sutter and Moss
10850Biochemistry: Sutter and Moss
CNKFEOMG L J H
A51 RA52R A53RA55R
designed for insertion of foreign DNA by homologous recombina-
tion. HindIII restriction endonuclease sites within the genome of
MVA are indicated at the top. The 480-bp Acc I-Acc I fragment that
overlapped thejunction ofdeletion III within the HindIII A fragment
was sequenced and compared to the published sequence of the
Copenhagen strain ofvaccinia virus (39). This comparison suggested
a deletion of 3501 bp affecting the five open reading frames A51R,
A52R, A53R, A54L, and A55R present in the Copenhagen strain of
vaccinia virus but partially or completely deleted from MVA. The
DNA sequences adjacent to the deletion (flanki and flank2) were
amplified by PCR and inserted into the left and right ends of the
multiple cloning site ofapGEM plasmid in order to make the transfer
vector pIII, which was used for the construction of the insertion
plasmids pIII LZ and pII
LZgpt. P11 and P7.5 refer to well-
characterized late and early/late vaccinia virus promoters, respec-
tively. kb, kilobase(s).
Schematic map of the genome of MVA and plasmids
lacking dense nucleoprotein bodies could be seen by electron
microscopic examination of human cells infected with MVA
(Fig. 2A). By contrast, mature brick-shaped particles with
complex internal structures were numerous in human cells
infected with WT (Fig. 2B) or CEF infected with MVA (Fig.
2C) or WT (Fig. 2D).
Construction and Isolation ofMVA Recombinants Express-
ing the E. coli lacZ Gene. The robust synthesis of viral late
proteins, despite the absence of infectious virus formation in
mammalian cells, suggested that MVA might be a useful
vector for expression of foreign genes. To test this strategy
and monitor gene expression, the E. coli lacZ gene regulated
by the well-characterized vaccinia virus late promoter P11
(21) was flanked by MVA DNA sequences to form the
plasmid insertion vector pIl LZ (Fig. 3). The foreign gene
was targeted precisely to the site of a deletion within the
MVA genome to avoid any further changes in its phenotype.
Recombinant vaccinia virus MVA LZ was formed in CEF
that were infected with MVA and transfected with pIl LZ.
Recombinant virus plaques were identified by screening for
f3-galactosidase synthesis with a chromogenic substrate (15).
A second plasmid, pIll LZgpt, contained the lacZ gene as
well as the E. coli gpt gene under the control ofvaccinia virus
early/late promoter P7.5 to provide antibiotic selection of
recombinant virus MVA LZgpt (16). In each case, multiple
plaque isolations were performed, and the correct insertion of
the foreign DNA and absence of parental virus was ascer-
tained by restriction enzyme analysis and Southern blot
viruses. (A) SDS/PAGE ofhuman 293 cells infected with MVA (lane
1), MVA LZ (lane 2), MVA LZgpt (lane 3), or vSC8 (lane 4). The
infected cultures were labeled with [35S]methionine at 6 h postinfec-
tion. Cell lysates were analyzed by electrophoresis on a 10%o poly-
acrylamide gel. Lane U, uninfected 293 cell extract. The numbers on
the left indicate the positions and molecular masses (in kDa) of
protein standards. The protein band representing the enzyme /3-ga-
lactosidase is marked by an arrowhead. (B) /3-Galactosidase activity.
CEF (1) orHeLa (m) cells were infected withMVA LZ, MVA LZgpt,
or vSC8 for 24 h. Cytoplasmic extracts were prepared, andP3-galac-
tosidase activities were determined. The0 time values, obtainedfrom
extracts made immediately after infection with MVA LZ, MVA
LZgpt, and vSC8 were 178, 132, and 61 units per mg, respectively.
Expression ofthe E. coli lacZgene by MVA recombinant
Metabolic labeling with [35S]methionine ofhuman 293 cells
infected with MVA LZ and MVA LZgpt revealed the late
synthesis of an additional protein of about 116,000 Da that
comigrated with03-galactosidasemade by vSC8 (15), a West-
ern Reserve strain recombinant virus that also contains a P11
promoter-lacZ gene cassette but that multiplies well in
human cells (Fig. 4A). Moreover, similar levels of /3-galac-
tosidase were detected by enzyme assays ofextracts ofHeLa
cells infected with the MVA and Western Reserve strain
recombinant viruses (Fig. 4B).
The structural and other abundant proteins made by vaccinia
virus are products of the late class genes, which are ex-
pressed only after viral DNA replication (23). The high level
of protein synthesis achieved with recombinant MVA in
nonpermissive human cells is consistent with our finding that
the block in virus assembly occurs after DNA replication.
This desirable result was not predicted because a large
numberofopen readingframes, including the K1L host-range
gene, are impaired in MVA (ref. 7; G.S., unpublished data).
Previous studies had indicated that a deletion including the
K1L gene from the Copenhagen strain of vaccinia virus
resulted in abortive expression of early viral genes followed
by a rapid inhibition of further viral and cellular protein
synthesis (11, 24). In addition, viral DNA replication was
inhibited, and there was no evidence of viral membranes or
immature particles in the nonpermissive human cells infected
with the K1L mutant (11). Viral DNA replication was also
severely inhibited in human cells infected with NYVAC, a
genetically engineered virus derived for recombinant vaccine
purposes from the Copenhagen strain by deletion of multiple
open reading frames including K1L and a second host-range
gene, C7L (25). Furthermore, naturally host range-restricted
avipoxviruses cannot carry out DNA replication in mamma-
lian cells (25). Nevertheless, the results obtained with MVA
are not entirely unprecedented since some host-range white
pock deletion mutants of rabbit poxvirus support DNA
replication and expression of late proteins in nonpermissive
Proc. Natl. Acad. Sci. USA 89(1992)
Proc. Natl. Acad. Sci. USA 89 (1992) Download full-text
pig kidney cells (26). The rabbit poxvirus mutants, however,
exhibited quantitatively decreased late protein synthesis in
nonpermissive cells and were blocked at a stage after the
proteolytic processing of core polypeptides (26). Thus, the
host-range phenotype ofMVA appears to be unique. In view
ofthe extensive deletions in MVA, further investigations are
needed to determine the genetic defects primarily responsible
for its phenotype. Since MVA still cannot multiply in human
cells after introduction ofan intact KML gene, the host-range
phenotype must be multifactorial (ref. 7; G.S., unpublished
LC16mO, an attenuated strain of vaccinia virus that still
replicates well in mammalian cells, including animal skin, has
been used to express foreign genes (27). In addition, a variety
of gene deletions that attenuate vaccinia virus to varying
degrees have been described, and some ofthese, individually
and in combination, have been used for construction of
expression vectors (25, 28-35). Avipox or other naturally
host-restricted poxviruses also have been advocated and
tested as nonreplicating vectors (22, 36-38). However, for
use as a gene expression vector, no other characterized
naturally derived or genetically engineered strain ofpoxvirus
is known to have a similar combination of desirable proper-
ties and to have been so extensively tested in humans as
MVA. The properties oftheMVA strain ofvaccinia virus that
make it so attractive as a vector include (i) high-level gene
expression in human and other mammalian cells, (ii) ability
to prepare high titer virus stocks in primary and established
ATTC CRL 1590 (G.S., unpublished data) CEF, (iii) inability
to produce infectious virus in most mammalian cells, (iv)
avirulence in a variety of animals even under immunosup-
pressive conditions, and (v) little or no local or systemic
reaction upon inoculation of humans including high-risk
individuals. Adoption of MVA-based vectors should reduce
the risk of infecting laboratory workers. Whether MVA
vectors will also be useful for live vaccine or therapeutic
applications remains to be determined.
We thank S. Isaacs and M. Buller for discussions and comments
on the manuscript. G.S. is supported by an AIDS Scholarship from
the Bundesministerium fur Forschung und Technologie, Federal
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