Rev. sci. tech. Off. int. Epiz., 1998 ,17 (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 o f t h e d y n a m i n s u p e r f a m i l y o f large g u a n o s i n e t r i p h o s p h a t a s e s . T h e s e p r o t e i n s h a v e a t t r a c t e d a t t e n t i o n b e c a u s e 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 examp le a g a i n s t m e m b e r s o f t h e o r t h o m y x o v i r u s ( i n f l u e n z a v i r u s ) f a m i l y o r t h e b u n y a v i r u s 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 r e s i s t a n t t o M x - s e n s i t i v e v i r u s e s , d e m o n s t r a t i n g t h a t M x p r o t e i n s a r e p o w e r f u l 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 v i ra l i n f e c t i o n s a n d m a y t h u s p r o m o t e r e c o v e r y f r o m d i s e a s e .

Keywords Ant iv i ra l agents - Disease resistance - Genet ics - Guanosine t r iphosphatase -Inf luenzavirus - Interferons - M x prote in - Transgenic an ima ls .

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 20 million people world-wide (37). 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 US$60 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, 73) .

Although influenza viruses belong to the group of best studied viruses, efficient control of the disease caused by these viruses is still not possible (77). 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 Mx 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 (42) . Screening of numerous inbred strains showed that this type of inborn

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resistance was present in just a few mouse strains, including A2G and SL/NiA (1 , 23 , 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 Mxl 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) (21 , 22) . 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 Mxl 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 Mxl 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 Mxl effect (25, 75) . 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 Mxl 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 Mxl genes. Once expressed, the Mxl protein immediately restricts further virus growth and the animal recovers. In susceptible mice carrying the Mxl gene defect, no antiviral Mxl 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 Mxl 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 immuno­fluorescence 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)

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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 Mxl 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. 2b). Obviously, Mxl 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 Mxl 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 Mxl system is indispensable for recovery from infection with otherwise deadly influenza viruses (5, 2 1 , 36 , 56) .

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 possessed a comparable host defence mechanism.

Human Mx genes The first evidence for an Mxl-related protein in man was obtained with a monoclonal antibody against Mxl 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 76 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 (21q22.3) , 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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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 interferon-regulated 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 (72) . 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 (26, 47, 51 , 72) . 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 (46, 59, 60) . 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 (45) (Fig. 3 ) . Those leucine repeats form amphipathic a-helices that are known to promote protein-protein interactions (40) . Targeted mutations which impaired the amphipathic character of these helices resulted in loss of antiviral activity of mouse Mxl protein (79). 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

basic region

Fig. 3 Functional domains of the human MxA protein

but maintained wild-type activity against influenza virus and Thogoto virus (12, 78) . 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 (72) . These large GTPases play important roles in fundamental cellular processes, such as endocytosis (76) , and intracellular vesicle transport in yeast (63) and plants (17) . 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 (35) . 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 Mxl protein (52) . 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, 35) .

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 (68) . Stably transfected cell lines were produced that expressed the Mx protein of interest constitutively and at high levels. For practical reasons, mouse 3T3 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 3T3 and Vero cells expressing either the human MxA protein in the cytoplasm or the mouse Mxl 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.

Many viruses have now been tested. Those that were found to be blocked by Mx do not have many properties in common: all are enveloped single-stranded RNA viruses, but their

224 Rev. sci. tech. Off. int. Epiz., 17(1)

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.

Influenza virus 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 (20) derived from the avian influenza A/Turkey/England/63 virus that causes fowl plague.

Influenza virus has a segmented, negative-stranded RNA genome and uses a complex replication strategy (37). 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

polymerase function (38, 55) . In contrast, the cytoplasmic human MxA protein does not inhibit primary transcription but blocks a post-transcriptional step that is still ill-defined.

Tick-borne orthomyxoviruses 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 (14, 25 , 75) . Thogoto virus was first isolated in Thogoto Forest near Nairobi, Kenya (19) 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) .

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

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Vesicular Influenza A stomatitis virus (strain FPV-B)

Neomycin

Human MxA protein

Mouse Mx1 protein

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 MxA protein, mouse M x l protein or the neomycin resistance gene only

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.

Vesicular stomatitis virus 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 Mxl 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.

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 Mxl protein, which is located in the nucleus, has no effect. However, human MxA protein, which

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 MxA protein, whereas viral mRNA levels have been found to be unchanged (65) . In human glioblastoma cells (U87), however, constitutive expression of MxA caused inhibition of viral RNA synthesis, most likely due to interference with primary transcription (64) . 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 to be cell-type specific, which indicates that MxA action may be host cell-dependent in general. Whether the interferon-inducible MxA protein plays an important role in measles virus pathogenicity by favouring the establishment of persistent infections remains to be investigated.

Bunyaviruses The Bunyavirus family comprises over 300 different viruses. Although 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. These viruses are transmitted to man from infected animals 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 with infected animals or by mosquito bite. In the 1977-1978 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 summer encephalitis in children in the midwestern USA (16) . Hantaan virus causes Korean haemorrhagic fever with a reported mortality rate of 10% to 15%, while Puumala virus causes a mild form of haemorrhagic fever with renal syndrome which is observed throughout the 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 1993 in the Four Corners region of the south-western USA (53) .

The authors have investigated the Mx-sensitivity of representative members of this virus family. When constitutively expressed in stably transfected Vero cells, MxA prevented the accumulation of viral transcripts and proteins of Hantaan virus (13) . La Crosse virus and Rift Valley fever virus were likewise inhibited (13) . A comparable protective effect was later found for Puumala virus (33) . 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 reported in the literature (57, 58) .

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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 (58). 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 positive-stranded 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 (39). 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 Mxl cDNA was sufficient to turn susceptible mice into resistant animals. The Mxl-transgenic mice survived large challenge doses with influenza A virus (4, 36) and Thogoto virus (25). These results provided irrefutable evidence for the fact that Mxl 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 Mxl 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

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 (56). 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 1992, 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 Mx 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

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emergence of new human pandemic strains of influenza that

may originate in these animal reservoirs.

Acknowledgements 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

Deutsche Forschungsgemeinschaft (HA 1582/1-2) , the

European Union Human Capital and Mobility Network

Programme (grant ERBCHRXCT940453), and the Federal

State of Baden-Württemberg (ZKF-Bl) .

Protéines Mx : médiat r ices de la résistance innée aux virus ARN 0. Haller, M . Frese & G. Kochs

Résumé Les pro té ines M x qui sont indui tes par l ' interféron a p p a r t i e n n e n t à la super fami l l e de la d y n a m i n e des g u a n o s i n e - t r i p h o s p h a t a s e s . Ces pro té ines ont évei l lé un grand in té rê t p a r c e que c e r t a i n e s d 'ent re e l les p r é s e n t e n t une act iv i té ant iv i ra le con t re les v i rus A R N p a t h o g è n e s , par e x e m p l e , con t re des m e m b r e s de la fami l l e des o r thomyxov i rus (virus in f luenza) ou de ce l le des bunyav i rus . Les ce l lu les t r a n s f e c t é e s e t les sour is t r a n s g é n i q u e s e x p r i m a n t les pro té ines M x sont t rès rés is tan tes aux v i rus sens ib les aux pro té ines M x , c e qui m o n t r e que c e s d e r n i è r e s cons t i tuen t de puissants a g e n t s ant iv i raux. Chez l ' h o m m e , on peut o b s e r v e r lors d ' in fect ions v i ra les au to l im i tan tes , la s y n t h è s e de la M x A , qui es t donc suscep t ib le de f a v o r i s e r la guér ison .

Mots-clés Agents antiviraux - Animaux transgéniques - Génétique - Guanosine-triphosphatase -

Interférons - Protéine M x - Résistance aux maladies - Virus influenza.

Proteínas Mx ; mediadoras de la resistencia innata a virus ARN 0 . Haller, M . Frese & G. Kochs

Resumen Las pro te ínas M x , c u y a génes is v i e n e induc ida por el i n t e r f e r ó n , p e r t e n e c e n a la super fami l i a de g r a n d e s guanos ín t r i fos fa tasas de la d i n a m i n a . El in terés que d e s p i e r t a n es tas p ro te ínas o b e d e c e a su ac t iv idad ant iv í r ica f r e n t e a v i rus 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 de las fami l i as de los or tomixov i rus (virus de la in f luenza) o los bunyav i rus . Las cé lu las 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 pro te ínas M x p r e s e n t a n una gran res is tenc ia a los v i rus sens ib les a las p ro te ínas M x , lo que v i e n e a d e m o s t r a r que és tas son p o t e n t e s a g e n t e s ant iv í r icos . En el ser h u m a n o se o b s e r v a s íntesis de 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 cas a u t o l i m i t a d a s , por lo que es posible que esta pro te ína f a v o r e z c a la c u r a c i ó n de la e n f e r m e d a d .

Palabras clave Agentes antivír icos- Animales transgénicos - Genética - Guanosín trifosfatasa -

Interferones - Proteína M x - Resistencia a la enfermedad - Virus de la influenza.

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