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Previous Article | Next Article 
J Virol, February 1998, p. 1662-1665, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Determinant for Central Nervous System
Persistence Localized in the Capsid of Theiler's Murine
Encephalomyelitis Virus by Using Recombinant Viruses
Cecilia
Adami,1,2,
Arthur E.
Pritchard,3
Todd
Knauf,1,2
Ming
Luo,4 and
Howard L.
Lipton1,2,*
Department of Biochemistry, Molecular Biology
and Cell Biology, Northwestern University,1 and
Division of Neurology, Evanston
Hospital,2 Evanston, Illinois;
Center
for Macromolecular Crystallography, University of Alabama at
Birmingham, Birmingham, Alabama 35294-00054; and
Department of Biochemistry, University of Colorado Health Sciences
Center, Denver, Colorado 802623
Received 18 June 1997/Accepted 20 October 1997
 |
ABSTRACT |
The demyelinating process in Theiler's murine encephalomyelitis
virus (TMEV) infection in mice requires virus persistence in the
central nervous system. Using recombinant TMEV assembled between the
virulent GDVII and less virulent BeAn virus cDNAs, we now provide
additional evidence supporting the localization of a persistence
determinant to the leader P1 (capsid) sequences. Further, recombinant
viruses in which BeAn sequences progressively replaced those of GDVII
within the capsid starting at the leader NH2 terminus suggest that a
conformational determinant requiring homologous sequences in both the
VP2 puff and VP1 loop regions, which are in close contact on the virion
surface, might underlie persistence.
| |
TEXT |
Recombinant Theiler's murine
encephalomyelitis viruses (TMEV) constructed by exchanging
corresponding genomic regions between the virulent GDVII and less
virulent BeAn or DA virus cDNAs have been used to map a determinant for
virus persistence to the leader P1 sequences encoding the leader and
capsid proteins (2, 4, 12, 18). However, reports regarding
whether this determinant can be contributed only by the less virulent
TMEV or whether highly virulent strains also contribute are conflicting
(4, 18). Direct assessment of GDVII virus persistence is
difficult because infected animals do not survive the acute period,
even at low inoculation doses, e.g., 1 50% lethal dose
(LD50) (8). Use of recombinant viruses for
finer-scale mapping of pathogenetic determinants within the TMEV capsid
where there is extremely tight packing of amino acids in protomers is
needed but may be problematic, especially considering the large number
of amino acids that differ between the parents. The recombinant nature
of such constructs tends to result in nonviable or growth-compromised
viruses (16, 19). We now provide further evidence supporting
the localization of a persistence determinant to the capsid as
suggested by McAllister et al. (12) and the existence of a
conformational determinant within the capsid requiring homologous
sequences in both the VP2 puff and VP1 loop regions, which are in close
contact on the virion surface (5, 9, 10).
The leader P1 (capsid) region is responsible for persistence.
Eleven TMEV recombinants were assembled to map the central nervous
system (CNS) persistence phenotype (Fig.
1). In recombinant 7B, the leader capsid
genes of BeAn virus were replaced with those of GDVII by using
restriction endonuclease sites SspI (within the NH2 terminus
of the leader gene) and XhoI (at the 1D/2A dipeptide cleavage site). Recombinant 3B, which was constructed with the same
restriction endonuclease sites, replaced the leader capsid genes of
GDVII with those of BeAn virus. Recombinant 7B was neurovirulent, and
no mice survived intracerebral (i.c.) doses of 103 PFU or
more (LD50, 
103 PFU). By contrast,
recombinant 3B produced no deaths at an i.c. dose of 106
PFU (LD50, 
106 PFU), and SJL mice inoculated
with 3B developed virus persistence in the CNS and demyelination. This
result supports a more precise localization of a TMEV persistence
determinant to the leader P1 (capsid) sequences than earlier studies
(12).
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FIG. 1.
The genome structures of parental and recombinant TMEV.
The organization of the TMEV RNA genome, showing the 5' untranslated
region (UTR), leader protein, and capsid proteins, is depicted at the
top. Filled bars, the highly virulent GDVII virus sequences; open bars,
the less virulent BeAn virus sequences. Recombinant cDNA clones were
assembled in pGEM-4 downstream of the T7 RNA polymerase promoter by
exchanging equivalent sequences from full-length parental clones
(2, 16). Leader P1 (capsid) exchanges were in subgenomic
clones with the restriction endonucleases shown here. Subgenomic
constructs were assembled into full-length clones by using the unique
XhoI site at 1D/2A and the ScaI site in pGEM-4.
An XhoI site, which was absent in wild-type GDVII, was
introduced by a T-to-C mutation by site-directed mutagenesis as
described elsewhere (16). Recombinant 33 was made by PCR
amplification and splicing by overlap extension to yield BeAn sequences
between nucleotides 1523 and 2219 (6). The 10% difference
in the nucleotide sequences of the two parental viruses enabled
confirmation of the origin of all exchanged genomic parts by cleavage
of cDNA clones with restriction endonucleases. In addition, the capsid
sequences across splice sites in the recombinants and the 2376 BeAn
nucleotides between the SspI and MluI sites in
recombinant 41 were verified by PCR amplification and dideoxynucleotide
sequencing of RNA isolated from viral stocks. (A) Parental viruses,
recombinant viruses in which the GDVII and BeAn leader capsid regions
were exchanged, and recombinant viruses that progressively replaced
GDVII with BeAn sequences within the capsid starting at the
SspI restriction endonuclease site in the NH2 terminus of
the leader; (B) TMEV recombinants that progressively replaced GDVII
with BeAn sequences within the capsid starting at the XhoI
restriction endonuclease site at the 1D/2A cleavage site.
|
|
Intracapsid exchanges reveal a minimal BeAn persistence
determinant.
To more finely map the persistence phenotype,
additional BeAn leader P1 (capsid) exchanges were assembled on a GDVII
background, with BeAn sequences progressively replacing those of GDVII
capsid genes starting at SspI in the 5' end of the leader
(Fig. 1A). We hoped that a persistent recombinant would be obtained
when the minimal BeAn determinant(s) of persistence was included.
Recombinants 38, 39, 40, and 41 produced progeny virus upon
transfection of BHK-21 cells and replicated in the mouse CNS (Table
1). Only CNS virus growth in mice
inoculated with recombinant 40 was severely compromised, since these
animals had virus titers that were >180-fold lower than those of BeAn
virus-inoculated mice. Recombinant 42 was not viable upon transfection
and could not be used for mapping. A complementary series of constructs
was also generated in which BeAn sequences progressively replaced their
GDVII counterparts starting at the XhoI site at the 3' end
of the capsid, e.g., recombinants 29, 35, 37, and 33 (Fig. 1B).
However, all but one construct, recombinant 29, which was as
neurovirulent as GDVII (data not shown), were nonviable and were not
informative for mapping.
In vitro growth characteristics of recombinant viruses.
The
progeny derived from the six recombinant constructs yielding viable
viruses were assessed for plaque size, virus RNA replication, and
growth kinetics. Recombinant 7B produced large plaques (>3-mm diameter), whereas 3B produced small plaques (<1-mm diameter). The
four recombinants in which BeAn leader P1 (capsid) sequences progressively replaced those of GDVII on a GDVII background all produced smaller plaques, e.g., similar to those of BeAn virus. Recombinants 38 and 40 produced plaques that were slightly larger and
much smaller, respectively, than those of the other two recombinants. Viral RNA replication (not shown) and one-step virus growth kinetics for recombinants 7B and 3B more closely paralleled those of the parent
that contributed the nonstructural genes, e.g., the 3D RNA polymerase
(Fig. 2A). Single-step growth kinetics
for recombinants 38 and 41 were similar to that of BeAn, with final
virus yields of 70 to 90 PFU/cell, whereas recombinants 39 and 41 were
growth delayed, with final yields of 10 PFU/cell (Fig. 2B). This
suggests that recombinants 39 and 40 are defective in RNA replication
or virion assembly, as demonstrated for other GDVII-BeAn capsid
recombinants (16).
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FIG. 2.
One-step growth kinetics of GDVII and BeAn parental
viruses and recombinant viruses in which the GDVII and BeAn
leader-capsid regions were exchanged (A) and TMEV recombinants that
progressively replaced GDVII with BeAn sequences within the capsid
starting at the SspI site at the NH2 terminus of the leader
protein (B). BHK-21 cell monolayers were infected at a multiplicity of
infection of 10 and incubated at 33°C until the indicated times, when
virus lysates were prepared.
|
|
The minimal BeAn determinant for CNS persistence requires
replacement of the entire capsid, excluding the carboxyl half of VP1
(1D).
Additional SJL mice were inoculated i.c. with parental BeAn,
and the recombinant viruses and monitored to days 60 to 90 postinfection (p.i.) (Table 2; Fig.
3). Of 14 mice inoculated with
recombinant 3B, 13 (92%) developed signs of chronic demyelinating
disease, e.g., a waddling, spastic gait, tremors, and neurogenic
bladders, and 3 of 3 mice examined had extensive inflammatory
demyelinating lesions throughout the spinal cord. Infectious virus was
detected in the CNS in 9 of 10 mice inoculated with 3B. None of the
mice inoculated with recombinants 38 or 39 developed demyelinating disease or virus persistence and demyelinating lesions; however, rare
mononuclear inflammatory cell infiltrates were seen in the spinal cord
white matter. While 1 of 19 mice inoculated with the recombinant 40 developed demyelinating disease, none of 8 mice had evidence of virus
persistence and only 1 of 10 had demyelinating lesions. The one
clinically affected recombinant 40-inoculated mouse had extensive
demyelinating lesions; spinal cord tissue from this
paraformaldehyde-perfused animal was not available for virus assay.
Considering the low acute CNS virus titers in recombinant 40-inoculated
mice, it remains unclear why this single animal developed CNS
persistence and demyelination.
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FIG. 3.
Virus titers in spinal cord homogenates from individual
mice killed at approximately 90 days after i.c. inoculation with BeAn
or recombinant viruses. The minimum detectable virus level of the assay
is 50 PFU (dashed line).
|
|
In contrast, 11 of 15 (70%) recombinant 41-inoculated mice developed
demyelinating disease, 4 of 4 mice examined had spinal cord
demyelination, and 10 of 10 developed CNS persistence (Table 2; Fig.
3). The inflammation and demyelination in recombinant 41-inoculated
mice were indistinguishable from those observed for parental BeAn
virus-inoculated mice. Therefore, the minimal BeAn determinant for CNS
persistence appears to require almost the entire replacement of the
capsid, excluding the carboxyl half of VP1.
Finer mapping of the TMEV persistence phenotype by assembling
recombinants in which GDVII was progressively replaced with BeAn
starting in the leader showed that persistence was restored only when
BeAn extended from the leader to approximately halfway through VP1 (169 of 276 VP1 residues replaced in recombinant 41). When BeAn sequences
extended only into the NH2 terminus (30 of 276 VP1 residues replaced in
recombinant 39) or one-fourth of the way through VP1 (65 of 276 VP1
residues replaced in recombinant 40), virus persistence was not
observed. There are at least two interpretations of this result. First,
one or more of the nine BeAn VP1 residues that are different in GDVII
between the MunI and MluI restriction
endonuclease sites might be required for virus persistence. Although
the use of recombinants between virulent and attenuated parental
viruses has served to map major pathogenic determinants in other
picornaviruses to a single structural amino acid (1a, 3, 15,
17), there are relatively greater amino acid differences between
GDVII and BeAn in the capsid (a total of 40 residue differences
[14]), arguing against the possibility that a single
residue underlies TMEV persistence. Nonetheless, we are currently
testing this hypothesis by mutating these GDVII residues to their BeAn
counterparts in the GDVII parent. Recombinants 29, 37, and 33, which
progressively replaced the GDVII capsid with BeAn starting at the 1D/2A
cleavage site and intended to resolve this issue, were
either neurovirulent or nonviable. Similar GDVII-DA
constructs replacing GDVII with DA sequences in VP1 have also been
nonviable (1). A second possibility is that since BeAn
sequences in the nonpersisting recombinants 39 and 40 ended upstream of
the VP1 loops, whereas in the persisting recombinant 41 BeAn
nucleotides extended through the VP1 loops, BeAn persistence depends on
a conformational determinant that requires homologous sequences in the
VP2 puff and VP1 loops, which closely interact on the virion surface
(5, 9, 10).
The idea of a conformational determinant that may involve these surface
loops is supported by recent observations on the persistence of another TMEV recombinant, GD1B-2C/DAFL3. This GDVII-DA
recombinant virus, which is partially neurovirulent, persists in the
CNS and produces demyelination (4, 18). GD1B-2C/DAFL3
contains GDVII sequences in the carboxyl half of VP2 (amino acids 152 to 267) and in VP3 and VP1 on a DA virus background and was constructed by using the NcoI site between the sequences encoding VP2
puff A and puff B. As a result, GD1B-2C/DAFL3 has a hybrid VP2 puff, with puff A containing DA sequences and puff B containing GDVII sequences. Recently, the GDVII NcoI-AatII or
1B-2C fragment was assembled into parental DA cDNAs constructed in
different laboratories (11, 13). It was shown that VP2
residue 141 on the tip of puff A was a Lys in one construct and an Asn
in the other (7). GD1B-2C/DAFL3 persisted only when DA VP2
141 was a Lys (7), highlighting the location of this residue
within the VP2 puff. The fact that progeny derived from the DA parental
clones persisted with either Lys or Asn in this position suggests that
mutations in residue 141 in the recombinant affect the conformation of
downstream GDVII sequences. Most likely, because of its proximity to
VP2 puff B, mutation of residue 141 changes the conformation of VP2 puff B and, indirectly, that of the VP1 loops.
Our recombinant 38, in which BeAn sequences in the capsid were replaced
by GDVII in the carboxyl terminus of VP2 (amino acids 237 to 267) and
in VP3 and VP1, is similar to GD1B-2C/DAFL3, except that the
NH2-terminal 24 amino acids of the leader protein are from GDVII and
that the VP2 puff and downstream VP2 nucleotides are entirely BeAn.
Recombinant 38 is also partially neurovirulent, but surviving mice do
not develop persistent CNS infections. The reason for the difference in
virus persistence between GD1B-2C/DAFL3 and recombinant 38 is not
readily evident, since the 24 NH2-terminal, less-virulent residues in
the leader protein are not required for persistence of GDVII-BeAn
recombinant 3B (this study) or GDVII-DA recombinant R2 (12).
However, changes in the region of the VP2 puff may affect the
conformation of its other parts or that of neighboring structures,
particularly VP1 loop 2, which interacts with VP2 puff B (5, 9,
10). For example, in BeAn, the carboxyl group of VP2 puff B Asp
170 forms a hydrogen bond with the side chain of Arg 172 and the side
chain of VP1 loop 2 Trp 95 (9). The latter is probably
mediated by a water molecule. Similar VP2 puff B and VP1 loop 2 interactions are found in the GDVII and DA structures (5,
10). These surface loop interactions might well be altered in the
recombinant viruses, yet general structural changes due to the
recombinant nature of the constructs cannot be predicted from the
parental structures. Such general structural changes may also affect a
putative conformational determinant. However, perhaps the most
persuasive argument for the involvement of a conformational determinant
of the VP2 puff and VP1 loops in persistence is the fact that both
GD1B-2C/DAFL3, which contains mostly GDVII capsid sequences, and
recombinant 41, which contains mostly BeAn capsid sequences, cause CNS
persistence.
| |
ACKNOWLEDGMENTS |
We thank Kristi Jensen and Alastair Lynn-Macrae for technical
assistance.
This research was supported by U.S. Public Health Service grants NS
21913 and NS 23349 from the National Institutes of Health and The
Leiper Trust.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Neurology, Evanston Hospital, 2650 Ridge Ave., Evanston, IL 60201. Phone: (847) 570-2168. Fax: (847) 570-1568. E-mail:
hllipton{at}merle.acns.nwu.edu.
Present address: Department of Experimental Medicine and
Biochemical Sciences, Section of Microbiology, University of Perugia Medical School, Perugia, Italy.
| |
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J Virol, February 1998, p. 1662-1665, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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