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Journal of Virology, January 2001, p. 910-920, Vol. 75, No. 2
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.2.910-920.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Comparison of Predicted Amino Acid Sequences of
Measles Virus Strains in the Edmonston Vaccine Lineage
Christopher L.
Parks,
Robert
A.
Lerch,
Pramila
Walpita,
Hai-Ping
Wang,
Mohinder S.
Sidhu, and
Stephen A.
Udem*
Department of Viral Vaccine Research,
Wyeth-Lederle Vaccines, Pearl River, New York 10965
Received 10 April 2000/Accepted 16 October 2000
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ABSTRACT |
Protein-encoding nucleotide sequences of the N, P, M, F, H, and L
genes were determined for a low-passage isolate of the Edmonston wild-type (wt) measles virus and five Edmonston-derived vaccine virus
strains, including AIK-C, Moraten, Schwarz, Rubeovax, and Zagreb.
Comparative analysis demonstrated a high degree of nucleotide sequence
homology; vaccine viruses differed at most by 0.3% from the Edmonston
wt strain. Deduced amino acid sequences predicted substitutions in all
viral polypetides. Eight amino acid coding changes were common to all
vaccine viruses; an additional two were conserved in all vaccine
strains except Zagreb. Comparisons made between vaccine strains
indicated that commercial vaccine lots of Moraten and Schwarz had
identical coding regions and were closely related to Rubeovax, while
AIK-C and Zagreb diverged from the Edmonston wt along slightly
different paths. These comparisons also revealed amino acid coding
substitutions in Moraten and Schwarz that were absent from the closely
related reactogenic Rubeovax strain. All of the vaccine viruses
contained amino acid coding changes in the core components of the
virus-encoded transcription and replication apparatus. This
observation, combined with identification of noncoding region
nucleotide changes in potential cis-acting sequences of the
vaccine strains (C. L. Parks, R. A. Lerch, P. Walpita, H.-P.
Wang, M. S. Sidhu, and S. A. Udem, J. Virol.
75:921-933, 2001), suggest that modulation of transcription and
replication plays an important role in attenuation.
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INTRODUCTION |
Measles is a highly contagious
disease that most commonly strikes children. The causative agent,
measles virus (MV), is generally transmitted by aerosolized secretions
deposited on upper-respiratory-tract mucosal surfaces. Exposure leads
to local respiratory tract replication; infection of regional lymphoid
tissues then occurs followed by viremia and systemic dissemination as
revealed by the characteristic skin rash. Most children recover
uneventfully from the illness, but serious complications can occur,
including pneumonia and involvement of the central nervous system
(17, 27, 28). Despite the highly contagious nature of the
disease, MV can be controlled effectively by immunization with live
attenuated vaccines. The effectiveness of MV vaccines is well
illustrated by the epidemiology of the disease in the United States.
Prior to 1963, before use of the earliest vaccines, there were over
500,000 reported cases per year. Twenty years later, MV incidence was
less than 2,000 cases per year (11, 28). The availability
of these effective vaccines has not eliminated the threat from MV, and
measles still causes significant levels of morbidity and mortality in
developing countries largely because of inadequate and unsustained
vaccination efforts (17).
Several effective MV vaccines were derived from a single clinical viral
isolate called the Edmonston strain (28, 66). Enders et
al. (20) developed the first MV vaccine by the classical approach (1) of propagating the pathogen in heterologous
cells and tissues. Specifically, MV was serially propagated in
semi-permissive chicken embryos and chick fibroblast cells. Variations
of the Enders approach have led to the development of a number of
independently derived but effective Edmonston-based vaccines (28,
66).
MV is a member of the genus Morbillivirus in the
Paramyxoviridae family and, like other members of this
family, it is an enveloped RNA virus that contains a single-strand,
negative-sense, nonsegmented genome (28, 47). The 16-kb MV
genome encodes eight known proteins from six nonoverlapping cistrons
arranged 3'-N-P-M-F-H-L-5'. The major structural polypeptide is encoded
by the N (nucleocapsid) gene. The N protein is essential for packaging
the genome into a ribonucleoprotein complex that serves as template for
transcription, replication, and packaging into progeny virions. The P
cistron specifies three polypeptides: P, C, and V. The P
(phosphoprotein) polypeptide is a subunit of the viral RNA polymerase.
P protein also acts as a chaperone that interacts with and regulates
the cellular localization of N protein and probably assists in
nucleocapsid assembly (28, 33, 70). The C and V
polypeptides are nonstructural proteins that are translated from P
mRNAs through the use of alternative reading frames; C protein is
synthesized from a downstream translation start signal, whereas V
protein is translated from an edited mRNA that contains an extra G
residue (28, 33, 70). The M gene encodes the matrix
protein that lines the inner surface of the viral envelope and
participates in virion maturation (28, 83). The F (fusion)
and H (hemagglutinin) genes encode envelope glycoproteins that mediate
cell surface recognition, membrane fusion, and virus entry (28,
83). Finally, the L (large) gene encodes the multifunctional catalytic subunit of the RNA-dependent RNA polymerase (28, 33, 70).
How changes in individual MV proteins may influence vaccine virus
attenuation is not well understood. Partial sequence data for MV
vaccine virus genomes clearly indicates that multiple mutations have
accumulated in more than one protein coding region, but these analyses
have so far failed to point to an underlying mechanism of MV
attenuation (28, 66). To facilitate further analysis of
the molecular basis of MV attenuation, we have determined the nucleotide sequence of all protein coding regions from an early-passage laboratory isolate of the original Edmonston virus (see Fig. 1, Edmonston wild type and reference (66) and five Edmonston
vaccine strains. Comparison of deduced amino acid sequences has
revealed amino acid coding substitutions common to all of the vaccines, as well as changes found only in subsets of the vaccine viruses. The
results of these comparisons identified a number of mutations that
appear to be strong candidates for attenuation determinants. In
addition, we also suggest a model that correlates modulation of gene
expression with the attenuated phenotype.
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MATERIALS AND METHODS |
Cells and virus.
Vero cells were maintained in Dulbecco
modified Eagle medium (Life Technologies) supplemented with 5% fetal
bovine serum. Stocks of MV were prepared by infection of Vero cell
monolayers at a multiplicity of infection of approximately 0.1 PFU per
cell. Infected cells were harvested by scraping the monolayer when the cytopathic effect was detectable in 70 to 80% of the cell monolayer. Harvested cells were collected by centrifugation and resuspended in
serum-free OPTIMEM (Life Technologies) and lysed by one freeze-thaw cycle. The Edmonston wt isolate (21, 28) was kindly
provided by Judy Beeler (Center for Biologics Evaluation and Research). Edmonston B (Rubeovax) (20, 28, 45), AIK-C
(52), Schwarz (28, 45, 69), and Zagreb
(28, 38) were generously provided by William Bellini and
Paul Rota (Centers for Disease Control and Prevention)
(66). Moraten (Attenuvax, Merck & Co.) (28, 31) and a second sample of Schwarz (Rimevax, SmithKline Beecham) (28, 45, 69) were obtained from commercially available
vaccine preparations. The Schwarz virus strain from each source was
analyzed independently.
Viral genome sequencing.
Sequencing was performed on DNA
fragments generated by reverse transcription and PCR amplification
(RT-PCR). Total RNA was extracted from infected Vero cells by the
guanidinium-phenol extraction procedure (12) using Trizol
reagent (Gibco-BRL). RT-PCR required first RT of approximately 1 µg
of total infected-cell RNA with avian myeloblastosis virus (AMV)
reverse transcriptase (Pharmacia) and random hexamer primers
(Pharmacia), followed by Taq DNA polymerase-mediated PCR
amplification (Amplitaq; Perkin-Elmer) with sequence-specific primers.
Some single-tube RT-PCR reactions were performed by RT in the presence
of gene-specific primers and AMV reverse transcriptase, followed by
amplification with the high-fidelity enzyme mixture in the Titan RT-PCR
kit (Roche Molecular Biology). Terminal fragments were either amplified
by RT-PCR performed across the junction formed after circularization of
the RNA genome with RNA ligase (71) or amplified by using
the RACE (rapid amplification of cDNA ends) procedure
(24). Amplified DNA fragments were purified for sequencing
by electrophoresis in low-melting-temperature agarose (FMC). DNA was
recovered from gel slices with the Wizard DNA Clean-Up System (Promega)
or by digestion of gel slices with 
-agarase (New England Biolabs).
Cycle sequencing (29, 44) was executed with dye-labeled
terminators and Taq DNA polymerase (Applied Biosystems), followed by analysis on an ABI Prism 377 automated sequence apparatus (Applied Biosystems). Primers for PCR amplification and sequencing of
both cDNA strands were designed based on published MV sequences (GenBank accession numbers K01711 and S58435). Sequence data were
analyzed using the MacVector (Oxford Molecular Group) and Lasergene
(DNAstar, Inc.) software packages. A rooted phylogram was prepared from
a CLUSTAL W alignment by the neighbor-joining method and plotted by
using NJplot (61, 79).
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RESULTS AND DISCUSSION |
Nucleotide sequence analysis.
MV coding region sequences were
analyzed to reveal genetic modifications in vaccine strains and thereby
identify candidate vaccine-specific coding changes for future studies
of MV attenuation. The virus strains sequenced are shown on the
Edmonston vaccine lineage in Fig. 1
(28, 66). Figure 1 also includes the passage history as
described by Rota et al. (66). The virus, referred to as
Edmonston wt (Fig. 1), was the lowest-passage stock available of the
original Edmonston clinical isolate. It had been passaged 13 times
(66) prior to our analysis. At several points in the passage history of this isolate, the virus has been shown to retain pathogenicity (2, 3). The virus samples obtained for
sequence analysis were passaged minimally (one to three times) in Vero cells to generate virus stocks, and these stocks were then used to
infect cells for the isolation of infected-cell RNA. Purified RNA was
used to generate RT-PCR products from all protein coding regions. These
PCR fragments were sequenced on both strands (see Materials and
Methods) to provide a "consensus" sequence representing the
population of viruses replicating within the infected cells. Direct
sequencing of these PCR products helped alleviate concerns associated
with sequencing cloned RT-PCR products that could represent a selected
subpopulation of viral genomes or include nucleotide substitutions
introduced by PCR amplification. Minor virus populations accumulated
during limited Vero cell passage should not notably affect consensus
sequence determinations since the RT-PCR products should reflect the
majority sequence in the viral RNA pool.
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FIG. 1.
Edmonston vaccine lineage. Passage history of the
Edmonston vaccines as described by Rota et al. (66) and
adapted with permission from Elsevier Science. The protein coding
region nucleotide sequence was determined for each virus highlighted in
the gray boxes.
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The compiled sequence data, summarized in Fig.
2 and
3,
demonstrated that the coding region nucleotide sequence of the vaccine
viruses differed from Edmonston wt by at most 0.3% (Fig.
2A).
Comparison of predicted amino acid sequences revealed substitutions
in
the five vaccine strains that differentiated these viruses
from the
low-passage Edmonston wt isolate (Fig.
2B). Eight amino
acid coding
substitutions were shared by all of the Edmonston
vaccine strains, and
two substitutions were found in all vaccine
strains except Zagreb (Fig.
3). Less-well-conserved amino acid
coding substitutions were also found
that affected one to three
of the vaccine strains. Generation of a
phylogram (Fig.
2C) indicated
that the Moraten and Schwarz vaccine
viruses contained identical
coding sequences and were closely related
to Rubeovax. Zagreb
and AIK-C were obviously distinguishable from this
group of vaccine
viruses.
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FIG. 2.
Comparison of MV protein coding region sequences. (A)
The total number of vaccine virus protein coding region nucleotide
substitutions is shown, and values are categorized further into coding
and silent substitutions. GenBank accession numbers are provided. (B)
Number of predicted amino acid changes in vaccine virus protein coding
regions. The size of each protein is given below the gene designation.
(C) The phylogram was generated using Edmonston wt as the out group.
The sequences were aligned using CLUSTAL W (79), and the
phylogram was generated by the neighbor-joining method using NJplot
(61). The scale represents the number of substitutions per
nucleotide.
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FIG. 3.
Comparison of Edmonston wt and vaccine virus genomes.
Nucleotide changes are shown for each coding region. Whether the
nucleotide substitution results in an amino acid change or is silent is
illustrated in the grid. Amino acid changes from wt are presented using
one-letter amino acid symbols. Yellow shading in the grid highlights an
amino acid substitution. Red shading of the amino acid position
indicates a residue that is substituted in four or five of the vaccine
strains. Blue shading in the grid without an amino acid symbol denotes
strains that contain a silent base change; a line through a blue box
indicates that the change is not silent in an overlapping reading
frame. The F protein amino acid numbers are given relative to the
predominant AUG codon at genomic nucleotide position 5458 (9). Abbreviations in the green header: NUC POS, MV
genomic nucleotide position; BASE SUB, nucleotide substitution; Wt-Vac,
wt and vaccine virus nucleotides; AA POS, amino acid position; W,
Edmonston wt; A, AIK-C; M, Moraten; R, Rubeovax; S, Schwarz; and Z,
Zagreb.
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The most highly conserved vaccine virus amino acid coding changes shown
in Fig.
3 (shaded in red) represent a "vaccine strain
signature"
that should prove useful for comparison with viruses
isolated during MV
outbreaks or isolated from suspected cases
of vaccine-related illness.
Although these coding changes are
characteristic of the vaccine
viruses, some of them may not be
entirely unique to Edmonston vaccine
strains. Several have been
detected in circulating wt virus strains and
in subacute sclerosing
panencephalitis brain tissue RNA (amino acid
positions 73 in C,
61 in M, 46 and 481 in H, and 1717 in L [data not
shown and references
4,
16,
43, and
67]). It is possible that
some degree
of cell culture adaptation during viral propagation
contributes
to this
observation.
The presence of amino acid substitutions shared by nearly all five
vaccine strains also implies a strong correlation between
at least some
of these genome changes and the attenuated phenotype.
These conserved
nucleotide and amino acid changes represent important
targets for
future studies aimed at understanding the molecular
basis of
attenuation. Additionally, it will likely be important
to consider some
of the less-well-conserved amino acid substitutions
in these studies.
These changes could in fact play important roles
in determining the
attenuation level of individual
strains.
How these genome modifications produce an attenuated phenotype is
unknown, but their origin must reflect selection that favors
growth in
the cells of animals other than the natural host (Fig.
1). An
indication that the particular tissue culture passage scheme
determines
the composition of genome modifications can be seen
in the sequence
data when it is examined in light of the details
of the MV lineage
(Fig.
1) (
66). The phylogram (Fig.
2C) showed
that
Moraten, Schwarz, and Rubeovax were the most closely related
vaccine
viruses, while AIK-C and Zagreb have distinguishing genetic
characteristics. In the lineage (Fig.
1) we can see that all of
the
vaccines were derived from the Edmonston-Enders strain; thereafter,
however, their passage histories differ. The Moraten-Schwarz-Rubeovax
group was propagated under very similar conditions, most notably
the
exclusive use of chicken cells. In contrast, AIK-C and Zagreb
were both
propagated in nonavian cell types that may account for
their divergence
from the Moraten-Schwarz-Rubeovax group. These
distinctions in the
lineage correlate well with the phylogram
displayed in Fig.
2C.
It was unexpected to find that Moraten and Schwarz contained identical
coding region nucleotide sequences given the fact that
they were
passaged independently. It was a further surprise to
find that the two
viruses also contained identical noncoding sequences
(
59).
It is possible that quasispecies subpopulation differences
in the
vaccines exist and were below the detection levels of consensus
sequencing; but even if this was the case, the results still indicated
that the two viruses were remarkably similar. Consensus sequence
analysis of a second Schwarz vaccine specimen was performed, confirming
the initial observation. The basis for the perplexing sequence
identity
of these two independently derived MV vaccine strains
is unknown.
Possibly, the convergence of sequence in the Moraten
and Schwarz
viruses reflects a highly selective and delimited
spectrum of
nucleotide changes imposed by extensive passage in
chicken embryo
fibroblasts at reduced temperatures (Fig.
1).
The process of adapting MV to semipermissive cell types obviously
generates strong selective pressure that favors the evolution
of viral
polypeptides that function more effectively in the new
host cell
environment. Although the following is speculative,
it seems likely
that early in the adaptation process the interaction
between some viral
proteins and proteins in the semipermissive
cell are inefficient. This
gives rise to slow viral growth and
selective pressure that favors
mutations leading to alterations
in viral polypeptides that enhance
functional interaction with
host cell proteins. A prime target for some
of the earliest mutations
would be the genes encoding the transcription
and replication
apparatus since these proteins must adjust the initial
stages
of viral replication (mRNA synthesis and positive-strand
synthesis)
to the semipermissive cell environment. A potential
disadvantage
associated with these earliest mutations is that they may
create
viral proteins that enhance interaction with the host cell at
the expense of other segments of the viral replication cycle.
For
example, a mutation that improves P protein interaction with
a
semipermissive host cell factor could have a negative effect
on another
function such as the P-N protein-protein interaction
or recognition of
template sequences by the P-L polymerase complex.
This will generate
additional selective pressure for "second-site
repressor" mutations
that help compensate for the effect of the
primary mutations. We could
imagine that these second-site repressor
mutations would evolve in
viral protein coding regions as well
as
cis-acting sequences
as the virus attempts to fine-tune the
replicative capacity to the
semipermissive cell environment during
the course of a prolonged
passage scheme. In theory, these genetic
adjustments give rise to the
best-fit virus for growth in the
semi permissive cells but ultimately
render the virus less effective
at interaction with the cellular
proteins of the natural host.
Thus, when this vaccine virus infects
permissive human host cells,
its accrued mutations lead to
less-effective virus-host cell protein
interaction and thereby reduce
virus replication efficiency. Adequate
time for the vaccine virus to
revert some of the genetic change
after vaccination is not available
before an immunocompetent host
clears the viral infection. We present
below the data analysis
for each gene region and describe how some of
the genetic changes
may relate to virus
attenuation.
N gene.
Analysis of the N gene identified three predicted
amino acid substitutions (Fig. 2B and 3); none of these changes were
conserved by all of the vaccine strains, and no single vaccine strain
contained all three substitutions. Although the N protein substitutions were not conserved by all vaccine strains, they still represent attractive candidates for attenuating modifications. N protein plays a
key role in the virus life cycle during genome packaging, genome
replication, and gene expression (28). The role of the N
protein in these diverse activities makes it seem probable that amino
acid substitutions would have some impact on the virus. The
nonconservative amino acid substitution found in the amino terminus of
the Moraten and Schwarz N protein (position 148, glutamic acid to
glycine) is a obvious candidate to alter N protein function. It
occurred in a region of N protein that likely plays a role in several
functions, including RNA binding (47), the formation of
the nucleocapsid structure, and interaction with P protein (Fig.
4A). Computer predictions also indicate
that this amino acid substitution would disturb an alpha-helical region
of the protein. Interactions with P protein may also be influenced by the amino acid substitutions in N protein at position 479 in Moraten, Rubeovax, and Schwarz, as well as the position 129 substitution in
AIK-C (Fig. 4A) (5, 50). No coding region changes were detected in the Zagreb N gene. This indicates that successful attenuation can occur without substitutions in N, but that the accumulated strain-specific changes in N may be important contributors to the degree of attenuation in viruses such as Moraten, Schwarz, and
AIK-C.
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FIG. 4.
Protein domains and vaccine virus amino acid
substitutions. Domain maps for N, P, V, and H proteins are shown along
with arrowheads marking the position of predicted amino acid
substitutions. Vaccine virus names are abbreviated as follows: AIK-C
(AIK), Moraten (Mor), Rubeovax (Rub), Schwarz (Sch), and Zagreb (Zag).
(A) The domain boundaries illustrated below the linear map of N protein
are derived from Bankamp et al. (5) and Liston et al.
(50). These include domains involved in N-P complex
formation and nucleocapsid formation and a region that affects protein
stability. (B) The linear maps of P protein and V protein drawn in
black illustrate sequences shared by these proteins. The unique
sequence in the carboxy terminus of V is designated with a
cross-hatched box. Domain boundaries include regions that promote
interaction with N protein, a region that affects the cellular
localization of N-P complexes, a region of P protein that promotes
interaction with L protein and the formation of P-P multimers, and the
carboxy-terminal domain of V that binds zinc (30, 34, 37, 51,
80). (C) Illustrated below the map of H protein are amino acids
that have been implicated in mediating binding and downregulation of
CD46 (7, 35, 49).
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P cistron.
Amino acid substitutions detected in the P gene
included a change shared by all of the vaccines at position 225 (Fig.
2B and 3). The amino-terminal portion of the P gene open reading frame (ORF) also encodes two-thirds of the V polypeptide; therefore, all of
the vaccine virus V proteins contain the same amino acid substitution.
The C protein coding region embedded within the P gene contained three
substitutions (Fig. 2B and 3) which were silent with respect to the
overlapping P and V ORFs.
P protein is a multifunctional polypeptide that is a component of
the polymerase complex. It also plays a role in viral RNA
encapsidation
and regulates the cellular localization of N protein
(
28,
33,
37,
47,
70). Given its pleiotropic activities,
substitutions in P
protein are likely to significantly impact
viral replication
efficiency. The position 275 and 439 substitutions
in AIK-C P (Fig.
3)
protein may be significant in the context
of this strain. They map
within P domains (Fig.
4B) that mediate
interaction with N protein as
well as promote its own multimerization
(
30,
37,
80). The
position 225 substitution (Fig.
3), found
in all of the vaccines,
replaced an Edmonston wt glutamic acid
with a glycine. Substitution of
this glutamic acid with a nonpolar
residue occurred in a region of P
protein that may be involved
in a chaperone function of P protein that
regulates the cellular
localization of N protein (
37,
80).
The fact that the N-P
protein-protein interaction is essential for
several functional
activities suggests that any mutations that alter P
protein and
influence the N-P interaction could affect gene expression,
replication
and ultimately the degree of
attenuation.
The 225 substitution also affects V protein (Fig.
3 and
4B). As
mentioned above, this substitution occurred within one of
the P protein
domains (Fig.
4B) that plays some role in the interaction
with N
protein (
30,
37). This region appears to play a similar
role in V protein (
80). Thus, it is possible that the
position
225 substitution could affect the interaction between the N
and
P proteins as well as between the N and V proteins. Perturbing
these interactions could lead to changes in the relative ratios
of N-P
and N-V complexes and possibly alter the effective availability
of N
protein for
encapsidation.
V protein is dispensable for growth in cultured cells
(
68), but several studies indicate that V protein may be a
virulence
factor. Sendai virus defective for V protein synthesis is
less
pathogenic in mice (
18,
36,
40,
41). Furthermore, a
recombinant
lab strain of Edmonston B that is defective for V protein
expression
replicates less efficiently in some experimental model
systems
(
56,
60,
80,
81). The connection between
pathogenicity
and V proteins suggests that mutations in the V ORF
should be
considered potential attenuation
determinants.
Finally, the variability in the P cistron also affected the C protein
ORF (Fig.
3). Like V protein, alterations that affect
C protein are
intriguing because C protein is dispensable for
MV growth in Vero cell
culture (
63) but may be an important
factor influencing
pathogenicity. In the SCID mouse model system,
transplanted human
thymic tissue supports less viral replication
if infection is performed
with a recombinant Edmonston B strain
that cannot express C protein
(
81). In addition, the C protein
defect appears to hinder
replication in cultured human peripheral
blood mononuclear cells
(
23). How C protein regulates growth
in these model
systems is not understood, but it can be inferred
from studies with
Sendai virus (
8,
15,
32) that MV C protein
may modulate
viral RNA synthesis. Also, in Sendai virus, C protein
seems to counter
innate immune responses to infection induced
by interferon
(
26), raising the possibility that MV C protein
may
perform a similar function. Care must be taken when drawing
these
comparisons between MV and Sendai virus C proteins given
that Sendai
virus encodes multiple C protein species (
48), while
MV
encodes only one known C protein. Yet it is interesting to
note that
Sendai virus C protein is dispensable for growth in
cell culture like
MV C protein and that Sendai virus defective
for C protein expression
is less virulent in mice (
25,
46,
57).
M gene.
Ten coding changes were identified in the vaccine
virus M genes (Fig. 2B and 3). Only two of these were common to all
vaccine strains (positions 61 and 89); these were two nonconservative changes that replaced a wt nonpolar glycine for an aspartic acid at
position 61 and a wt glutamic acid for lysine at position 89. The
substitution at position 61 has been detected also in some circulating
wt strains (data not shown and references 16 and 67).
Changes in M protein could influence the level of attenuation by
perturbing M protein function during virion maturation
(
83)
or transcriptional repression (
77). In
addition, the accumulation
of M gene mutations is one characteristic of
latent genomes indicating
that changes in the M gene can contribute to
an atypical virus
life cycle (
10).
F and H glycoproteins.
Since the glycoproteins are important
determinants of MV host range and cell tropism (39, 76) it
was expected that some mutations would accumulate in these genes after
serial passage in cells of nonprimate origin. In addition, the role
played by the viral glycoproteins in cell entry, cell fusion, and virus maturation (28, 83) raises the possibility that some of
the changes in F and H may influence the cell-to-cell spread of the virus and contribute to the attenuated phenotype. The F protein coding
region contained a number of codon changes, but none of these were
conserved in all vaccine strains (Fig. 3). The specificity of nine
codons varied in the H ORF. Three H amino acid substitutions were
conserved among all of the vaccine viruses, and a fourth differed in
all vaccines strains except Zagreb (Fig. 2B and 3). None of the amino
acid substitutions should affect the glycosylation pattern of H
(28).
The H gene actually accumulated the highest number of
substitutions that were shared by all vaccine viruses (Fig.
3). This
may reflect the fact that H protein is the receptor component
of the
virus envelope and significant changes were required to
adapt H protein
for effective infection of the heterologous cell
types used during
vaccine passage (
22). These changes in H protein
may also
contribute to attenuation if they render the virus less
efficient at
infecting human cells. In fact, several of the amino
acid residues that
were changed in the vaccine viruses have received
considerable
attention recently because studies indicate that
they play an important
role in binding to one of the cellular
receptors for MV (Fig.
4C). The
amino acids at positions 211 and
481 are both important for interaction
with one of the cellular
receptors (CD46) for MV (
7,
35,
49). Virus isolates with
a wt asparagine residue at position 481 do not readily infect
monkey kidney cell lines but efficiently infect a
transformed
marmoset lymphoid cell line (derivatives of B95-8 cells)
(
42,
54). In contrast, tyrosine at position 481 enhances
the ability
to infect monkey kidney cell lines. Interaction between H
protein
and CD46 also promotes clearance of CD46 from the infected-cell
surface. This phenomenon also depends on amino acids at positions
211 and 481. H proteins containing the vaccine virus amino acids
at these
positions displayed an enhanced ability to remove CD46
from the cell
surface (
7,
49). This has led to speculation
that more
potent clearance activity of vaccine H protein may contribute
to
attenuation. Recombinant MV used in primate studies probably
will be
necessary to definitively determine the role of H protein
in
attenuation.
L gene.
Nine amino acid coding changes were detected in the L
gene. The only one shared by all vaccines was at position 1717, where a
wt glutamic acid was substituted with an alanine. The L gene had one of
the lowest frequencies of amino acid substitution (Fig. 3), presumably
reflecting the intolerance of functional domains of this enzyme to
amino acid substitution.
To better assess the changes in L protein, they were displayed on
a domain map (Fig.
5). Domains of
homology exist among various
RNA-dependent RNA polymerases, including
the paramyxovirus L proteins
(
6,
53,
62,
72). It has been
proposed that these homologous
regions (RNA-dependent RNA polymerase
domains I to VI) (
6,
53,
62,
72) are functional domains.
Further comparison of
morbillivirus L proteins has led to a somewhat
different model
of three large domains (morbillivirus domains 1 to 3)
and two
hinge regions (
53). Curiously, only one vaccine
virus substitution
was located within the RNA-dependent RNA polymerase
domains I
to VI. This relatively conservative isoleucine-to-threonine
substitution
at position 331 was found in Moraten and Schwarz. This
region
of L protein is within a domain that interacts with P protein
(
34), suggesting that this amino acid substitution may
affect
L-P protein-protein interaction. This suggestion needs to be
examined
experimentally since a recent report indicates that a valine
substitution
found at this site in some circulating virus strains does
not
seem to affect the interaction between the L and P proteins
(
4).
The remaining amino acid coding changes affecting L
protein occurred
outside the boundaries of RNA-dependent RNA polymerase
domains
I to VI. This may indicate that domains I to VI indeed contain
regions essential for enzymatic function and that these regions
are
less tolerant of amino acid substitutions. Furthermore, it
is
attractive to speculate that the vaccine virus L protein amino
acid
substitutions occurred in regions of the protein that may
modulate
enzymatic activity rather than directly affect a region
that contains
an active site.
View larger version (50K):
[in this window]
[in a new window]
|
FIG. 5.
Position of amino acid substitutions relative to the
domain map of the L protein. The domain organization deduced from
analysis of RNA-dependent RNA polymerase sequences is labeled with the
roman numerals I to VI (6, 53, 62, 72), and the domain and
hinge structures predicted for morbillivirus L proteins are labeled 1 to 3 (53). The location of amino acid substitutions is
marked with an arrowhead, and circles identify changes from the
Edmonston wt sequence.
|
|
Silent mutations.
Silent nucleotide substitutions were
identified at 17 positions (Fig. 3). Three of these base substitutions
were common to all of the vaccine strains (one each in the M, F, and L
coding regions). An additional silent substitution in P and V is
conserved in all vaccines, but it is not truly silent; three of the
four silent base changes in the P and V coding regions result in amino acid substitutions in the overlapping C ORF. Identification of several
silent mutations that were conserved in multiple vaccine strains could
support the idea that some of these base changes provide a
cis-acting advantage such as increased mRNA stability or
more favorable secondary structure for translation or may reflect selection due to codon bias. More likely it simply reveals that some
silent base substitutions were incorporated early during vaccine virus
passage. These are tolerated after incorporation and can be maintained
in the genome if they do not affect the fitness of the virus.
It was somewhat surprising to find that so few silent changes have
accumulated in light of the fact that the negative-strand
RNA virus
polymerases have a relatively high error rate (
19)
and the
vaccine viruses had been passaged extensively (Fig.
1).
Although this
is difficult to explain, it is not unique to the
Edmonston vaccines.
Biologically derived respiratory syncytial
virus vaccine candidiates
also have been noted to accumulate relatively
few nucleotide
substitutions after extensive passage in culture
(
13).
Comparison of AIK-C sequences.
The complete genomic sequence
of AIK-C was carefully analyzed previously in 1993 by Mori et al.
(55). Comparison of the coding and noncoding
(59) sequences generated by this laboratory (GenBank
accession number AF266286) to the earlier AIK-C sequence (GenBank
accession number S58435) revealed 21 nucleotide differences. Ten of
these predict amino acid substitutions, six were silent, and five were
located in noncoding regions. A likely explanation for many of these
differences lies in the types of tools and methods used to analyze the
virus genomes. Since 1993, sequencing technology, including the
enzymatic steps and gel systems, has advanced dramatically and now
allows for increased resolution of sequences containing secondary
structure. Inspection of regions containing the nucleotide differences
indicated that about 10 of the 21 discrepancies lie in areas of locally
high G+C content or contain sequences that may form intramolecular
duplexes. These regions would likely complicate sequence determination
and explain some of the differences between the presented sequence and
that of Mori et al. (55). A second source of variation may
reflect the fact that Mori et al. analyzed cloned cDNA fragments,
whereas we employed direct sequencing of RT-PCR fragments. Finally, a
third difference lay in the passage history of the sequenced AIK-C
viruses. Here, the virus was passaged a limited number of times in Vero
cells, whereas Mori et al. used chicken embryo cells. Taken together,
these technical variations probably account for the majority of
nucleotide differences in the two sequence determinations.
Only two of the nucleotide differences between our sequence and
that of Mori et al. (
55) affected amino acids that
distinguish
between Edmonston wt and the currently proposed AIK-C
sequence.
Amino acid 362 in F protein was a serine in Edmonston wt and
a
tyrosine in AIK-C, while the sequence of Mori et al. predicted
no
amino acid change. Similarly, in F protein, we found that all
of the
viruses except AIK-C encoded an H residue at position 419,
while AIK-C
encoded an N. The sequence of Mori et al. again predicted
no change
from the
wt.
Edmonston vaccine attenuation.
Comparison of a low-passage
isolate of the Edmonston wt strain with five vaccine virus derivatives
revealed distinguishing genetic changes in all coding (Fig. 3) and
noncoding regions (59). Finding genetic change in multiple
genes and noncoding regions may indicate that viral replication in
semipermissive cells requires modification of several components of the
virus replication cycle, including cell entry, gene expression, genome
replication, and virus maturation. Although a constellation of genomic
changes may be essential for adaptation to semipermissive growth
conditions, it remains to be determined whether all of these changes
are indeed necessary for effective attenuation. Clues from other
negative-strand RNA vaccine viruses and vaccine virus candidates
suggest that only a subset of these genome changes may be essential for
attenuation and that modification of the gene expression and
replication apparatus is a particularly critical target. For example,
mutations in the L polymerase gene of RSV have been found to
effectively modulate the degree to which live virus vaccine strains are
attenuated (82). Similarly, cold-adapted parainfluenza
virus type 3 vaccine candidate strains contain polymerase gene
mutations and base substitutions in the 3' transcription promoter
region that are attenuating (73, 74). Live influenza virus
vaccines generated by replacing the genes for the hemagglutinin and
neuraminidase glycoproteins retain an attenuated phenotype specified by
mutations within the components of the replication apparatus
(75). Taken together, these studies imply that polymerase
mutations, as well as cis-acting signal mutations, are
important contributors to the attenuated phenotype.
Consistent with this notion, all Edmonston vaccine viruses were
found to contain mutations in the L and P genes (Fig.
3) as
well as
within the leader region (
59). That polymerase gene
mutations in combination with
cis-acting signal mutations
significantly
contribute to attenuation by altering gene expression is
a relatively
simple and attractive hypothesis. Modulating the abundance
of
viral gene expression to achieve suitable immunogenicity while
limiting virus replication, dissemination, and injury is an essential
element of an optimally attenuated
virus.
Modulation of MV gene expression as a mechanism of attenuation may
involve more than the core polymerase complex and
cis-acting
signals in the leader. This concept also was proposed by Takeda
et al.
(
78) after they found that the coding differences between
a pathogenic MV strain and a Vero cell-adapted derivative resided
in
the core polymerase genes (L and P), as well as in the V and
C coding
regions. As described above, the vaccine viruses of the
Edmonston
lineage also have substitutions outside of the core
polymerase genes
that appear to have the potential to affect gene
expression. These
included substitutions within the N gene, the
genes encoding accessory
proteins (V, C, and M), and additionally
in noncoding regions that may
have important
cis-acting functions
(
59).
The possibility that regulation of gene expression and replication
plays a role in attenuation gains support from our analysis
and can be
illustrated by sequence comparison between the underattenuated
Rubeovax
strain and the identical genomes of the desirably attenuated
Moraten
and Schwarz vaccine viruses (Fig.
6).
Presumably, some
of the genetic differences between these viruses were
responsible
for the underattenuated phenotype of Rubeovax. Comparison
of these
virus genomes revealed differences in the L protein, the N
protein
(Fig.
3 and
6), and several putative
cis-acting
sequences. The
noncoding region changes (Fig.
6) (
59)
included nucleotide substitutions
in sequences corresponding to the
long untranslated region of
the F mRNA (nucleotides 4608 and 5308), the
M gene translation
start codon context (nucleotide 3431), and the gene
end signal
of the F gene (7234). It is also worth noting that most of
the
changes that distinguish Rubeovax from Moraten and Schwarz were
due
to substitutions in Moraten and Schwarz that did not occur
in the
Rubeovax genome. Thus, it is possible that the slightly
more wt
genotype in parts of the gene expression apparatus of
Rubeovax is
responsible for its underattenuated phenotype.
View larger version (14K):
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[in a new window]
|
FIG. 6.
Comparison between Moraten, Schwarz, and Rubeovax.
Substitutions that distinguish between wt and this group of vaccine
viruses are illustrated in the boxes representing each gene region. The
amino acid positions are indicated above the columns of amino acids.
Noncoding region changes (59) in the intergenic regions
that distinguish Moraten and Schwarz from Rubeovax are shown along with
the nucleotide position below the boxed gene designations. Black
highlighting identifies substitutions that differentiate the identical
genomes of Moraten and Schwarz from Rubeovax. Morat/Schw, identical
genomes of Moraten and Schwarz.
|
|
An attractive feature of the hypothesis that links gene expression to
attenuation is the pathway it suggests for rational
negative-strand RNA
virus vaccine design
that downregulating viral
RNA synthesis in human
cells will effectively decrease replication
and contribute to
attenuation. Current molecular technology (
14,
58,
64,
65)
will facilitate the development of recombinant
measles viruses with
targeted mutations in different elements
of the gene expression
apparatus that can be used to test this
model. If a recombinant MV can
be developed that displays altered
gene expression, reduced replicative
ability, and safe levels
of attenuation, it may be possible to apply
these findings to
a wider range of
paramyxoviruses.
| |
ACKNOWLEDGMENTS |
We thank William Bellini and Paul Rota (CDC) for providing
vaccine strains, and Judy Beeler (CBER, FDA) for providing the Edmonston wt virus isolate. We also thank Martin Billeter and the
anonymous journal reviewers for their thoughtful reviews and suggestions. We are grateful to Shuo Lin for assistance with sequence comparisons.
The early part of this research was supported by NIH grant AI35286 to
S.A.U.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Viral Vaccine Research, Wyeth-Lederle Vaccines, 401 North Middleton
Rd., Pearl River, NY 10965. Phone: (845) 732-5450. Fax: (845) 732-5727. E-mail: udems{at}war.wyeth.com.
| |
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Journal of Virology, January 2001, p. 910-920, Vol. 75, No. 2
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.2.910-920.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
