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JOURNAL OF VIROLOGY,
0022-538X/99/$04.00⫹0
Nov. 1999, p. 9568–9575 Vol. 73, No. 11
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Observation of Measles Virus Cell-to-Cell Spread in
Astrocytoma Cells by Using a Green Fluorescent
Protein-Expressing Recombinant Virus
W. PAUL DUPREX,
1
* STEPHEN MCQUAID,
2
LARS HANGARTNER,
3
MARTIN A. BILLETER,
3
AND BERT K. RIMA
1
School of Biology and Biochemistry, The Queen’s University of Belfast, Belfast BT9 7BL,
1
and Neuropathology
Laboratory, Royal Group of Hospitals Trust, Belfast BT12 6B1,
2
Northern Ireland, United Kingdom,
and Institut fu¨r Molekularbiologie, Universitat Zu¨rich-Irchel, 8057 Zu¨rich, Switzerland
3
Received 21 April 1999/Accepted 22 July 1999
A recombinant measles virus (MV) which expresses enhanced green fluorescent protein (EGFP) has been
rescued. This virus, MVeGFP, expresses the reporter gene from an additional transcription unit which is
located prior to the gene encoding the measles virus nucleocapsid protein. The recombinant virus was used to
infect human astrocytoma cells (GCCM). Immunocytochemistry (ICC) together with EGFP autofluorescence
showed that EGFP is both an early and very sensitive indicator of cell infection. Cells that were EGFP-positive
and ICC-negative were frequently observed. Confocal microscopy was used to indirectly visualize MV infection
of GCCM cells and to subsequently follow cell-to-cell spread in real time. These astrocytoma cells have
extended processes, which in many cases are intimately associated. The processes appear to have an important
role in cell-to-cell spread, and MVeGFP was observed to utilize them in the infection of surrounding cells.
Heterogeneity was seen in cell-to-cell spread in what was expected to be a homogeneous monolayer. In tissue
culture, physical constraints govern the integrity of the syncytia which are formed upon extensive cell fusion.
When around 50 cells were fused, the syncytia rapidly disintegrated and many of the infected cells detached.
Residual adherent EGFP-positive cells were seen to either continue to be involved in the infection of sur-
rounding cells or to remain EGFP positive but no longer participate in the transmission of MV infection to
neighboring cells.
Measles virus (MV) is a morbillivirus which belongs to the
family Paramyxoviridae and is therefore a member of the
Mononegavirales, all of which have largely similar replication
strategies (39). Its single-stranded RNA genome is composed
of 15,894 nucleotides and contains a total of six transcription
units, which are separated by intergenic trinucleotide spacers.
The transcription units encode the six major structural proteins
of the virus. Due to RNA editing and the use of multiple
translation start sites, at least two further proteins, designated
V and C, are generated from the genome (4, 21). Transcription
occurs by sequential, interrupted copying from the six genes. A
gradient of transcripts, which decrease in abundance depend-
ing on the distance of the gene from the single 3⬘ terminal
promoter, is generated (10). Alterations in the MV transcrip-
tion gradient have been observed in infected human astrocy-
toma cells (44), and astrocytes have been shown to be infected
by MV in vivo (1, 29, 33). How these cells become infected is
unclear. Neurones are also infected in vivo, and transynaptic
spread of the virus in the central nervous system (CNS) has
been suggested but remains unproved (1).
Full-length infectious clones are available for a number of
negative-stranded RNA viruses, including MV (11, 22, 40, 48,
57). Reverse genetics has demonstrated that the genomes of
members of the Mononegavirales, for example, vesicular sto-
matitis virus, can tolerate substantial rearrangements in gene
order (56). Additional transcription units (ATUs) have been
added to respiratory syncytial virus and vesicular stomatitis
virus, and the foreign genetic material is stably retained after
many cycles of multiplication in cultured cells (7, 47). Recently,
interleukin-12 has been incorporated into the MV genome.
The additional sequences represent an additional 20% of the
genome, and they were retained for at least 10 passages (50).
These observations indicate that negative-stranded genomes
exhibit a remarkable degree of flexibility in both their length
and organization. In addition, the MV rescue system (40) has
been used to investigate the function of the M, F, H, P, V, and
C virus proteins (8, 9, 41, 43); pathogenesis (8, 17, 52, 54); and
virus maturation and assembly (49).
Green fluorescent protein (GFP) has recently become a
widely used reporter gene. The protein’s autofluorescence has
been enhanced by mutating amino acids surrounding the chro-
mophore to alter the excitation peak (23), and its expression
levels have been increased by changing the codons of the gene
to those which are frequently represented in human genes (58).
These modifications resulted in the production of an enhanced
GFP (EGFP) variant. Both GFP and EGFP genes have been
inserted into a number of viral genomes, for example, those of
herpes virus 1 and mouse hepatitis virus (19, 20). Simian vari-
cella-zoster virus pathogenesis and latency have been studied
in experimentally infected animals following GFP insertion by
homologous recombination (31). Additionally, GFP has been
fused to the Tat protein of human immunodeficiency virus type
1 to study trafficking and intracellular localization (51).
It is generally assumed that MV is propagated in the CNS in
the absence of budding, possibly by cell-to-cell fusion, and the
intimate relationship which is known to exist between astro-
cytes and neurones (16, 25) may facilitate cell-to-cell spread. In
* Corresponding author. Mailing address: School of Biology and
Biochemistry, The Queen’s University of Belfast, Medical Biology
Centre, 97 Lisburn Rd., Belfast BT9 7BL, Northern Ireland, United
Kingdom. Phone: 01232 272060. Fax: 01232 236505. E-mail: p.duprex
@qub.ac.uk.
9568
this paper we report on the utilization of a recombinant MV
which expresses EGFP to examine astrocytoma cell infection.
We show that EGFP is a very early indicator of cell fusion and
infection compared to the immunocytochemical (ICC) detec-
tion of nucleocapsid protein, the most abundant MV protein.
Finally, we show that the cellular processes of these glial cells
mediate MV spread in tissue culture.
MATERIALS AND METHODS
Cells and viruses. The MV rescue cell line, 293-3-46, was maintained as
previously described (40). Vero cells were grown in Dulbecco’s modified Eagle
medium (DMEM; Gibco) containing 8% newborn-calf serum (NCS; Gibco).
These were used for the growth of recombinant MVs, which were propagated in
DMEM containing 2% NCS. Stocks of rescued, plaque-purified viruses were
produced by passage in Vero cells. Titers were obtained by 50% endpoint
dilution assays and are expressed in 50% tissue culture infectious doses deter-
mined by the method of Reed and Muench (42). Human astrocytoma cells
(GCCM) were obtained from an anaplastic astrocytoma grade IV and were
grown in DMEM supplemented with 5% fetal calf serum (Gibco).
Plasmids and virus rescue. Plasmid pMeGFPNV includes the antigenome of
MV with an insertion containing the open reading frame of EGFP flanked by the
3⬘ and 5⬘ untranslated regions of the N gene. The cloning strategies used in the
generation of pMeGFPNV will be reported elsewhere (23a). The ATU is com-
posed of 852 nucleotides, and pMeGFPNV conforms to the rule of six (28). The
insertion was made into the full-length infectious clone of MV, p(⫹)MV
(EMBL accession no. Z66517), between the 3⬘ end and the gene encoding the
nucleocapsid protein. Recombinant virus was recovered from this plasmid by
using the 293-3-46 rescue cell line, which stably expresses T7 RNA polymer-
ase and the N and P proteins of MV (40). Briefly, the cell line was transfected
with pMeGFPNV (5 g) and pEMCLa (10 ng), which expresses the MV
polymerase protein under the control of the T7 promoter, using a calcium
phosphate transfection procedure. The cell sheets were monitored microscopi-
cally each day for the appearance of syncytia. Autofluorescence within these
syncytia, indicating EGFP expression, was verified with an inverted UV micro-
scope (Leica). Virus stocks were produced following plaque purification, and
titers of approximately 5 ⫻ 10
5
50% tissue culture infectious doses/ml were
obtained. The stocks were stored at ⫺70°C. Fluorescence microscopy was used
routinely to determine that EGFP expression was retained upon virus passage
and to ensure that mutants which had lost the ability to express EGFP were
present below the limit of detection.
Immunofluorescence and confocal microscopy. GCCM cells were grown on
glass coverslips to 80% confluency. The monolayers were rinsed with mainte-
nance medium, and the cells were infected with MVeGFP at a multiplicity of
infection (MOI) of 0.01 and incubated for1hat37°C. After this time, unad-
sorbed virus was removed, maintenance medium containing 2% NCS was added,
and the cells were incubated for 50 to 60 h at 37°C. The presence of EGFP-
positive cells was verified by UV microscopy, and the cells were then fixed for 10
min in 4% paraformaldehyde. Infected cells were observed at a time point similar
to that following the parental Edtag virus infection, and it appears that the
presence of EGFP in an ATU does not significantly impede virus replication
(23a). Anti-MV nucleocapsid (N) monoclonal antibody (Seralabs) was diluted
1:1,000 in phosphate-buffered saline (PBS). Anti-tubulin monoclonal antibody
(Sigma) was diluted 1:1,500 in PBS. Human hyperimmune serum was obtained
from a patient with confirmed subacute sclerosing panencephalitis (SSPE) and
was used at a dilution of 1:1,000 in PBS. Primary antibodies were added to the
cells on the glass coverslips and incubated for1hat37°C. Incubation was
followed by three 5-min washes with PBS. Secondary antibodies, CY3-conju-
gated sheep anti-mouse (Sigma; 1:40) and CY5-conjugated goat anti-human
(Amersham; 1:40), were diluted in PBS and added to the coverslips, which were
then incubated for1hat37°C. The coverslips were washed three times in PBS
and mounted with Citifluor (Amersham). A Leica TCS/NT confocal microscope
equipped with a krypton-argon laser as the source for the ion beam was used to
examine the samples for fluorescence. CY5-stained samples were imaged by
excitation at 647 nm with a 664- to 696-nm-long-pass emission filter, and CY3-
stained samples were imaged by excitation at 568 nm with a 564- to 596-nm-
band-pass emission filter. EGFP was visualized by virtue of its autofluorescence
by excitation at 488 nm with a 506-538 band-pass emission filter.
Vital fluorescent microscopy. Astrocytoma cells (GCCM) were cultured to
60% confluence in 25-cm
3
tissue culture flasks. The cells were infected at an MOI
of 0.01 with MVeGFP. An inverted UV microscope was used to monitor the
monolayers for the appearance of single infected cells. The flasks were oriented
on the microscope stage, which was marked to permit the repeated observation
of the chosen groups of infected cells in the monolayers. Autofluorescent images
of the cells were collected by confocal scanning laser microscopy (CSLM). All
settings on the microscope were kept constant, and the laser intensity was
adjusted, if necessary, to maintain similar output levels. The flasks were handled
with extreme care to avoid disruption of the infected cell structures. Immediately
after the image was recorded, the infected monolayers were returned to 37°C for
further incubation. The stored images were used throughout the experiment to
precisely reorient the cells. Observations were made over a period of 50 h at
intervals of between 2 and 4 h.
RESULTS
Recombinant MVeGFP infects astrocytoma cells. Recombi-
nant MV expressing EGFP was recovered 6 days posttransfec-
tion. Figure 1 shows a schematic representation of the plasmid
pMeGFPNV, from which this virus was derived. In addition to
the complete MV genome pMeGFPNV contains an ATU
FIG. 1. Schematic representation of the full-length MV plasmids used for virus rescue. The solid lines indicate the positions of the intergenic trinucleotide spacers.
The positions of the T7 promoter, hepatitis delta ribozyme (␦), and T7 terminator (T⌽) are indicated (not to scale). Open reading frames encoding the virus structural
genes are shaded, and their flanking untranslated regions are represented as open boxes. The genome length of each virus is given in nucleotides (nt). (A) Structure
of p(⫹)MV encoding the MV Edmonston B strain antisense genome (Edtag). The sizes of the 3⬘ and 5⬘ untranslated regions of the N gene are indicated. (B) Structure
of pMeGFPNV encoding the MV Edmonston B strain antisense genome with an ATU which is composed of the open reading frame of EGFP flanked by sequences
based on the 3⬘ and 5⬘ untranslated regions of the N gene. A total of 852 additional base pairs are present in pMeGFPNV. The sizes of the untranslated regions are
indicated below and above the EGFP and N gene segments respectively.
VOL. 73, 1999 MEASLES VIRUS CELL-TO-CELL SPREAD 9569
composed of the complete EGFP open reading frame flanked
by 3⬘ and 5⬘ untranslated regions which are based largely on
those of the nucleocapsid gene. The ATU is located in the 3⬘
terminal region of the genome, prior to the N gene. Infectious
virus was recovered at a time point similar to that for Edtag
virus (control), indicating that the high-level expression of the
reporter gene and surrounding untranslated sequences has no
major effects on virus replication.
In this study the recombinant virus, MVeGFP, was used to
infect GCCM cells in order to assess the distribution of EGFP
and viral antigens with respect to the cytoskeleton. The latter
was visualized with an anti-tubulin monoclonal antibody. Fig-
ure 2A shows a single infected astrocytoma cell surrounded by
uninfected cells. Diffuse EGFP autofluorescence was detected
throughout the cytoplasm and seemed to accumulate in the
nucleus. The figure is a composite of several images represent-
ing different depths within the tissue. Examination of the in-
dividual sections indicated that EGFP was present throughout
the nucleus. Intranuclear localization of the reporter protein to
any particular area of the nucleus, or to the nuclear membrane,
was not observed. MV antigens were detected with an SSPE
antiserum and visualized with an anti-human CY5-conjugated
secondary antibody. A punctuate staining pattern was ob-
served, which is characteristic of cytoplasmic inclusion bodies
which contain N protein encapsidating viral RNA (1). The
inclusion bodies were seen to localize in the perinuclear region
of the infected cell. Tubulin was visualized indirectly with a
CY3-conjugated secondary antibody. This gave an indication
of the overall GCCM cell morphology, and extended astrocytic
processes, which are typical of an astrocytoma cell line, were
seen to connect the cells. Observing tubulin in conjunction with
EGFP indicated that every part of the cytoplasm contained the
reporter protein. No effects on the cytoskeleton were observed
within the infected cells.
EGFP is an early indicator of MV cell infection. From initial
observations of infected cells it appeared that EGFP was
present in cells which were negative for MV antigen. One of
two mechanisms could cause a cell to be EGFP positive and
MV antigen negative. First, due to the location of the EGFP
gene at the 3⬘ end of the genome and the consequent high
levels of expression, detection of autofluorescence could be
predicted to be sensitive in comparison to MV antigen detec-
tion by ICC methods. In this instance detection of EGFP
would sensitively reflect the rate of replication within the in-
fected cell. In a second scenario EGFP may passively enter
from earlier infected cells into new cells soon after MV-in-
duced cell fusion due to the contiguity of the cytoplasm. In this
case, the presence of EGFP would not reflect the amount of
viral replication within the newly infected cell. In order to
distinguish between these two possibilities, we made a direct
comparison between EGFP autofluorescence and the standard
FIG. 2. Immunoreactivity and autofluorescence in astrocytoma cells infected
with MVeGFP. GCCM cells were infected with MVeGFP at an MOI of 0.01 for
50 h. The cells were fixed and examined by CSLM for autofluorescence and
immunoreactivity. The micrographs represent an 8- to 10-m composite optical
section. The images were obtained in double- or triple-excitation mode. MV
antigens were detected with either human SSPE antiserum or a monoclonal
antibody which recognizes the MV nucleocapsid protein. EGFP was detected by
virtue of its autofluorescence, and the cell cytoskeleton was visualized with a
monoclonal antibody specific for tubulin. (A) A single cell infected with
MVeGFP. MV antigens, detected with SSPE antiserum (blue), tubulin (red), and
EGFP (green), are shown. (B) A single cell infected with MVeGFP (arrow) in
close proximity to a large syncytium; MV antigens, detected with SSPE antiserum
(blue), tubulin (red), and EGFP (green), are shown. MV antigen was not ob-
served in the single infected cell. (C) Cells in the early stages of MVeGFP
infection. MV nucleocapsid antigen (red) and EGFP (green) are shown. Arrow
a indicates two EGFP-positive and MV antigen-negative cells. Arrow b indicates
an EGFP-positive cell which also stains positive for a small number of MV
cytoplasmic inclusion bodies. The three cells indicated by arrows are connected
to the syncytium, in which relatively large amounts of viral antigen are detected.
Magnification, ⫻40.
9570 DUPREX ET AL. J. VIROL.
ICC detection of MV in infected cells. The most abundant
virus proteins, N and P, were visualized by indirect immuno-
fluorescence in GCCM cells infected with MVeGFP, as de-
scribed above. A triple-labelled composite image is shown in
Fig. 2B. Viral antigen and EGFP autofluorescence were ob-
served within the syncytium. A single infected cell, which is
negative for viral antigen, is indicated. Due to the cytoskeletal
staining, it is clear that this cell is isolated from the nearby
syncytium and is in the initial stages of infection. This type of
cell was frequently observed by confocal microscopy. The pos-
sibility that EGFP can passively enter recently fused cells is
also indicated in this figure. A cell was observed, at the pe-
riphery of the syncytium, which appeared to be antigen nega-
tive. To clarify this situation we used ICC to stain cells infected
with MVeGFP. Nucleocapsid protein was detected with a
highly sensitive anti-N monoclonal antibody recognized by a
CY3-conjugated sheep anti-mouse secondary antibody. CY3 is
a very stable fluorescent conjugate with the additional advan-
tage that, unlike CY5, it can be visualized directly by UV
microscopy. Figure 2C also shows that EGFP is detected in
MVeGFP-infected cells which are negative for MV N protein.
Two cells are shown in the very early stages of infection. Fusion
appears to have only recently taken place, as the cells are
physically joined to the syncytium. Antigen-containing intracy-
toplasmic inclusions are usually found surrounding the nuclei,
and these were clearly observed in the main body of the syn-
cytium. A low level of positive staining for nucleocapsid was
also observed in cells on the periphery of the syncytium (Fig.
2C). The protein is present in smaller amounts, and we believe
that these cells, like the N protein-negative/EGFP-positive
cells, are in early stages of infection. Three phases of MV
infection are therefore shown in Fig. 2C. First are the cells
within the syncytium which are producing large amounts of
MV proteins. Second are the cells which are EGFP positive but
have much lower levels of N antigen than those within the main
body of the syncytium. In this case EGFP autofluorescence
clearly shows that these cells are infected even though viral
antigen levels are low. Third are the cells which lack the most
abundant viral protein, N, but, because of the presence of
EGFP, must be fused to the syncytium. This was confirmed by
phase microscopy. Detection of these cells at this early stage of
infection has not been possible previously.
Processes mediate the rapid spread of MVeGFP from cell to
cell. From the above-mentioned results it appears that EGFP
autofluorescence within fixed tissue is a more sensitive indica-
tor of infection than ICC. Fixed tissues, however, do not allow
cell-to-cell spread of the virus to be examined. The availability
of the recombinant virus, MVeGFP, gives the first opportunity
of making observations of the spread of MV from one individ-
ual cell to another in real time.
GCCM cells were infected at a low MOI, and single infected
cells were observed by UV microscopy from 60 h postinfection.
Three representative time courses of MVeGFP-infected cen-
ters illustrating the variation in progression of the virus from
cell to cell are shown in Fig. 3. Observations were made at
appropriate time points over a total period of 50 h. In Fig. 3A,
two infectious centers are shown. The upper left center is
simply a single infected cell, whereas in the lower-right infec-
tious center fusion with a neighboring cell has already oc-
curred. MV-infected cells have fused with uninfected cells via
the connecting processes, leading to the formation of a syncy-
tium which is composed of two cells. Where these processes
are in intimate contact with other cells, the fusion is rapid, and
it can be seen that after 8.5 h many more cells are infected
(74.5 h post infection [p.i.]). In many cases extended cellular
processes of uninfected cells passed above or below the in-
fected GCCM cells, and these seemed to be refractile to in-
fection. This may indicate that intimate cell contact with the
end of the processes is a prerequisite for fusion. As time
proceeds and more cells are recruited into the syncytium, phys-
ical constraints lead to its breakdown. By 95 h p.i., the lower-
right syncytium has lysed and only a few residual infected cells
remain. By this time point the two infectious centers have
merged.
A different pattern of spread was observed when the initial
infection occurred in a cell which was in more intimate contact
with the cell bodies of the surrounding cells (Fig. 3B). In this
instance, by 66 h p.i. fusion of the surrounding cells produced
a syncytium in which the brightly fluorescent nuclei clustered in
the center. Diffuse EGFP autofluorescence was visible in the
cytoplasm of the syncytium. More cells were infected at this
time point, particularly at the periphery of the syncytium, than
in the time course presented in Fig. 3A. By 71 h p.i., cytoplas-
mic bridges had formed. These were derived from the con-
tracted cytoplasm of fused cells and do not represent astrocyte
processes. As time proceeds (76.5 h p.i.), more cells become
infected via interconnecting processes and the cytoplasmic
bridges are seen to become narrower. By 79 h p.i., the cyto-
plasmic bridges ruptured, causing the fused structure to disin-
tegrate, and a hole was left in the monolayer. The majority of
the surrounding infected cells remained adherent, and these
residual cells were observed for a further 37 h. The infection
proceeded in a fashion similar to that shown in Fig. 3A by
further outward spread via processes. Observations of other
infected centers indicated that in some cases these residual
cells, which remained after a syncytium had burst, could no
longer fuse with surrounding cells, and the infection process
appeared to terminate (data not shown).
Finally, a very different situation was observed for one of the
infectious centers which had been selected for observation at
66 h p.i. (Fig. 3C). In this case, repeated observations over 24 h
showed no progression of the infection, despite the presence of
surrounding GCCM cells which were readily observed by
phase microscopy. During this period, slight differences were
observed in the overall shape of the infected cells, indicating
that they had moved slightly with respect to each other (66, 77,
and 90 h p.i.). At 90 h p.i., the cells were no longer observed
every 2 to 4 h. Interestingly, in the final observation, at 116 h
p.i., many of the surrounding cells were seen to be infected.
This indicates that at some stage the impediment to infection
of these cells was surmounted and a burst of infection followed
as detailed in the series in Fig. 3A.
Partially autofluorescent cells were never observed in repeat
observations. It appears that upon fusion an influx of EGFP
from the infected cells causes the rapid dissemination of EGFP
into the cell cytoplasm. Autofluorescence is subsequently de-
tected, first in the nucleus, possibly due to nonspecific accu-
mulation, and secondly throughout the cytoplasm, rather than
at the site of cell fusion. This agrees with the observations
which were made in fixed cells (Fig. 2). All the main stages in
the spread of MVeGFP infection are shown in Fig. 4, and the
role of cellular processes is particularly evident. At 66 h p.i. two
infected cells were visible, and these were connected by an
astrocytic process. Four hours later (70 h p.i.), a further two
cells were infected, and the extended process from one of these
cells is indicated. By 74 h p.i., the neighboring cell was in the
very early stages of infection, as indicated by EGFP autofluo-
rescence in the nucleus. Cytoplasmic staining was not ob-
served. Five hours later, EGFP autofluorescence in this cell
was much more intense and a process from the cell was now
visible. Two hours later (81 h p.i.), the processes were more
defined, although at this stage in the time course no new cells
VOL. 73, 1999 MEASLES VIRUS CELL-TO-CELL SPREAD 9571
FIG. 3. Variation in the cell-to-cell spread of MVeGFP in astrocytoma cells (see text for details). GCCM cells at 60% confluency were infected with MVeGFP at
an MOI of 0.01. Infected cells were identified by UV microscopy, and the positions of the infectious centers in the monolayer were marked to aid in their reidentification
throughout the time course. Three representative time course experiments are shown (A, B, and C), and each demonstrates the variation in the virus spread from cell
to cell. The images were collected in a single optical section by CSLM in single-excitation mode. The number of hours postinfection at which each autofluorescent image
was collected is indicated. EGFP autofluorescence is shown in false-white color. Magnification, ⫻10.
9572
had been infected. Finally, by 87 h p.i. a number of connected
cells were infected and an extended process was visualized by
EGFP autofluorescence. This series clearly shows that pro-
cesses play an important role in cell-to-cell spread, especially
as it should be noted that uninfected cells were present be-
tween the autofluorescent cells.
DISCUSSION
Astrocyte infection by morbilliviruses has been demon-
strated both in vivo and in vitro (1, 5, 12, 26, 30, 33, 38, 45, 55).
The predominant cell types infected in SSPE are neurones and
oligodendrocytes (24). Infected glial cells have been detected
by immunohistochemistry, albeit at lower frequencies (1, 30,
33), but the degree of astrocyte infection in vivo remains con-
troversial (6, 18, 29, 32). Canine distemper virus, another mor-
billivirus, also infects astrocytes. The virus persists in the CNS,
replicating and spreading in astrocytes without eliciting an
inflammatory response (5, 26, 55). MV infects rat astrocytes in
tissue culture (45). A distinct alteration in the transcription
gradient of MV monocistronic messages is observed in exper-
imentally infected rodents (46), and this mirrors the in vivo
situation in brain tissue from patients with SSPE and measles
inclusion body encephalitis (10). Alterations in the transcrip-
tion gradient also occur in cultured human astrocytoma cells
(44). In light of these observations, we were first interested to
examine if MVeGFP could infect the human astrocytoma cell
line GCCM and, if so, at what stage in the infection EGFP
autofluorescence could be detected. Secondly, we wished to
examine, in real time, the cell-to-cell spread of MV in this glial
cell line.
GCCM cells were found to be readily infectable by
MVeGFP, and a diffuse cytoplasmic autofluorescence was ob-
served (Fig. 2). We have also verified that nontransformed
fetal astrocytes could be productively infected with MVeGFP.
The infection was highly fusogenic (data not shown). It was
evident that EGFP autofluorescence served as a very early
indicator of GCCM cell infection. Indeed, EGFP autofluores-
cence occurred in cells in which MV antigen was not detectable
by ICC staining with a highly specific monoclonal antibody
against the most common viral antigen, the nucleocapsid pro-
tein. This can be explained in two ways: (i) the influx of EGFP
from infected cells upon fusion or (ii) the ability to sensitively
detect the reporter gene by confocal microscopy soon after
FIG. 4. Process-mediated cell-to-cell spread in GCCM cells infected with MVeGFP. Astrocytoma cells at 60% confluency were infected with MVeGFP at an MOI
of 0.01. (A) Two infected cells were identified by UV microscopy at 66 h p.i. and observed for a further 21 h at approximately 4-h intervals. (B) The arrow indicates
an extended astrocyte process of a newly infected cell. (C) The arrow indicates the weakly autofluorescent nucleus of a cell in the very early stages of infection. (D and
E) The arrows indicate the same nucleus 5 and 7 h later. (F) The arrow indicates an extended astrocytic process emanating from the cell indicated in panels D and
E. EGFP-autofluorescent images were collected as single optical sections by CSLM and are shown in false-white color. Magnification, ⫻15.
VOL. 73, 1999 MEASLES VIRUS CELL-TO-CELL SPREAD 9573
primary infection before viral antigens reach detectable levels.
Both were demonstrated. Therefore, expression of EGFP by
MVeGFP serves to locate cells which are in the early stages of
infection, and this virus will be an invaluable tool to investigate
MV spread in vitro and possibly in vivo.
EGFP was seen to accumulate in the nuclei of GCCM cells
at a very early stage of cell infection. When nonenhanced GFP
was expressed in BHK21 cells with the Semliki Forest virus
system it was present diffusely throughout the cytoplasm and
was also seen to enter the nucleus (2). Our results seem to
indicate that a nonspecific mechanism may lead GCCM cells to
accumulate GFP in the nucleus. Recombinant EGFP-express-
ing pseudorabies virus has also been generated (27). In this
system it does not appear that EGFP accumulates in the nu-
cleus, although different cell lines were used. A monoclonal
antibody was used to detect EGFP by indirect immunofluores-
cence in MDBK cells infected with this recombinant virus.
Interestingly, when EGFP autofluorescence was compared
with ICC, the fluorescein isothiocyanate-labelled fluorescence
was much brighter despite the use of a strong gG promoter.
This limits the use of the EGFP reporter in this system.
In SSPE it is assumed, although not yet proven, that MV
spreads transneuronally from cell to cell, as no budding is seen
from the surfaces of infected cells (14, 37). Virus isolated from
various parts of infected brains of patients with SSPE is clonal,
suggesting that the virus entered the brain at a particular time
point with concomitant spread through the nervous system (3).
In a recent report MV was observed to spread through axonal
pathways from initial point infections in the olfactory bulbs in
the brains of C57BL/6 mice (53). Progression along olfactory
pathways was enhanced in mice which lacked the transporter
associated with antigen presentation (TAP) gene. Conversely,
in ependymal cells, which lack processes, lateral cell-cell con-
tacts have been suggested as the main means of MV propaga-
tion in the CNS of transgenic mice which lack the alpha-beta
interferon receptor and express CD46 (34).
Heterotypic coupling between glial cells in the CNS is
thought to be common, and this is thought to coordinate the
activities of the interconnected cells (59). Astrocytes are
known to be closely associated with neuronal synapses and to
provide trophic support for synapses (16, 25). Recent studies
suggest that astrocytes and neurones communicate reciprocally
through nonsynaptic mechanisms (35, 36). Astrocyte-astrocyte
contact is so extensive that these cells have been postulated to
form a generalized functional syncytium which extends for
large distances within the CNS (13). Gap junctions couple
astrocytes, and although it would be difficult to imagine MV
crossing the junction, the fusogenic nature of the virus may
permit cell-to-cell infection at these junctions without a re-
quirement for virus budding. Therefore, we tried to mimic the
process-to-process contacts of astrocytes within the brain by
utilizing subconfluent GCCM cells for the cell-to-cell spread
experiments. EGFP autofluorescence indicates that MV repli-
cation and gene expression is occurring within a cell or group
of fused cells. Using EGFP, it was possible to make noninva-
sive observations of MV cell-to-cell spread in real time. In
these in vitro experiments, processes were seen to connect the
cells, and these mediated the spread of the virus from cell to
cell (Fig. 3 and 4). Even though this study was carried out with
a single homogeneous monolayer of cells, three different types
of infection courses were observed (Fig. 3). The presence of
the process-to-process connections appears to produce a con-
nected group of fused cells rather than a typical MV syncytium.
In some of these experiments we observed infected cells which
were EGFP positive but which did not infect neighboring cells
(Fig. 3C). The reason for this is unclear. These cells were
clearly surrounded by others which seemed both to be in close
contact and to have connecting processes which would be ex-
pected to facilitate cell infection. The heterogeneous response
in what would be considered to be a homogeneous cell mono-
layer was an unexpected new observation.
The approaches used in this study will be very useful for
observing virus spread between neurones in vivo and may allow
the mechanism to be elucidated. We have used a mouse model
of MV-induced encephalitis to examine determinants of neu-
rovirulence in the H gene (15). The infection is predominantly
neuronal, and in the future it may be possible to utilize this
model to examine cell-to-cell spread in vivo with brain slices
from animals infected with a recombinant, neurovirulent virus
which also expresses the EGFP protein. Here, the virus could
function as a tracer of neuronal connections and may indicate
whether the hypothesis that MV spreads along specific ana-
tomical pathways is valid. One of the difficulties of studying
cell-to-cell spread by MV is that no cell pathogenic effect is
observed prior to the obvious fusion and formation of syncytia.
MVeGFP now provides an opportunity to study the early
stages of MV infection, allowing infected cells to be quickly
identified without the need for fixation.
This study has shed light on MV cell-to-cell spread in this
important glial cell type. We have shown that the autofluores-
cence of the reporter gene EGFP is a more sensitive indicator
of cell infection than standard ICC methods. Finally, it is clear
that MVeGFP will be an invaluable tool for future in vivo
studies of MV pathogenesis because of the high levels of tran-
scription which can be obtained upon infection.
ACKNOWLEDGMENTS
We thank Gudrun Christiansen for helpful discussions and invalu-
able advice on the establishment of the MV rescue system. Addition-
ally, we thank Roy Creighton for photographic work and Paula Had-
dock for excellent technical assistance. We acknowledge the help of
Uta Gassen in critical reading of the manuscript.
This work was supported by the Wellcome Trust (grant 047245) and
the Swiss National Science Foundation (no. 31-43475.95).
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VOL. 73, 1999 MEASLES VIRUS CELL-TO-CELL SPREAD 9575
