D9193.PDF

Rev. sci. tech. Off. int. Epiz., 1 9 9 8 , 1 7 (1), 220-230
Mx proteins: mediators of innate resistance
to RNA viruses
0. Haller (1), F r e s e
(2)
& G. K o c h s
(1)
(1 ) Department of Virology, Institute for Medical Microbiology and Hygiene, University of Freiburg,
Hermann-Herder-Strasse 11, 79104 Freiburg, Germany
|2) Division of Plant Biology, The Scripps Research Institute, 10666 N. Torrey Pines Road, La Jolla, CA 92037,
United States of America
Summary
M x p r o t e i n s a r e i n t e r f e r o n - i n d u c e d m e m b e r s of t h e d y n a m i n s u p e r f a m i l y o f large
guanosine triphosphatases. These proteins have attracted attention
because
s o m e d i s p l a y a n t i v i r a l a c t i v i t y a g a i n s t p a t h o g e n i c RIMA v i r u s e s , f o r e x a m p l e
against m e m b e r s of t h e o r t h o m y x o v i r u s (influenzavirus) f a m i l y or t h e bunyavirus
f a m i l y . T r a n s f e c t e d c e l l s a n d t r a n s g e n i c m i c e e x p r e s s i n g M x p r o t e i n s a r e highly
resistant t o Mx-sensitive viruses, demonstrating that M x proteins are powerful
a n t i v i r a l a g e n t s . In h u m a n s , s y n t h e s i s o f M x A is o b s e r v e d d u r i n g s e l f - l i m i t i n g viral
infections and may thus promote recovery from disease.
Keywords
Antiviral agents -
Disease resistance -
Genetics -
Guanosine triphosphatase -
Influenzavirus - Interferons - M x protein - Transgenic animals.
Influenza: an ongoing major
health problem
Influenza is a highly contagious, acute respiratory illness that
is still a major health problem around the world today.
Epidemics caused by influenza A or B viruses occur regularly,
and often lead to high mortality in the elderly. In addition,
influenza A viruses can cause devastating pandemics in
humans: the pandemic of 1918 killed an estimated 2 0 million
people world-wide ( 3 7 ) . Likewise, influenza A viruses are
responsible for considerable morbidity and mortality in
domestic animals. Avian influenza viruses occur around the
world and outbreaks may cause tremendous economic losses.
For example, in 1983 an influenza outbreak occurred in
chickens and turkeys in Pennsylvania and Virginia, United
States of America (USA), and twelve million birds were lost at
a cost of more than U S $ 6 0 million (34). An avian influenza
virus currently circulating among domestic birds in Hong
Kong made headline news because of its potential to infect
and kill humans (7, 7 3 ) .
Although influenza viruses belong to the group of best studied
viruses, efficient control of the disease caused by these viruses
is still not possible ( 7 7 ) . To compound the problem,
immunisation regimes are continually being confronted with
the extreme antigenic variability of influenza A viruses which
is caused by antigenic drift and shift. New approaches and
reagents to control influenza are badly needed.
For this reason and others, the study of the M x system has
become extremely attractive. This system, originally described
as the inborn resistance of mice to influenza viruses (42, 43),
is now clearly present in other species (including man) which
also have antivirally active Mx proteins. Unlike antibodies, Mx
proteins work intracellularly, by interacting with the viral
replication machinery in the infected cell. These proteins are
active against all influenza A viruses, irrespective of origin or
antigenic composition. Detailed analysis of their mode of
action will provide new insights into the intricacies of the
influenza virus life-cycle and should allow the identification of
targets for novel and specific antiviral drugs. This article
reviews the current understanding of Mx proteins and
discusses possible applications in the light of earlier and more
recent findings.
Reasons for mouse resistance to
influenza viruses
Many years ago, researchers observed a mouse strain which
proved resistant to large doses of influenza virus that were
lethal to ordinary laboratory mice ( 4 2 ) . Screening of
numerous inbred strains showed that this type of inborn
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Rev. sci. teck Off. int. Epiz. 17(1)
resistance was present in just a few mouse strains, including
A2G and SL/NiA ( 1 , 2 3 , 4 4 ) . Resistance was inherited as a
single, dominant trait and was specific for orthomyxoviruses
(43). Subsequent work revealed that this resistance was
conferred by a single gene, M x l , localised on chromosome 16
(61). The M x l gene consists of 14 exons spreading over a
stretch of more than 55 kilobase pairs of genomic DNA (29).
The Mxl gene is normally not expressed. However, gene
expression is rapidly induced upon viral infection through the
action of interferon type I ( a and B) ( 2 1 , 2 2 ) . Unexpectedly,
the rare resistance allele turned out to represent the wild-type.
In influenza-susceptible mice, the M x l gene is defective.
Thus, most inbred strains of mice carry non-functional M x l
alleles (69). The reason why intact M x l genes are absent in
most inbred mouse strains is still an open question, but
researchers working with such mice should be aware of the
fact that they are using animals with a genetic defect. The
mouse genome contains a second Mx gene, designated Mx2
(70), which also maps to chromosome 16 and appears to be
closely linked to M x l . Interestingly, this gene is also defective
(70).
Functional M x l genes are found in wild mice and in some
mouse strains derived from wild mice ( 1 , 2 3 , 2 4 ) . This shows
that resistance is not an artifact of inbreeding and, more
importantly, indicates that M x l resistance must serve a
function in wild mouse populations. This is puzzling because
wild mice are not natural hosts for influenza viruses: why then
should mice have an interferon-regulated gene that confers
resistance to these viruses? The answer is most probably that
the Mxl response in wild mice is targeted to rodent pathogens
other than influenza A and B viruses. Likely candidates are
tick-transmitted orthomyxoviruses, which are distantly
related to the influenza viruses and which produce sporadic
infections in rodents and larger mammals, including humans
(54). The authors recently demonstrated that some of these
viruses are indeed susceptible to the M x l effect ( 2 5 , 7 5 ) . In
fact, Thogoto virus exhibits an extraordinarily high sensitivity
to the inhibitory effect of Mx proteins, as shown below.
Activation of Mx resistance
The assumption that resistant mice simply cannot be infected
with influenza viruses is incorrect. On the contrary, influenza
virus can infect resistant mice perfectly well if, at the time of
infection, the M x l protein is not yet in place. As a
consequence, the animals do become ill and may develop
influenza-like symptoms with high fever. However, resistant
animals eventually recover from disease, whereas susceptible
animals die. What is the reason for this difference in the
outcome of infection? Both types of animals produce large
quantities of interferon in the early stages of infection. But, as
is now known, only resistant animals have functional M x l
genes. Once expressed, the M x l protein immediately restricts
further virus growth and the animal recovers. In susceptible
mice carrying the M x l gene defect, no antiviral M x l protein is
produced and the infecting virus can continue to grow
unhindered, even in the presence of large amounts of
interferon.
To illustrate the inducibility of M x l protein, mouse cells were
treated with interferon type 1. The newly produced M x l
protein accumulates in the cell nucleus where it can be
detected by using specific antibodies in an immuno­
fluorescence assay (11). Figure 1 shows that M x l protein was
produced in interferon-treated embryo fibroblasts from
resistant mice (Fig. la) but not in similarly treated fibroblasts
from susceptible animals (Fig. l b ) . During an acute viral
infection, M x l protein is induced by interferon in a similar
way in infected organs. For example, resistant mice were
infected intranasally with a lethal dose of a mouse-adapted
Fig. 1
Mouse Mx1 protein accumulates in the nucleus of interferon-treated fibroblasts from influenza virus-resistant congenic BALB.A2G-Mx1 mice (a) but
not in interferon-treated fibroblasts from virus-susceptible BALB/c mice (b)
Rev. sci. tech. Off. int. Epiz., 17 (1|
222
influenza A/PR8 virus. Lungs were harvested 4 days later and
cryostat sections were prepared. The tissue sections were then
double labelled by indirect immunofluorescence with
antibodies to influenza A virus and antibodies to M x l protein.
Figure 2 shows a characteristic lesion found in infected lungs.
Influenza virus antigens could be detected in a restricted area
due to initial virus growth (Fig. 2a). The viral lesion is
surrounded by non-infected cells that express the antiviral
Mxl protein (Fig. 2 b ) . Obviously, M x l is produced at
precisely the sites of infection. Infected cells thus become
surrounded by a barrier of protected cells blocking further
infection of neighbouring cells. This leads to a drastic delay in
further spread of the virus and provides enough time for
additional defence mechanisms to join in and to help clear the
virus from the body.
In summary, proper recovery from influenza virus infection in
mice depends on the early availability of an M x l defence
mechanism. If this early blocking mechanism fails for genetic
or other reasons, the virus takes over and the infected animal
has no chance to recover. Numerous experiments with
Mxl-congenic or Mxl-transgenic mice have established this
scenario and have shown beyond doubt that the M x l system
is indispensable for recovery from infection with otherwise
deadly influenza viruses (5, 2 1 , 3 6 , 5 6 ) .
The course of disease described here for resistant mice reflects
quite well the characteristics of an uncomplicated acute
influenza virus infection, as also observed in normal,
immunocompetent human patients. The next point of
enquiry, therefore, was whether humans
comparable host defence mechanism.
possessed a
Human Mx genes
The first evidence for an Mxl-related protein in man was
obtained with a monoclonal antibody against M x l that
cross-reacted with an interferon-induced protein in human
cells (67). Surprisingly, the human homologue was localised
in the cytoplasm of cells, not in the nucleus, thus the ability of
this cytoplasmic protein to inhibit influenza virus replication,.
which is known to occur in the nucleus of infected cells,
seemed uncertain. Interferon stimulated the synthesis of two
distinct but related Mx proteins in human cells, now called
MxA and MxB. Subsequently, the complementary DNAs
(cDNAs) of the two human Mx genes were isolated and were
shown to code for cytoplasmic proteins of 7 6 kiloDaltons
(kDa) and 73 kDa, respectively (2, 28). Interestingly, both Mx
genes were found to be located in close proximity to each
other on the distal part of the long arm of chromosome 21
( 2 1 q 2 2 . 3 ) , which is syntenic to mouse chromosome 16 (15,
27). Later research demonstrated that human MxA protein
had antiviral activity not only against influenza viruses but
also against a broad range of RNA viruses, which multiply in
the cytoplasm of infected host cells. The human MxB protein
lacked detectable antiviral activity. Similarly to the situation in
mice, MxA protein is transiently expressed in humans during
an acute self-limiting viral infection (62). However, whether
MxA is as helpful in fighting disease in humans as Mxl
Fig. 2
Influenza viral lesion in the lung of an influenza virus-resistant mouse
Double immunofluorescence staining shows infected cells (a) surrounded by protected cells expressing the antiviral Mx1 protein (b)
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Rev. sci. tech. Off. int. Epiz.. 17(1)
protein is in mice, remains to be seen. In any case, MxA is now
well established as a useful marker to monitor type I
interferon action in vivo, for example during interferon
therapy of cancer patients (30).
Work in many laboratories has now shown that Mx proteins
are found in at least eight vertebrate species, including birds
and fish. However, only some of these proteins seem to
possess antiviral activity. Others, such as human MxB, rat
Mx3, duck Mx and fish Mx, seemingly have no effect on the
otherwise Mx-sensitive viruses. Therefore, these interferonregulated proteins probably serve another purpose in the
realm of the interferon system ( 3 ) . Alternatively, the full
antiviral profile of Mx proteins may not have been discovered
so far, and Mx may in fact inhibit in each species a set of
species-specific pathogens.
Characteristics of Mx proteins
The available sequence data reveal considerable conservation
among Mx proteins from different species. Mx proteins seem
to be organised in two functional domains; an N-terminal
regulatory domain and a C-terminal effector domain ( 7 2 ) .
Thus, in the N-terminal region, the proteins have three
sequence elements characteristic of guanosine-5'-triphosphate
(GTP)-binding proteins. In fact, Mx proteins have been
shown to bind and hydolyse GTP ( 2 6 , 4 7 , 5 1 , 7 2 ) . GTP
binding seems to be crucial for antiviral function because
mutations within the N-terminal GTP-binding domain result
in a loss of antiviral activity ( 4 6 , 5 9 , 6 0 ) . In addition, the
C-terminal part of Mx proteins seems to play an important
role, and contains a stretch of basic amino acids and two
highly conserved leucine zipper motifs ( 4 5 ) (Fig. 3 ) . Those
leucine repeats form amphipathic a-helices that are known to
promote
protein-protein
interactions
( 4 0 ) . Targeted
mutations which impaired the amphipathic character of these
helices resulted in loss of antiviral activity of mouse M x l
protein ( 7 9 ) . Furthermore, a single amino acid exchange
within the distal leucine zipper motif changed the antiviral
specificity of human MxA protein, so that the mutant protein
lost antiviral activity against vesicular stomatitis virus (VSV)
GTP-binding elements
highly conserved tripartite guanosine-5'-triphasphate (GTP)-binding
consensus elements
putative leucine zippers
variable 'hinge' region
but maintained wild-type activity against influenza virus and
Thogoto virus ( 1 2 , 7 8 ) . The importance of the C-terminal
region for the antiviral activity was also suggested by
comparing the inactive rat Mx3 with the active rat Mx2
protein (31). When two residues in the C-terminal domain of
Mx3 were replaced with those of Mx2, partial activity was
re-established (32). Moreover, co-expression of an antivirally
inactive C-terminal fragment of MxA interferes with wild-type
MxA in a dominant-negative manner (60). In summary, these
findings indicate that the C-terminal half may expose domains
that interact directly with viral target structures.
Sequence alignments revealed that Mx proteins belong to the
newly defined dynamin superfamily of high molecular weight
GTPases found in yeast, plant and animal cells ( 7 2 ) . These
large GTPases play important roles in fundamental cellular
processes, such as endocytosis ( 7 6 ) , and intracellular vesicle
transport in yeast ( 6 3 ) and plants ( 1 7 ) . The GTP-bound
conformation of dynamin is known to self-assemble around
tubular membrane invaginations (74), and the hypothesis that
the ability to form helical arrays around tubular templates
might be a functional link between all dynamin-like large
GTPases has been proposed ( 3 5 ) . In fact, MxA protein also
forms aggregates of about 3 0 molecules that adopt a helical
structure in solution (G. Kochs, U. Aebi and O. Haller,
unpublished data). Furthermore, C-shaped and ring-like
structures have been described for mouse M x l protein ( 5 2 ) .
Thus, there is a temptation to speculate that antivirally active
Mx proteins might inhibit viruses by wrapping around helical
structures, such as viral nucleocapsids (5, 3 5 ) .
Which viruses are inhibited by
Mx?
The antiviral potential of Mx proteins has generally been
assessed using cDNA transfection experiments. The most
effective method of performing these experiments was to
express the relevant Mx cDNAs under the control of a
constitutive promoter. Use of this method enabled the authors
to test Mx activity independent of the action of other
interferon-induced cellular proteins ( 6 8 ) . Stably transfected
cell lines were produced that expressed the Mx protein of
interest constitutively and at high levels. For practical reasons,
mouse 3 T 3 cells (derived from a susceptible Mxl-negative
mouse strain) or monkey Vero cells (known to be susceptible
to many viruses) were used, with much the same results.
Figure 4 shows an immunofluorescence picture of 3 T 3 and
Vero cells expressing either the human MxA protein in the
cytoplasm or the mouse M x l protein in the nucleus. Figure 5
illustrates the antiviral effect of human MxA and mouse M x l
protein against the rhabdovirus VSV and the influenza A virus
fowl plague virus B (FPV-B) in a plaque reduction assay.
basic region
Fig. 3
Functional domains of the human MxA protein
Many viruses have now been tested. Those that were found to
be blocked by M x do not have many properties in common:
all are enveloped single-stranded RNA viruses, but their
Rev. sci. tech. Off. int. Epiz., 17(1)
224
Fig. 4
Intracellular localisation of human MxA and mouse Mx1 protein in stably transfected mouse 3T3 or monkey Vero cell lines
replication strategies are quite distinct. Therefore, the
mechanisms by which the MxA GTPase is able to inhibit such
a diverse group of viruses is far from being understood.
polymerase function ( 3 8 , 5 5 ) . In contrast, the cytoplasmic
human MxA protein does not inhibit primary transcription
but blocks a post-transcriptional step that is still ill-defined.
Influenza virus
Tick-borne orthomyxoviruses
Natural isolates of influenza A viruses are not readily
pathogenic for mice; only upon serial passage do they become
virulent for this species. Thus, for most work on Mx
resistance, influenza virus strains have been used whose
history includes many mouse passages. Of particular interest
was the TURH hepatotropic variant ( 2 0 ) derived from the
avian influenza A/Turkey/England/63 virus that causes fowl
plague.
Some of the many tick-borne viruses have now been
recognised to be distantly related to orthomyxoviruses,
namely, Thogoto virus and Dhori virus. Both were recently
found to be inhibited in A2G mice and to be blocked by Mxl
protein in tissue culture ( 1 4 , 2 5 , 75). Thogoto virus was first
isolated in Thogoto Forest near Nairobi, Kenya ( 1 9 ) and has
since been found in a variety of ticks and their vertebrate hosts
in central Africa, Egypt, Iran, Sicily and Portugal (9). The virus
replicates both in the tick and in the vertebrate, and
antibodies have been found in rats, buffaloes, camels,
donkeys, cattle, sheep and man. In sheep, infections may be
associated with febrile illness and possibly abortions (8).
Thogoto virus infections in humans have also been reported
but may be rare (48).
Influenza virus has a segmented, negative-stranded RNA
genome and uses a complex replication strategy ( 3 7 ) . After
uncoating, the viral nucleocapsids migrate rapidly into the cell
nucleus. There, the parental viral genome is transcribed into
messenger RNAs (mRNAs) by the virion-associated
RNA-dependent RNA polymerase, in a process known as
primary transcription. Genome replication is initiated
following translation of the primary viral mRNAs, with the
help of newly synthesised viral proteins, after their
transportation into the nucleus. Finally, genomic RNAs are
recognised with high affinity by viral proteins, exported to the
cytoplasm and packaged into new nucleocapsids. The nuclear
Mxl protein has been shown to interfere with the process of
primary transcription, most likely by disturbing the
The similarities of these tick-borne orthomyxoviruses to
influenza virus are based on several molecular characteristics.
For example, Thogoto virus comprises six segments of
single-stranded, negative sense RNAs and is able to produce
reassortants in vivo (10). Thogoto virus has a nuclear phase in
its multiplication and seems to use a complex replication
strategy similar to that used by influenza viruses. The authors
were able to demonstrate recently that both M x l and MxA
Rev- sci. tech. Off. int. Epiz., 17(1)
Vesicular
stomatitis virus
225
Influenza A
(strain FPV-B)
accumulates in the cytoplasm, has antiviral activity against
measles virus, at least in certain cell types. Thus, measles virus
glycoprotein synthesis has been found to be inhibited in a
human monocytic cell line constitutively expressing human
Neomycin
MxA protein, whereas viral mRNA levels have been found to
be
unchanged ( 6 5 ) . In human glioblastoma cells (U87),
however, constitutive expression of MxA caused inhibition of
viral RNA synthesis, most likely due to interference with
primary transcription ( 6 4 ) . In contrast, no antiviral effect of
MxA
has been detected in similarly transfected Vero cells
(U. Meier-Dieter and O. Haller, unpublished findings). The
antiviral activity of MxA protein against measles virus seems
Human
MxA protein
to be cell-type specific, which indicates that MxA action may
be host cell-dependent in general. Whether the interferoninducible MxA protein plays an important role in measles
virus
pathogenicity
by
favouring
the
establishment
of
persistent infections remains to be investigated.
Bunyaviruses
Mouse
Mx1 protein
The Bunyavirus
Although
family comprises over 3 0 0 different viruses.
only a few are presently
known
as human
pathogens, others have now been recognised as a source of
significant health problems. The current prospect of so-called
emerging viruses in this family is a cause for public concern.
Fig. 5
Plaque formation of vesicular stomatitis virus and influenza A virus
(strain FPV-B or fowl plague virus B) in monolayers of permanently
transfected Vero cells expressing human M x A protein, mouse M x l
protein or the neomycin resistance gene only
These viruses are transmitted to man from infected animals
proteins inhibit an early step in the viral multiplication cycle
(12, 25; unpublished findings) and the mechanistic details of
this inhibition at the molecular level are presently under
further investigation.
with infected animals or by mosquito bite. In the 1 9 7 7 - 1 9 7 8
Vesicular stomatitis virus
summer encephalitis in children in the midwestern USA
that constitute a huge natural reservoir. Some viruses cause
sudden and severe epidemics: for example, Rift Valley fever
virus causes epizootics among domestic animals in many
parts of sub-Saharan Africa and also in Egypt. During such
outbreaks, the virus can be transmitted to humans by contact
Vesicular stomatitis virus is a relative of the rabies virus (an
important human pathogen), and is of agricultural
importance, infecting primarily horses. The VSV genome is a
single strand of a negative sense RNA, which is transcribed,
through a stop/start mechanism, into five individual mRNAs.
VSV multiplies exclusively in the cytoplasm of infected cells.
As expected, growth of VSV is not affected by the nuclear M x l
protein. However, cytoplasmic MxA inhibits transcription of
VSV in cell culture (71) and in an in vitro transcription system
(66). Experiments with the related virus, rabies, have not yet
been published.
epidemic in Egypt, over 2 0 0 , 0 0 0 people contracted the
disease and at least 6 0 0 people died (41). La Crosse virus is
transmitted by mosquitoes and is an important cause of
(16).
Hantaan virus causes Korean haemorrhagic fever with a
reported mortality rate of 1 0 % to 1 5 % , while Puumala virus
causes a mild form
syndrome
which
of haemorrhagic
is observed
fever with
throughout
the
renal
Eurasian
continent. Moreover, Sin Nombre virus is responsible for
hantaviral pulmonary syndrome, a severe and often fatal form
of adult respiratory distress first recognised in 1 9 9 3 in the
Four Corners region of the south-western USA (53).
The
authors
representative
have
investigated
members
of
this
the
Mx-sensitivity
virus
family.
of
When
constitutively expressed in stably transfected Vero cells, MxA
Measles virus
Measles virus is a human pathogen, causing acute febrile
infections and (rarely) subacute sclerosing panencephalitis
and inclusion body encephalitis. Measles virus has a
non-segmented negative strand RNA genome and belongs to
the Paramyxoviridae
family. Measles virus replicates entirely
in the cytoplasm of infected cells, and it is therefore not
surprising that mouse M x l protein, which is located in the
nucleus, has no effect. However, human MxA protein, which
prevented the accumulation of viral transcripts and proteins
of Hantaan virus ( 1 3 ) . La Crosse virus and Rift Valley fever
virus were likewise inhibited ( 1 3 ) . A comparable protective
effect was later found for Puumala virus ( 3 3 ) . These data
indicate that humans have developed a mechanism for
controlling these viruses irrespective of differences in viral
coding strategies, which may explain the excellent protective
effect of interferon against bunyaviruses
literature (57, 5 8 ) .
reported
in the
226
If humans are equipped with such a potent defence system,
why are these virus infections sometimes not self-limiting?
The reason is probably that, depending on virus strain, dose
and circumstances, the infecting virus does not induce MxA
protein rapidly enough to reach sufficiently high levels in the
right place to halt the spread of the virus. A rapid expression
of MxA at the sites where the virus multiplies is probably
crucial for proper recovery. If this is the case, then why is
interferon not established as a therapeutic agent? A possible
explanation is that interferon therapy is, by necessity, always
given too late (18). When patients are symptomatic, virus
growth and spread has already occurred. If interferon were
given along with the infection, the patients might benefit, as
has been observed in animal models ( 5 8 ) . New laboratory
tests for rapid viral diagnosis are expected to allow trials of
early interferon treatment in the near future.
Semliki Forest virus
Semliki Forest virus (SFV) is the first example of a positivestranded RNA virus to be inhibited by Mx. The virus has a
broad host range and can cause encephalitis in man. Most
recently, Pavlovic and co-workers have shown that human
MxA protein inhibited virus growth by interfering with the
viral replicase ( 3 9 ) . Most interestingly, this effect was
observed only in human cells and not in cells from other
species, which suggests the involvement of cellular cofactors,
as is the case with measles virus.
The role of human MxA protein
in disease protection
To study the potential of human Mx protein to give protection
against disease, several transgenic mouse lines were
generated. The various approaches used to generate
Mx-transgenic mice have recently been described in a
comprehensive review (5) and will not be discussed here. The
results obtained were most revealing: transgenic expression of
a mouse M x l cDNA was sufficient to turn susceptible mice
into resistant animals. The Mxl-transgenic mice survived
large challenge doses with influenza A virus (4, 3 6 ) and
Thogoto virus ( 2 5 ) . These results provided irrefutable
evidence for the fact that M x l is the key mediator of inborn
resistance in mice.
How does this relate to the situation in man? Does MxA
protein play a similar role in the antiviral defence of humans?
This question cannot be answered directly, but one means of
testing the possible defence in humans involved the
introduction of the human MxA protein into mice. Mouse
strains carrying defective M x l genes were used as MxA
recipients. These animals are unable to produce their own
Mxl protein and are therefore highly susceptible to infection
with influenza A virus or Thogoto virus. Accordingly, they
provide an ideal test system to probe the antiviral efficacy of
human MxA protein in vivo. The transgenic animals, which
Rev. sci. tech. Off. int. Epiz., 17(1)
permanently expressed the human MxA protein in various
organs, were highly resistant to challenges with Thogoto vims
and showed reduced susceptibilities to influenza A virus and
VSV ( 5 6 ) . A typical experiment with Thogoto virus is
summarised in Table I. Transgenic and control animals were
infected with 100 plaque-forming units (PFU) of Thogoto
virus by the intraperitoneal route. All eleven transgenic
animals survived, whereas all eight nontransgenic C57BL/6
mice died within days after infection. These results
demonstrate that the human MxA protein is a powerful
antiviral agent in vivo and can be used for 'intracellular
immunisation' (4, 6 ) . The results suggest that MxA may
likewise protect humans from the fatal effects of infection with
certain viral pathogens.
Table I
Resistance of MxA-transgenic mice to Thogoto virus infection
Mouse strain
Mortality (No. dead/No. infected)
MxA-transgenic
0/11
C57BL/6
8/8
Mice of the MxA-transgenic line t and non-transgenic C57BL/6 control mice were infected
with 100 plaque-forming units (PFU) of Thogoto virus. Susceptible mice died within four to
eight days after infection
Conclusion: is Mx useful for
producing disease resistant
livestock?
As early as 1 9 9 2 , the research group led by Müller and Brem
reported the first successful introduction of mouse Mxl
transgenes into pigs (49). Pigs do possess Mx genes (50), but
are susceptible to influenza and provide a substantial reservoir
for swine influenza viruses. By introducing the potent mouse
Mxl transgene, researchers hoped that pigs with increased
influenza-resistance could be obtained. However, none of
these pigs expressed detectable amounts of transgenic Mxl
protein. Nevertheless, these animals represent a first step in
the direction of generating virus-resistant livestock.
What of the prospects for using this technology to produce
disease-resistant livestock? Mx proteins are host-derived
antiviral proteins whose corresponding genes are present in
most vertebrates. Hence, deliberate expression of Mx proteins
in domestic animals only makes sense when the animal has a
comparably weak M x system. Chickens, for example, have
Mx proteins that appear to be devoid of antiviral activity.
Avian influenza A viruses cause devastating epidemics in
domestic birds which are a major threat to the poultry
industry. Thus, introduction of an active Mx protein may
yield chickens with increased resistance. If used widely, such
transgenic chickens and other domestic animals with
increased resistance might in turn help to control the
227
Rev. sci. tech. Off. int. Epiz., 17 (1)
emergence of new human pandemic strains of influenza that
may originate in these animal reservoirs.
Deutsche
Forschungsgemeinschaft
European Union Human
(HA
Programme (grant ERBCHRXCT940453),
Acknowledgements
1582/1-2),
the
Capital and Mobility Network
and the Federal
State of Baden-Württemberg (ZKF-Bl).
The authors thank Jovan Pavlovic and Peter Staeheli for many
stimulating discussions and Eva-Christa Heimer for help with
the manuscript. This work was supported by grants from the
Protéines M x : médiatrices de la résistance innée aux virus ARN
0. Haller, M . Frese & G. Kochs
Résumé
Les p r o t é i n e s M x qui s o n t induites p a r l'interféron a p p a r t i e n n e n t à la s u p e r f a m i l l e
de la d y n a m i n e d e s g u a n o s i n e - t r i p h o s p h a t a s e s . C e s p r o t é i n e s o n t éveillé u n
g r a n d i n t é r ê t p a r c e q u e c e r t a i n e s d ' e n t r e elles p r é s e n t e n t u n e activité antivirale
c o n t r e les virus A R N p a t h o g è n e s , p a r e x e m p l e , c o n t r e d e s m e m b r e s d e la f a m i l l e
des o r t h o m y x o v i r u s (virus influenza) o u d e c e l l e d e s b u n y a v i r u s . Les c e l l u l e s
t r a n s f e c t é e s e t les souris t r a n s g é n i q u e s e x p r i m a n t l e s p r o t é i n e s M x s o n t t r è s
r é s i s t a n t e s a u x virus s e n s i b l e s a u x p r o t é i n e s M x , c e qui m o n t r e q u e c e s d e r n i è r e s
c o n s t i t u e n t d e puissants a g e n t s antiviraux. C h e z l ' h o m m e , on p e u t o b s e r v e r lors
d ' i n f e c t i o n s v i r a l e s a u t o l i m i t a n t e s , la s y n t h è s e d e la M x A , q u i e s t d o n c
s u s c e p t i b l e d e f a v o r i s e r la g u é r i s o n .
Mots-clés
Agents antiviraux - Animaux transgéniques - Génétique - Guanosine-triphosphatase Interférons - Protéine M x - Résistance aux maladies - Virus influenza.
Proteínas M x ; mediadoras de la resistencia innata a virus ARN
0 . Haller, M . Frese & G. Kochs
Resumen
Las p r o t e í n a s M x , c u y a g é n e s i s v i e n e i n d u c i d a p o r el i n t e r f e r ó n , p e r t e n e c e n a la
s u p e r f a m i l i a d e g r a n d e s g u a n o s í n t r i f o s f a t a s a s d e la d i n a m i n a . El i n t e r é s q u e
d e s p i e r t a n e s t a s p r o t e í n a s o b e d e c e a s u a c t i v i d a d antivírica f r e n t e a virus A R N
p a t ó g e n o s , por e j e m p l o m i e m b r o s d e las f a m i l i a s d e los o r t o m i x o v i r u s (virus d e la
i n f l u e n z a ) o los b u n y a v i r u s . Las c é l u l a s t r a n s f e c t a d a s y los r a t o n e s t r a n s g é n i c o s
q u e e x p r e s a n p r o t e í n a s M x p r e s e n t a n u n a g r a n r e s i s t e n c i a a los virus s e n s i b l e s a
las p r o t e í n a s M x , lo q u e v i e n e a d e m o s t r a r q u e é s t a s s o n p o t e n t e s a g e n t e s
antivíricos. En el s e r h u m a n o s e o b s e r v a síntesis d e M x A d u r a n t e i n f e c c i o n e s
v í r i c a s a u t o l i m i t a d a s , p o r lo q u e e s posible q u e esta p r o t e í n a f a v o r e z c a la
c u r a c i ó n d e la e n f e r m e d a d .
Palabras clave
Agentes antivíricos- Animales transgénicos - Genética -
Guanosín trifosfatasa
Interferones - Proteína M x - Resistencia a la enfermedad - Virus de la influenza.
-
228
Rev. sci. tech. Off. int. Epiz., 17 (1)
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