D N A SiqiiwL? - Tire 1 0 i i 1 ? i d ifS0711mcing nrid Mti}yrrr~, Vol. 7(3/4), pp. 239-242 Reprmts available directly from the publisher Photocopying pennitted by license only

C 19Y7 OI’A (Overseas I’ublishers Aswoation) Amsterdam B.V. Published in The Nethcrlands by

Harwood Academic I’ublihherb Printed 111 Malaysia

Short Communication

Nucleotide Sequence of Equine MxA cDNA

Database Accession No.: U55216

A 2.6 kb cDNA species has been isolated from a cDNA library prepared from interferon-a stimulated equine peripheral blood leucocytes and the nucleotide sequence determined. The cDNA has a single open reading frame potentially encoding a 660 amino acid polypeptide showing a high degree of homology with known mammalian Mx proteins, including the posses- sion of three consensus GTP-binding motifs. The pro- tein has a calculated pl = 6.1 and in accordance with proposed nomenclature we have designated it equine MxA.

Kryiuortls: cDNA sequence, equine Mx, Mx protein

Mx was originally discovered in mice to confer resistance to influenza virus. However, the mech- anism of the antiviral action of Mx protein remains unclear (reviewed in Sen and Ransohoff, 1993 and references within). Mx proteins have been detected in many mammalian species (Horisberger and Gunst, 1991) and in chickens (Schumacher ef al., 1994). These proteins are pro- duced in response to interferon (IFN) and contain a GTP binding domain including three consensus motifs (Muller et al., 1992) (Fig. 1).

Horisberger and Gunst (1991) have demon- strated the presence of Mx protein homologues in extracts of IFN stimulated cells from a num- ber of mammalian species using 2D gel electro- phoresis, immunoprecipitation and Western blot-ELISA and have determined the cellular location of these homologues by immuno- staining fixed cells. Equine cells lines were found to contain two Mx protein species after IFN treatment. In order to further characterise equine Mx genes we have made a cDNA library in h-Zap (Stratagene, La Jolla, USA) from mRNA isolated from equine peripheral blood leucocytes treated in u i f r o with human recombi- nant IFN-a. We have isolated and sequenced a cDNA of length 2585bp excluding the poly(A) tail (fig. 1) by screening the A-Zap library with a 536bp equine Mx DNA probe (fig. 1, nucleo- tides 915 to 1451) amplified from the library by PCR using oligonucleotide primers annealing to sequences conserved among mammalian Mx genes. The Mx consensus translation start sequence. (Muller et al . , 1992) and the polya-

*Corresponding author. Tel.: (..44-171) 468 5299. Fax: (..44-171) 388 2342. E-mail: nedingto@rvc.ac.uk.

239

Mito

chon

dria

l DN

A D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y T

he U

nive

rsity

of

Man

ches

ter

on 1

2/08

/14

For

pers

onal

use

onl

y.

A G G A C C T O G C C C T O C C n ; C C G C C G T W L T C G G G G A C C A D L A L P A I A V I [ G D Q S S G K S S V

T G C T O O A A G C T C T G C c c T T c c ~ ~ m ~ ~ l ! c L E A L S G V A L P R G S G I V T R C P

CTCTGGTGCTGAAAC~GACTGGTGAXlGAAGAlWGTGGAAAGGCAPAGTCAGCT L V L K L K R L V K E D E W K G K V S Y

A C C G G G A T A T C G R G G T T G A G A T T T C A A A T G C T T T G G A T G T R D I E V E I S N A L D V E E Q V R K A

C C C A G A A T G T C C T T G C T T ~ ~ G ~ A ~ G C ~ ~ A C T C ~ G Q N V L A G E G V G I S Q E L V T L E V

T C A G C T C T C C T C A n ; T C C C G O A T C n ; A C C C T O A T C D A C C T ~ C ~ ~ ~ ~ S S P H V P D L T L I D L P G I T R V A

C C G ~ A A ~ G C C A ~ C ~ C A T C ~ C G ~ ~ ~ ~ C l C A T C ~ ~ G ~ V G N Q P A D I G R Q I K T L I R K Y I

T C C A G A O O C A A G A G A C O A T C A A C C n ; O T G G T G O T C C C C A G Q R Q E T I N L V V V P S N V D I A T T

C G G A G G C G C T G A G C A n ; G C T C A G G A O G T G G A C C C C C ~ ~ ~ C C A T ~ T C T E A L S M A Q E V D P E G D R T I G I L

T G A C A A A G C C T G A C C T G G T A C C ~ ~ ~ T A ~ C G T G G T G C ~ -1 L V D K G T E E Q V V D V V R N

A C C T C A T C T G C C A C C T G A A G A A G G G T T A T A T G A T C G T C A A L I C H L K K G Y M I V K C R G Q Q D I

T C c A G G A C C G A C T G A G C C T T G A . G G c T C T G C A ~ G G c C T T C ~ Q D R L S L A E A L Q R E K A F F E E N

A C C C A T A T T T C A G G G G C C T T C T G G A G G A A G G A A G A G C C T C P Y F R G L L E E G R A S V P C L A E R

G G C T O R C C A C T G A A C T C A ~ C G C A C A ~ A G ~ T C ~ ~ C C C ! N i ~ l C ~ L T T E L I T H I S K S L P L L E N Q I

T A A A G G A A A G T T A C C T A ~ C G A G ~ ~ ~ C C ~ ~ T C C ~ K E S Y Q N L S D E L Q K Y G T D I P E

A A G A T G A A A C ~ G T T C T T C C n ; A T A G T G A A A A T D E T E K T F F L I V K I T T F N Q N I

T C A C C T C T T T G ~ ~ C C ~ ~ ~ ~ ~ ~ T ~ T S F V Q G E E L V G P N D T R L F N K

60

120 6

180 26

240 46

300 66

360 86

420 106

480 12 6

540 146

600 166

660 18 6

720 206

7 80 226

840 246

900 266

960 286

1020 306

1080 326

1140 346

1200 366

12 60 386

1320 406

FIGURE 1 This figure shows the nucleotide sequence of the equine MxA gene with the deduced aa sequence below it. The nucleotide sequence was determined using an ABI 373A (version 1.2.1) DNA sequencer employing Taq polymerase with fluorescently labelled dideoxy chain termination nucleotides. A directed sequencing strategy was adopted using, in addition to Mx specific primers, a T7 22-mer, an SK 20-mer (Stratagene) and an M13 reverse 17-mer primer. Sequencing primers were synthesised by Pharrnacia Biotech except where indicated. The sequence of both strands was determined from 46 separate sequences; 21 covering the strand shown and 25 the complementary strand, p i n g an average depth of coverage of 5.96. Sequence analysis was carried out using DNASTAR programs. Consensus GTP binding sequences are shown boxed. The MY consensus translation start sequence and the polyadenylation signal are underlined (once and twice respectively).

Mito

chon

dria

l DN

A D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y T

he U

nive

rsity

of

Man

ches

ter

on 1

2/08

/14

For

pers

onal

use

onl

y.

EQUINE Mx cDNA SEQUENCE 241

A A A T C C G A C A G G A G T T C C A G A A A T G G A G T G G G G T G A T T G A T G I R Q E F Q K W S G V I E N N F R K G G

G C G A A G C T A T C C G T A G A C A G A T C n ; G A C A T ~ ~ T C ~ T A T c G ~ G A ~ ~ T A C E A I R R Q I W T F E N Q Y R G R E L P

C A G G A T T T G T G A A T T A C A ~ ~ T T T ~ ~ C ~ ~ ~ ~ G C ~ T C C A T G C T G ~ G ~ ~ T ~ G F V N Y R T F E T I I K Q Q I Q L L E

A A G A G C C A G C C A T T G A T A T G C n ; C A C A G G A T A A G T G A T C T E P A I D M L H R I S D L V R D T F T K

AAGTTTCAGAAAAAAATTTCAGTGAATTTTTCAACCTC~~~C~C~GTCCAAAC V S E K N F S E F F N L H R T T K S K L

T T G A A O A C A T T A A A T T T ~ G C ~ ~ ~ ~ ~ C ~ C ~ ~ C T T C C E D I K L E Q E N E A E K S I R L H F Q

AAATGGAGAAGATCGTCTACTGCCAAGACCACGT~ACC~~CGTTA~GAA~TCA M E K I V Y C Q D H V Y R G T L Q K V R

G A G A G A A T G A A A T G G A G A A T C ~ C G T C T ~ T C ~ C A E N E M E E E K K K K T I N V W G Q N T

C T T C C A C A G A G T C C T C G A n A ~ T C T ~ ~ A T C T C A A C G C C T A C ~ G C A C ~ ~ S T E S S M A E I L E H L N A Y Q H E A

CCGGCAACCGCCTCTCGACCCACATCCCCCTTGATCATCCAGT~CTTCGTCC~CAGACAT G N R L S T H I P L I I Q F E V L Q T F

TCGGCCAGCAGCTGCAGAAGTCCATGCTGCAT~TGCAGC~CT~~CA~ACACCTACGACT G Q Q L Q K S M L Q L L Q D R D T Y D W

G G C T C C T G A A G G A G C G C A A C G A C A C C T G T G A C A A G A ~ G ~ C C T ~ ~ ~ G L L K E R N D T C D K R K F L K E R L A

CTCGGCTGGCCCAGGCTCGGCCGGTTAGCCAAGTTCCC~TTAAATC~TCTCTG R L A Q A R R R L A K F P G

TCTCAGCCTCATGTCTCCATGCACATCTCCAGGGAGCGGAGGCCCAGCATCTCTCCCC~

CAGCCACACCATCATTAGTTACCCATTCACAGATACCCGAGCGGTTAC~TCA~C~G

G T G G T C A C T G T C T G T G C T n ; T C C T T ~ A G G A T G

CGATTTGGTTTCAAGCATPGAGACTAGAGCCCCGCCC~CTT~TG~AGCATA~TTTAGACT

G A A T G A G C A G T G C C A T T T T C ~ T T A ~ ~ T ~ T T T T C T A C C C C C A ~ T ~ ~ C T

C G T C T C C A G A C T C T C T C C A T C T C T T T A T C A G A C C G A T G

G G T T A T T T T C G G T T T T T T T G T G T ~ T C C C T

G C T G C T T G T G T G A A T G C T T G T A ~ ~ C A T C T T T T T T T C T

A C T A G P

FIGURE 1 (Contrrzued)

1380 42 6

1440 446

1500 466

1560 486

1620 506

1680 526

1740 546

1800 566

1860 586

1920 606

1980 62 6

2040 646

2100 660

2160

2220

2280

2340

2400

2460

2520

2580

2608

denylation signal are both indicated in approx. 75.6kDa in accordance with the MR range Fig. 1. Within this sequence there is a single of known Mx proteins (70 to 80 kDa; Horisberger open reading frame from nucleotides 105 to and Gunst, 1991) and shows a high degree of 2087 potentially encoding a 660 amino acid (aa) homology with known Mx proteins, including polypeptide. The deduced aa sequence of this human MxA (75.2”/,); human MxB (60%); Mouse polypeptide (Fig. 1) determined using a DNAS- Mxl (63.2%); mouse Mx2 (73.9%); pig M x l TAR translation program, has a calculated MR of (74.4%); pig Mx2(74%); sheep Mx (72.3%); rat

Mito

chon

dria

l DN

A D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y T

he U

nive

rsity

of

Man

ches

ter

on 1

2/08

/14

For

pers

onal

use

onl

y.

242 P. M. CHESTERS et al.

Mxl (63.9%); rat Mx2 (72.2%); and rat Mx3 (71.9%). In contrast equine Mx showed only 46.5% homology with chicken Mx. A comparison of a11 known Mx proteins has shown an average homology of 67.5% (Muller et al., 1992). The three consensus GTP binding motifs that are highly conserved amongst known Mx proteins (Muller et aI., 1992) are shown boxed in Fig. 1.

The sequence data indicate that equine Mx is typical of known Mx proteins in terms of sequence homology and MR. Most known Mx proteins accumulate in the cytoplasm after IFN induction, only rat Mxl, mouse Mxl, hamster MxA2, and Guinea pig Mx proteins being found in the nucleus (Horisberger and Gunst, 1991; Muller et al., 1992; Samuel, 1991). Both rat Mxl and mouse Mxl possess a nuclear localisation signal in the carboxy terminal region (Muller et al., 1992). No such signal exists in equine Mx, indicating its cytoplasmic location in agreement with the findings of Horisberger and Gunst (1991).

Mouse Mxl protein appears to selectively inhibit influenza virus replication while leaving unaffected the replication of other viruses, including vesicular stomatitis virus (VSV). In contrast, human MxA protein appears to protect cells against influenza virus and VSV. Human MxB, which is also cytoplasmic, does not appear to confer protection against any virus, including influenza and VSV. While the different cellular localisation of human and mouse Mx proteins would indicate a different mode of action of these proteins, it does not seem to provide the explana- tion for their different specificities. Evidence for this comes from investigation of the expression of rat Mx cDNAs in cells cultured in vitro. Rat Mxl, which is nuclear, inhibits both influenza virus and VSV, rat Mx2, which is cytoplasmic, inhibits VSV, but not influenza virus and rat Mx3, which is cytoplasmic, inhibits neither virus (reviewed in

Samuel, 1991). The specificity of human MxA protein against both influenza virus and VSV appears to be conferred by a peptide sequence (. . .RKFLKERLARL.. .) in the carboxy terminal region (Zurcher et al., 1992), a sequence also pre- sent in equine Mx protein (fig. 1, peptides 638-648). Furthermore, since both human MxA and equine Mx are localised in the cytoplasm (Horisberger and Gunst, 1991) and show a high degree of homology (75.2%) it seems likely that equine Mx might have similar antiviral proper- ties to human MxA.

Horisberger and Gunst (1991) have proposed a system of nomenclature for Mx proteins based on the isoelectric point, PI, such that acidic proteins (PI < 7) are designated MxA and basic proteins (PI 2 7) are designated MxB. The Mx protein described here has a calculated PI = 6.1 and we propose to designate it equine MxA.

Acknowledgements

This work was funded by a grant from the Horserace Betting Levy Board.

References

Horisberger, M. A. and Gunst, M. C . (1991) "Interferon- induced pr0teins:identification of Mx proteins in various mammalian species", Vir., 180, 185-190.

Muller, M., Winnacker, E-L. and Brem, G. (1992) "Molecular cloning of porcine Mx cDNAs:New members of a family of interferon-inducible proteins with homology to GTP-bind- ing proteins", J. Interferon Res., 12,119-129.

Samuel, C. E. (1991) "Antiviral actions of interferon. Interferon-regulated cellular proteins and their surpris- ingly selective antiviral activities", Vzr., 183,l-11.

Schumacher, B., Bernasconi, D., Schultz, U. and Staeheli, P. (1994) "The chicken Mx promoter contains an ISRE motif and confers interferon inducibility to a reporter gene in chick and monkey ce!Is", Vir., 203, 144-148.

Sen, G. C. and Ransohoff, R. M. (1993) "Interferon-induced antiviral actions and their regulation", Advances in Virirs Research, 42,57-102.

Ziircher, T., Pavlovic, J. and Staeheli, P. (1992) "Mechanism of human MxA protein action:variants with changed antiviral properties", EM50 I., 11(4), 1657-1661.

Mito

chon

dria

l DN

A D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y T

he U

nive

rsity

of

Man

ches

ter

on 1

2/08

/14

For

pers

onal

use

onl

y.