JOURNAL OF VIROLOGY, Mar. 1992, p. 1709-17160022-538X/92/031709-08$02.00/0Copyright C) 1992, American Society for Microbiology

Resistance to Influenza Virus Infection of Mx TransgenicMice Expressing Mx Protein under the Control of

Two Constitutive PromotersE. KOLB,t* E. LAINE,t D. STREHLER,§ AND P. STAEHELIII

Institute for Immunology and Virology, University of Zurich, CH-8028 Zurich, Switzerland

Received 27 August 1991/Accepted 25 November 1991

Transgenic mice constitutively expressing in the brain the influenza virus resistance protein Mxl controlledby the HMG (3-hydroxy-3-methylglutaryl coenzyme A reductase) promoter showed specific resistance againstthe neurotropic influenza A virus strain NWS. Control mice of the A2G strain express Mxl protein in allorgans, but only after induction by interferon type I upon or without viral infection. The extent of specificresistance in transgenic mice of the best-expressing line reached about two-thirds that of controls, most likelybecause of considerably less total-body Mx protein activity in the transgenic mice. Thus, the theoreticaladvantage in these mice of the continuous presence of Mx protein with early inhibitory potential to viralreplication was apparently offset by restricted organ expression. Strong evidence that the Mxl protein on itsown is a specific anti-influenza A virus agent and that its efficiency in the experimental setting is independentof interferon actions could be derived from the treatment of experimental and control mice with anti-interferonantibodies at the time of virus tests. Whereas in A2G mice, Mxl mRNA and Mxl protein synthesis wereabolished and viral resistance was markedly reduced or abolished, resistance in the transgenic mice persistedto almost the same degree. Transgenic mice generated with a mouse albumin/Mxl cDNA construct showedliver-specific expression. However, in two expressing transgenic lines, Mxl protein synthesis was suppressedafter a few months. The mechanism of suppression could not be elucidated, but increasing methylation of thetransgene's coding region was not the cause. It is possible that continuous Mxl protein expression in the liveris less well tolerated than that in the brain. Whether this partial suppression and, with the HMG promoter,restricted organ expression are the organism's responses to interference of Mxl with normal cellular activitiessuch as nucleocytoplasmic transport of RNA and proteins cannot be determined until the molecularmechanisms of antiviral activity of Mxl protein are understood.

Viruses and double-stranded RNAs induce in experimen-tal animals and humans a number of reaction sequences,starting with interferons and ending in an antiviral state (forreviews, see references 15 and 27). In some mouse strains,establishment of this state of readiness for multiprongeddefense requires synthesis of the protein Mxl (for a reviewof Mx proteins, see reference 2). The presence of Mxlspecifically prevents growth in vivo and in vitro of theinfluenza A virus and other orthomyxovirus family mem-bers.

Interferon-inducible, Mx-cross-reactive proteins weremore recently found in cells of many other species (15studied so far [12, 13, 32]), often present as two homologs ortruncated/mutated derivatives thereof, not all of which areactive. The proteins, named Mxl and Mx2 in mice (14,28-31) and MxA and MxB in humans (1), are always locatedin the cytoplasm, not in the nucleus, in species other thanmice and rats. Mx protein accumulates in both compart-ments in rats and exclusively in cell nuclei in mice (19).

* Corresponding author.t Present address: Department of Maxillo-Facial Surgery, Uni-

versity Hospital, CH-8091 Zurich, Switzerland.t Laboratory for Experimental Pathology, Institute of Pathology,

University of Zurich, CH-8091 Zurich, Switzerland.§ Present address: Department of Internal Medicine, Regional

Hospital, CH-8620 Wetzikon, Switzerland.11 Present address: Institute for Medical Microbiology and Hy-

giene, University of Freiburg, D-7800 Freiburg im Breisgau, Ger-many.

Furthermore, the range of antiviral activity in some humanand rat Mx homologs was recognized to comprise, in addi-tion to influenza virus, the rhabdovirus vesicular stomatitisvirus (20, 22a), and that list may not be complete.DNA sequence technology has established a close struc-

tural homology between Mx species from different sources(1) but unexpectedly also led to the discovery of unequivocalsequence similarities in the N-terminal third between Mxand two essential structural proteins; one, the yeast vacuolarprotein sorting protein VPSlp (25), responsible for thechanneling of protein products into the appropriate vacuoles(Golgi or lysosomal); the other, dynamin from rat brain (5,22, 24), is involved with vectorial motility within the micro-tubular system in eukaryotic cells. How are these structuralsimilarities to be interpreted? Do these undeniable factsunseat Mx from its antiviral position? We do not think so, forthe results presented below show beyond doubt that Mx1 onits own, as a constitutive transgene product, is an efficientanti-influenza virus effector in the mouse. The most recentlydiscovered, seemingly unsettling structural relationshipsmay in our view relate to the mechanism of action ratherthan the function of Mx1, that is, indicate the means, not thegoal, of its activity.

MATERIALS AND METHODS

Structures of injected DNA fragments mHMG/Mxl andmAlb/Mxl. The construct mHMG/Mxl consisted of thepromoter region of the mouse 3-hydroxy-3-methylglutarylcoenzyme A reductase (mHMG) gene, the mouse Mx1-

1709

Vol. 66, No. 3

on June 1, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

1710 KOLB ET AL.

HMG Mx I PAII (SV40)

Not I Not

Promoter Exon5.5 kb

Intron2.2 kb

from pCL642

Mx (pos. 147-2314)

B

Not Alb promoter

Pst Pst

'Probe (Pvu II)

Mx

from pCL642

l-l

IpASWO0(183 bp) Not

Pst

pA B-globin

CI |[.

2.3 kb 3.7 kb_40-.4.

from p Alb e/p from p SP64 Mx from p Alb e/p

FIG. 1. Two cDNA constructs used to generate transgenic mice and structure of the radioactive probe used for Southern analyses. (A)Plasmid derivation of sequences 5' and 3' to the Mx-coding region in first transgene construct (mice of Fig. 2). HMG represents a promoterexpected to give non-organ-restricted expression. (B) PvuII restriction fragment contained in the Mx-coding region, used as a radioactiveprobe. (C) Plasmid derivation of sequences 5' and 3' to the Mx-coding region in second transgene construct (mice of Fig. 3). Alb e/p,enhancer/promoter sequences of the murine albumin gene, expected to give constitutive, liver-restricted expression; SV40, simian virus 40.

coding region, and a simian virus 40-derived polyadenylationsignal (Fig. 1A). The construct was obtained as follows. The2.2-kb BamHI-BamHI fragment (positions 147 to 2314 [30])of plasmid pHG 327 Mx was cloned into the unique EcoRVsite of plasmid pCL 642 (7), also termed pHMG. Thisplasmid contains the promoter region, the first noncodingexon, the complete first intron, and the splice acceptor siteof the second exon of the mHMG gene. The sequence usedfor pronuclear injection was cut out of plasmid pHMG-Mxwith restriction endonuclease NotI (NotI sites in pCL 642closely flank the original EcoRV site).A plasmid (Alb e/p) containing the mouse albumin (mAlb)

enhancer/promoter element (23) was used as the acceptor ofthe Mxl cDNA fragment with the adjacent 0-globin-derivedpolyadenylation site, cut with XbaI and BamHI out ofplasmid pSP 64 (21). After cloning, the desired mAlb/MxlDNA construct was obtained by cutting the Alb e/p-Mxlplasmid at the NotI site 5' to the albumin enhancer/promoterregion and at the YpnI site 3' to the inserted poly(A) segmentwith the respective endonucleases (Fig. 1C).

Generation of transgenic mice. The mHMG/Mxl (about 8kb long) or the mAlb/Mxl (about 6 kb long) fragment wasinjected at a concentration of 3 or 2 ,ug/ml, respectively, intoone of the pronuclei of fertilized mouse eggs essentiallyaccording to Gordon and Ruddle (8) and Hogan et al. (11).The fertilized eggs were taken from (C57BL/6 x SJL)F2hybrid mice. Injections were made about 15 h after fertiliza-tion. The DNA-injected embryos were transferred topseudopregnant Swiss albino mice 2 to 3 months of age.

Identification of transgenic mice by Southern blot analysis.Genomic DNA was isolated from the tail skin of about3-week-old mice grown from injected embryos as describedby Epstein et al. (6). In the case of mice derived frommHMG/Mxl DNA-injected eggs, the genomic DNA wasdigested with endonuclease PstI and subjected to Southern

kb A BC D E F G H I K L M N 1 2 3 4

3.5 -

1.9-

1.61.3 -

_E.

0.8 - *

0.6 -

9% W-I;

-f' ;.&

ft'? "-.,z#V ,:4TI

04.10- -- *,-. -,,- 40 0 o

se.. ..A

..

so .#4"W_ ''!.-'p- .oeeqex-0

.* *@.

o .* 0 .,. s. *Z. w. .

FIG. 2. Southern blot analysis of all 13 mHMG/Mxl transgenicmice (lanes A to N), of an A2G KK cross (lane 3), and of appropriatecontrols. DNA was digested with PstI. Lane 1, Mxl-negativecontrol with an endogenous 3.2-kb band; lane 2, pattern of strainA2G naturally Mxl positive (bands of 4.5 and 3.2 kb); lane 4, lineKK, homozygous for the transgene, derived from line K. Note thedouble band: upper half (3.2 kb) is endogenous, lower half (3.0 kb)is transgene derived. Compare the intensity of the 3.0-kb band withthat of the 3.2-kb band: in KK mice, the latter is twice as intense asthe former; in K mice, the two bands are equally intense. Thisrelationship was a useful indicator of Mxl homozygosity in KKbreeding. The 3.0-kb band probably represents an end fragment ofthe tandem repeat transgene integration complex. Marker dots:small, two endogenous bands (the 3.2-kb band was always taken asan indicator of the amount of DNA loaded); large, transgene-derivedbands. The 1.3-kb band was used to estimate the number oftransgene copies integrated (between 1 and about 20).

A

Kpn

MIMIRMI 7= r-I L-

J. VIROL.

__0

on June 1, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

RESISTANCE TO INFLUENZA VIRUS OF Mx TRANSGENIC MICE 1711

blot analysis (26) essentially as described by Maniatis et al.(17). PstI fragments obtained from the inserted Mxl cDNAare shown in Fig. 1B. The fragments, after electrophoreticseparation in 0.8% agarose gel, were transferred to a Gene-Screen (NEN Research Products, Du Pont) sheet by 2-hvacuum blotting (LKB Products, Uppsala, Sweden). In thecase of mice derived from mAlb/Mx1 DNA-injected eggs,genomic DNA was digested with PstI or StuL. The probeused in all cases was a 1.0-kb PvuII fragment of pMx 34 (30)labeled by random priming (Fig. 1B). Hybridization condi-tions were those described by Staeheli et al. (30) except that1% sodium dodecyl sulfate (SDS) was substituted for 0.2%SDS and membranes were washed in 0.2% SSC (SSC is 0.15M NaCl plus 0.015 M sodium citrate) instead of 0.1% SSC.

Detection of Mxl protein in frozen tissue sections withfluorescent antibodies. Organs to be analyzed were quick-frozen by immersion in 2-methylbutane, which was cooled inliquid nitrogen. Frozen sections (5 ,um thick) were cut, airdried, fixed with 3% paraformaldehyde (10 min), and perme-abilized (2 min) with 0.5% Triton X-100 (30). Each slide wasthen incubated for 30 min with the AP5 rabbit antiserumspecific for the C terminus of mouse Mxl (19) and, afterwashing, incubated with tetramethyl rhodamine isothiocy-anate-labeled goat anti-rabbit antiserum (Nordic). The slides

were washed again and covered with Mowiol (Hoechst)before being sealed with coverslips. The tissue sections wereviewed with a Reichert-Jung Polyvar microscope equippedfor UV incident light fluorescence microscopy (30).Virus infections. One virus preparation used for resistance

tests was the neurotropic NWS influenza A virus strain usedpreviously (16). Stock virus was a preparation of allantoicfluid from 12-day-old chicken embryos that had been in-fected 2 days earlier with a 10-6-diluted brain extract of aninfluenza A virus (NWS)-infected mouse. The infectivity ofthe allantoic fluid had been previously determined. Its 50%lethal dose (LD50) was 10-6-5 (dilutioh, 1:3 x 106). The testsolutions mentioned in the text were dilutions in phosphate-buffered saline of the stock allantoic fluid solution. Theywere applied in a volume of 0.03 ml.The hepatotropic influenza A virus designated All 40 (9)

was originally derived from strain A/Turkey/England/63(Havl, Nav3; Langham strain). The virus was propagated inchicken embryos. Stock solution was an allantoic fluidpreparation with an LD50 of 10-65. Virus dilutions wereapplied intraperitoneally (i.p.). Animals surviving 500 LD50had been classified earlier as resistant.

Antibodies against interferon. Sheep antibodies to mouseinterferon were supplied by I. Gresser, Villejuif, France (10).

Il

FIG. 3. Immunofluorescence staining of Mxl protein in the brain. Nuclei of neurons in hemispheres show a punctate Mxl protein pattern.(a and d) Mice of the A2G strain (a) and of the transgenic line A2GKK (d), both stimulated with poly(rI) poly(rC) (100 p.g i.p.) 24 hpreviously; (b) Mxl-negative control mouse; (c) mouse of the transgenic line KK (Mxl homozygous), nonstimulated (constitutive Mxlexpression). Note the similar degrees of Mxl protein expression in panels a and c and additive expression in panel d (A2GKK is doubly Mxlhomozygous). Bar = 50 p.m.

VOL. 66, 1992

on June 1, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

1712 KOLB ET AL.

Sera were kept at -80°C. An antiserum of tested activitywas used at a dilution of 1:3 (0.02 ml) and applied intracere-brally together with virus.

RESULTS

Mice with incorporation of the mHMG/Mxl cDNA. Twelvetransgenic mice with germ line integration of constructmHMG/Mxl and one with only somatic incorporation wereobtained. Of all mice born after embryonal injection, about20% carried the transgene. Southern blot analysis mostlyshowed incorporation of multiple copies (between 1 andapproximately 20) (Fig. 2). Mx protein was expressed inthree mice, mainly in the brain. The corresponding lines,named D, E, and K, showed low, intermediate, and highlevels of expression, respectively. Some Mx protein expres-sion could also be demonstrated in heart and testis, but notin other organs, in these three mice. The following organswere tested: liver, lung, kidney, intestine, pancreas, skeletalmuscle, uterus, and ovary. This result was rather surprisingconsidering that cholesterol synthesis controlled by theHMG enzyme takes place in all cells but mostly in the liver.According to the literature, the HMG gene promoter usuallyaffords transgene expression in most organs (18), althoughnonuniform expression is not unusual (4). Constitutive Mxexpression in the brain with a patchy distribution reachedabout one-fourth to three-fourths the extent observed afterinterferon induction in mice of the A2G strain (Fig. 3). LineK was bred to be homozygous (KK) for the transgene. Thisstatus was first judged from the intensity of the bands inSouthern blots and then confirmed by backcrossing.Mice with incorporation of the mAlb/Mxl construct. Nine

transgenic mice with germ line incorporation of constructmAlb/Mxl were obtained, with an efficiency of about 7%. Inone line, named S, transgene incorporation occurred in twodifferent loci (Fig. 4) which segregated with further breeding.This and another line, named P, expressed Mx proteinexclusively in the liver, as expected. The two lines were bredto contain the transgene in at least two loci or to behomozygous for it, respectively; these lines were named SSand PP. Constitutive expression ofMx protein in these casesamounted to about one-half to three-fourths that of mice ofthe A2G strain stimulated with poly(rI) poly(rC) (Fig. 5).Unexpectedly, mice of the PP line and individuals with an SSpattern showed reduced Mx expression in the liver afterabout 3 months from the second generation onward (Fig. 5).The question of whether this development was due tomethylation of the transgene was approached by studyingthe pattern of restriction fragments after digestion of liver-derived genomic DNA with the methylation-indicative re-striction enzymes HpaII and MspI. The results showed thatmethylation within the Mx region of most but not all copiesof the transgene occurred early but did not change with time(Fig. 6) and therefore could not explain the loss of expres-sion with time. However, it is possible that the albuminpromoter, which was not probed for, was increasingly meth-ylated. A similar observation in a different transgene systemhas been reported by Mehtali et al. (18).

Preliminary tests with a hepatotropic influenza A virusindicated some resistance (not shown), but in later tests withmice 3 to 6 months of age, all except one mouse died a fewdays after virus infection, probably because of Mx proteinsuppression.

Resistance of mice with the mHMG/Mxl transgene testedwith the neurotropic influenza A virus strain NWS or tested inthe presence of anti-interferon antibodies. (i) Tests with

BAkb 0 P 0 R S T U V W 1 2 3 4

o ..:.:

,x-

5.0-

4.3-

3.5 -

1.9 -

1.6 -

1.3 -

FIG. 4. (A) Southern blot analysis of all nine mAlb/Mxl trans-genic mice (lanes 0 to W) and a nontransgenic control (lane 1).Chromosomal DNA was cut with PstI. Small dots, endogenous Mxbands, present in all mice. The intensity of 3.2-kb fragment isindicative of the amount of DNA loaded. Large dots, transgenicbands. The 0.6-kb fragment stems entirely from within the Mxl-coding region (see Fig. 1B). It is the same as that in the mHMG/Mxlmice. The 1.7-kb fragment probably corresponds to the one adjacentto it in the sequence; it also must contain some non-Mxl-codingnucleotides from plasmid pSP 64. From the 1.7-kb fragment, thenumber of transgene copies integrated was estimated (betweenabout two and eight). (B) Genomic DNA digested with Stul,allowing detection of the two patterns of segregation (lanes 3 and 4)in offspring of founder S, who incorporated the transgene into twosites. The pattern of founder S is shown in lane 2, containing all ofthe bands seen in lanes 3 and 4.

(C57BL/6 x SJL)F3 mice. Figure 7 shows the survival ratesof the KK transgenic mice to be 46 and 42%, res ectively,when tested with virus dilutions of 10-2 and 10-'. Controlmice with the same background and of similar age showedthe same lack of discrimination between the two virus doses.The average survival rates in these controls tested with thetwo doses were 4.4 and 4.3 days, respectively. These find-ings were attributed to the inhomogeneous genetic back-ground of all mice. Therefore, to reduce this heterogeneity,the KK mice with long-term survival were used for furtherbreeding. Mice of the next generation were again tested. Anincrease in long-term survival from 46 to 67% was obtained(Table 1); this result seemed to confirm our assumption.

(ii) (C57BL/6 x SJL)F3 mice tested in the presence ofanti-interferon antibodies. Virus dilutions (intracerebral) ofi0' and 10-4 and antibodies were given at the same time.Comparisons with the experimental group KK betweensurvival without and with antibodies show that survivalwithout antibodies was about 70% and survival with anti-bodies was somewhat lower, 63 and 50% at the two virusdoses given (better with the higher virus dose) (Table 2).Overall, this means that between 50 and 70% of the micesurvived, regardless of whether interferon activity wasblocked or free. Yet the fact that survival tended to beslightly better with interferon present probably indicates thata fraction of the resistance phenomenon is attributable tounspecific, interferon-induced antiviral mechanisms which

A

M, M;iv"-,i-:iR

J. VIROL.

a

on June 1, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

RESISTANCE TO INFLUENZA VIRUS OF Mx TRANSGENIC MICE 1713

I

I

FIG. 5. Immunofluorescence staining of Mxl protein in the liver of mice with the mAlb/Mxl transgene. Photographs show Mxl proteinexpression decreasing with time. (a) Mouse of the A2G strain stimulated with poly(rI) poly(rC) (100 ,ug i.p.) 24 h earlier. Abundant synthesisof Mxl protein with punctate pattern in liver cell nuclei is seen. (b) Mxl-negative littermate of transgenic mice. (c and e) Five-week-old miceof the SS and PP lines, respectively, carrying two transgenic Mxl alleles in different (SS) or the same (PP) loci. There is stronger Mxl proteinexpression in mouse SS than in mouse PP, with mouse SS reaching almost the level of A2G mice. (d and f) Mice of the SS and PP lines,respectively, at the age of 6 months. They were from F2 generations with respect to S and P founders. Note the decrease of Mx proteinexpression from panels c to d and e to f. Bar = 50 aLm.

seemed to be stronger at the higher virus dose (dilution of feron, which counteracted the antibodies and allowed 60% of10-3). the mice to survive. This result was in contrast to those ofA similar observation was made for the A2G controls, in earlier experiments (10) in which no A2G mice survived in

which the higher virus dose seemed to induce more inter- the same setting. At the lower virus dose, less interferon was

VOL. 66, 1992

I

on June 1, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

1714 KOLB ET AL.

A

kb

3.5 - 0

1.9

1*6

1.3 -

B C

1 2 3 4 5 6 7 8 9 101112_

~~ ~ ~ ~

control

10 -2

control- 4

1 0

=~~ ~~~ ffi s~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~........

transgenic-2

_ _ 0IO= ~<f_ fi>B_-

FIG. 6. Southern blot analysis of DNA isolated from liver di-gested with three different restriction enzymes (one methylationsensitive) of transgenic mice carrying the mAlb/Mxl cDNA con-struct, performed at 3 to 5 weeks or 5 to 6 months of age. (A)Digestion with PstI and HpaII (which exclusively cuts the nonme-thylated form of cDNA); (B) digestion with PstI and MspI (which isan isoschizomer of HpaII but cuts both methylated and nonmethy-lated forms of cDNA); (C) digestion with PstI alone. Lanes: 1, 5,and 9, young mice of transgenic line S; 2, 6, and 10, older individualsof the same line; 3, 7, and 11, young mice of transgenic line P; 4, 8,and 12, older individuals of the same line. The analysis shows thesame patterns in all mice, irrespective of line and age. Panel Ashows that only a small fraction of the transgene, the Mxl part, wasnonmethylated (faint band of 1.5 kb) at any of the times studied.

probably induced and was apparently inactivated by theantibodies; thus, neither Mx protein nor sufficiently strongother antiviral activities could be induced, and all of the micedied. Without anti-interferon antibodies, all of the A2G micesurvived. The discrepancy mentioned above with respect tothe efficacy at the higher virus dose of a given anti-interferon

TABLE 1. Survival of selected' KK transgenic mice andunselected K transgenic mice after infection with the neurotropicinfluenza A virus strain NWS (10-2 dilution of stock solution6)

Line No. of Day of Long-term survivalmice death (survivors/total [%l)

KK 10 10/15 (67)2 73 6

K 12 12/36 (33)1 109 93 7

11 12Controls 16 5.4 (avg) 0/16 (0)Mx BALB/c congenic controls 10 10/10 (100)BALB/c controls 6 5.5 (avg) 0/6 (0)

Bred with surviving individuals from tests shown in Fig. 7.Stock solution was an allantoic fluid mixture with an infectivity of 106.5

LD50. Application volume was 0.03 ml.

transgenic

sX wff IT,AM,,4.E 'gA.' 4

________________ 4E

= 1~~~~~~~~~~~~0

I -4eI

3 4 5 6 7 30FIG. 7. Survival of KK transgenic mice (homozygous for the

transgene) after injection with the neurotropic influenza A virusstrain NWS. Each bar represents one mouse. The scale at thebottom indicates days. The stock solution of virus (allantoic fluidwith infectivity of 106.5 LD50) was diluted 10-2 and 10'. Survivalwith the higher and lower doses was almost the same in the controls(on average, 4.4 and 4.3 days, respectively). In the transgenicgroups, 6 of 13 (46%) of the mice survived with the higher virus doseand 5 of 12 (42%) survived with the lower dose.

antibody preparation was most likely due to an unnoticedinfection in our present A2G colony, causing some inter-feron background. This view was supported by the recentobservation on a tissue extract from A2G mice of this colonyshowing, by Western immunoblotting, a low level of Mxprotein activity without external induction.

In a comparison of KK and A2G mice, KK performancewas remarkably good in view of the fact that the extent ofconstitutively expressed Mxl protein did not reach the levelsof induced Mx of A2G mice.

In an Mxl-negative control group (BALB/c), all mice diedirrespective of the presence or absence of antibodies. Thisfinding indicates that nonspecific, interferon-induced antivi-ral mechanisms were rather weak and that the absence ofMxl determined the outcome.

DISCUSSION

Recently, Mx1 transgenic mice with a construct contain-ing the murine Mx1 promoter coupled to the structural Mx1cDNA were generated (3). In this setting mimicking thestatus of naturally influenza A virus-resistant mice of theA2G strain, a well-expressing line (interferon induced Mx1

-., -., ..-

MMM"r,"I"

M-9030=7--iEss3mE=

anoia

3MM

0M

r-,

MIM..

mmm;mrl---71 1.11

opilgAMBBZMmmmvrAmr-llln.

ompmzmgmmm

annommmommmxmwmm=m

J. VIROL.

on June 1, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

RESISTANCE TO INFLUENZA VIRUS OF Mx TRANSGENIC MICE 1715

TABLE 2. Survival of selecteda KK transgenic mice after infection with the neurotropic influenza A virus strain NWS and concomitanttreatment with mouse anti-interferon antibodies (10-i and 10-' dilutions of virus stock solutionb)

Line Treatment Virus dose No. of mice Day of death Long-term survival(survivors/total [%])

KK Anti-interferon antibodies 10-3 19 5, 5, 6, 6, 6, 6, 17 12/19 (63)_c 10-3 10 5, 5, 8 7/10 (70)

Anti-interferon antibodies 10-4 10 5, 7, 8, 10, 10 5/10 (50)10-4 11 7, 7, 7 8/11 (73)

A2G Anti-interferon antibodies 10-3 5 9, 9 3/5 (60)10-3 5 5/5 (100)

Anti-interferon antibodies 10-4 4 3, 15, 17, 22 0/4 (0)10-4 4 4/4 (100)

BALB/c control Anti-interferon antibodies 10-3 5 3, 3, 3, 3, 3 0/5 (0)-1l0-3 4 3, 4, 5, 6 0/4 (0)

a.b See Table 1, footnotes a and b.c, no treatment.

mRNA and Mxl protein in different organs to a level ofabout two-thirds that of A2G mice) showed a degree ofresistance against the neurotropic influenza A virus strainNWS similar to that of A2G mice, thus confirming theexpected effect of the introduced construct. The results ofour experiments, however, show that the transgene-encodedMx protein expressed in the brain of line KK, uncoupledfrom induction by interferon, unequivocally conferred on thegenetically Mxl-negative mice resistance against the neuro-tropic influenza A virus, even in the presence of anti-interferon antibodies which prevented induction of anyadjuvant antiviral factors. These findings were in agreementwith results from cultured 3T3 cells transfected with simianvirus 40/Mxl cDNA (30). The experiments with antibodiesto interferon type I indicated that in experimental mice,there was probably a small contribution to resistance byadditional interferon-induced factors.The degree of resistance in transgenic mice amounted to

about one-half to two-thirds that of naturally resistant mice.This was probably due to somewhat less abundant expres-sion of Mxl in the brain of the transgenic mice, as estimatedby the number of cells with Mx fluorescence. Fluorescenceintensity per cell was similar in transgenic and A2G mice.The fact that in the experimental mice Mx protein wasmainly expressed in the brain, whereas it was induced inmost organs in the A2G strain, probably had no effect onsurvival in view of this virus strain's neurotropism. Indeed,by using fluorescent antiviral antibodies, virus could bedemonstrated only within, never outside, the brain in bothtransgenic and A2G mice (not shown).The predominant expression of Mx protein in the brain

and the limited (heart, testis) or absent expression in otherorgans was unexpected, since according to the literature (18)transgenes under the control of the HMG gene promoter areexpressed in most organs. Apparently, in our case, the organdistribution of transgene expression was also influenced bythe coding region. Considering the similarity of the expres-sion patterns in three transgenic lines, the site of integrationcould have influenced the level of expression but veryunlikely the organ pattern.The studies of transgenic mice with the mAlb/Mxl con-

struct in two expressing lines (Mxl protein constitutivelypresent in the liver) gave complex results in more than onerespect. First, there were at least two different integrationsites in one line (S) which segregated by further breeding.Mice with two integration sites (SS) or homozygous for thetransgene (PP) [the extent of expression here was at first

about three-fourths that of poly(rI) poly(rC)-stimulatedA2G mice] were bred for resistance tests. To our surprise,Mxl expression markedly diminished during this time (about3 months), and resistance against a hepatotropic influenza Avirus almost vanished. The mechanism of this phenomenonwas not elucidated, but it was observed that methylation ofthe Mx part of the transgene took place early on, and it couldwell be that with time, methylation extended also to thealbumin promoter and increased there with time, as noted byMehtali et al. (18) with the HMG gene promoter in anHMG/chloramphenicol acetyltransferase construct. Fromthe results with both types of cDNA constructs, the follow-ing conclusions can be drawn. Widespread and permanentexpression of Mxl protein seems not to be easily admittedby the organism. In addition, one may even speculate thatour modest success rate of transgenification in the case ofmAlb/Mxl (7% versus up to 25% with some other cDNAs inour hands) was partly due to death of transgenic fetusesresulting from Mxl interference with essential developmen-tal steps. This impression of negative effects by the forcedcontinuous presence of Mx protein together with our first-hand proof that Mxl on its own in the mouse is the majoranti-influenza virus effector (normally dormant, as we know,but rapidly mobilizable) will help in seeing recently discov-ered structural relationships, unsettling to some, in an un-strained perspective.

ACKNOWLEDGMENTS

The gifts of plasmid mHMG by P. Gerlinger, University ofStrasbourg, Strasbourg, France, and R. Lath6, University of Edin-burgh, Edinburgh, Scotland, of plasmid Alb e/p by D. Palmiter,University of Washington, Seattle, and of anti-interferon serum byI. Gresser, Institut de Recherche Scientifique sur le Cancer,Villejuif, France, are greatly appreciated.

This study was supported by the Swiss National Science Foun-dation (grants NF 3.411.86 and NF 3100-026330), by the HartmannMuller Foundation for Medical Research, and by the Emdo Foun-dation.

REFERENCES1. Aebi, M., J. Fah, N. Hurt, C. E. Samuel, D. Thomis, L.

Bazzigher, J. Pavlovic, O. Haller, and P. Staeheli. 1989. cDNAstructures and regulation of two interferon-induced human Mxproteins. Mol. Cell. Biol. 9:5062-5072.

2. Arnheiter, H., and E. Meier. 1990. Mx proteins: antiviralproteins by chance or by necessity? New Biol. 2:851-857.

3. Arnheiter, H., S. Skuntz, M. Noteborn, S. Chang, and E. Meier.1990. Transgenic mice with intracellular immunity to influenza

VOL. 66, 1992

on June 1, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

1716 KOLB ET AL.

virus. Cell 62:51-61.4. Bchini, O., A. C. Andres, B. Schubaur, M. Mehtali, M. LeMeur,

R. Lathes, and P. Gerlinger. 1991. Precocious mammary glanddevelopment and milk protein synthesis in transgenic miceubiquitously expressing human growth hormone. Endocrinol-ogy 128:539-546.

5. Dever, T. E., M. J. Glynias, and W. C. Merrich. 1987. GTP-binding domain: three consensus sequence elements with dis-tinct spacing. Proc. Natl. Acad. Sci. USA 84:1814-1818.

6. Epstein, C. J., K. B. Avraham, M. Lovett, S. Smith, 0. Elroy-Stein, G. Rotman, C. Bry, and Y. Groner. 1987. Transgenic micewith increased Cu/Zn-superoxide dismutase: animal model ofdosags- effects in Down syndrome. Proc. Natl. Acad. Sci. USA84:801 h-8048.

7. Gauthier, C. M., M. Mehtali, and R. Lathe. 1989. A ubiquitousmammalian expression vector, pHMG, based on a housekeep-ing gene promoter. Nucleic Acids Res. 17:8389.

8. Gordon, J. W., and F. H. Ruddle. 1983. Gene transfer intomouse embryos. Production of transgenic mice by pronuclearinjection. Methods Enzymol. 101:411-433.

9. Haller, O., M. Acklin, and P. Staeheli. 1987. Influenza virusresistance of wild mice: wild type and mutant Mx alleles occurat comparable frequencies. J. Interferon Res. 7:647-656.

10. Haller, O., H. Arnheiter, I. Gresser, and J. Lindenmann. 1979.Genetically determined, interferon-dependent resistance to in-fluenza virus. J. Exp. Med. 149:601-612.

11. Hogan, B., F. Costantini, and E. Lacy. 1986. Manipulating themouse embryo: a laboratory manual. Cold Spring Harbor Lab-oratory, Cold Spring Harbor, N.Y.

12. Horisberger, M. A. 1988. Homologs of the interferon-inducedmouse Mx protein in 14 animal species. J. Interferon Res.8(Suppl. I):S102.

13. Horisberger, M. A. 1988. The action of recombinant and bovineinterferons on influenza virus replication correlates with theinduction of two Mx-related proteins in bovine cells. Virology162:181-186.

14. Hug, H., M. Costas, P. Staeheli, M. Aebi, and C. Weissmann.1988. Organization of the murine Mx gene and characterizationof its interferon- and virus-inducible promoter. Mol. Cell. Biol.8:3065-3079.

15. Lindenmann, J. 1981. The role of interferon in natural resis-tance, p. 1-12. In J. Gresser, K. Cantell, E. DeMaeyer, M.Landy, M. Revel, and J. Vilcek (ed.), Interferon 1981. Aca-demic Press, London.

16. Lindenmann, J., C. A. Lane, and D. Hobson. 1963. The resis-tance of A2G mice to myxoviruses. J. Immunol. 90:942-951.

17. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1989. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

18. Mehtali, M., M. LeMeur, and R. Lathe. 1990. The methylation-free status of a housekeeping transgene is lost at high copynumber. Gene 91:179-184.

19. Meier, E., J. Fah, M. S. Grob, R. End, P. Staeheli, and 0.

Haller. 1988. A family of interferon-induced Mx related mRNAsencodes cytoplasmic and nuclear proteins in rat cells. J. Virol.62:2386-2393.

20. Meier, E., G. Kunz, 0. Haller, and H. Arnheiter. 1990. Activityof rat Mx proteins against a rhabdovirus. J. Virol. 64:6232-6269.

21. Noteborn, M., H. Arnheiter, L. Richter-Mann, H. Browning, andC. Weissmann. 1987. Transport of murine Mx protein into thenucleus is dependent on a basic carboxy-terminal sequence. J.Interferon Res. 7:657-669.

22. Obar, R. A., C. A. Collins, J. A. Hammarback, H. S. Shpetner,and R. B. Vallee. 1990. Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homol-ogy with a new family of GTP-binding proteins. Nature (Lon-don) 347:256-261.

22a.Pavlovic, J., T. Zurcher, 0. Haller, and P. Staeheli. 1990. Resis-tance to influenza virus and vesicular stomatitis virus conferredby expression of human MxA protein. J. Virol. 64:3370-3375.

23. Pinkert, C. A., D. M. Ornitz, R. L. Brinster, and D. Palmiter.1987. An albumin enhancer located 10 kb upstream functionsalong with its promoter to direct efficient, liver-specific expres-sion in transgenic mice. Genes Dev. 1:268-276.

24. Rothman, J. H., C. K. Raymond, T. Gilbert, P. O'Hara, and T.H. Stevens. 1990. A putative GTP binding protein homologous tointerferon-inducible Mx proteins performs an essential functionin yeast protein sorting. Cell 61:1063-1074.

25. Segev, N., J. Muholland, and D. Botstein. 1988. The yeastGTP-binding YPT1 protein and a mammalian counterpart areassociated with the secretory machinery. Cell 52:915-924.

26. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

27. Staeheli, P. 1990. Interferon-induced proteins and the antiviralstate. Adv. Virus Res. 38:147-199.

28. Staeheli, P., P. Dreiding, 0. Haller, and J. Lindenmann. 1985.Polyclonal and monoclonal antibodies to the interferon-induc-ible protein Mx of influenza virus-resistant mice. J. Biol. Chem.260:1821-1825.

29. Staeheli, P., R. Grob, E. Meier, J. G. Sutcliffe, and 0. Haller.1988. Influenza virus-susceptible mice carry Mx genes with alarge deletion or a nonsense mutation. Mol. Cell. Biol. 8:4518-4523.

30. Staeheli, P., 0. Haller, W. Boll, J. Lindenmann, and C. Weiss-mann. 1986. Mx protein: constitutive expression in 3T3 cellstransformed with cloned Mx cDNA confers resistance to influ-enza virus. Cell 44:147-158.

31. Staeheli, P., and G. Sutcliffe. 1988. Identification of a secondinterferon-regulated murine Mx gene. Mol. Cell. Biol. 8:4524-4528.

32. Staeheli, P., Y.-X. Yu, R. Grob, and 0. Haller. 1989. Adouble-stranded RNA-inducible fish gene homologous to themurine influenza virus resistance gene Mx. Mol. Cell. Biol.9:3117-3121.

J. VIROL.

on June 1, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from