A Naturally Occurring Variant of Porcine Mx1 Associated with Increased Susceptibility

[Mingus]

Mingus'note:

They have apparantly found a swine gene variant responsible to an increase suceptibility to influenza.
The first step in the quest to find the so-said genetic factor that human also may have.

It's so far the first serious hint pointing toward that path that I see.

__________________________________________________ _____

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17203407&query_hl=1&itool=pubmed_docsum

<table border="1" cellpadding="2"> <tbody> <tr class="header"> <td>Biochemical Genetics</td></tr> <tr> <td>? Springer Science+Business Media, LLC 2006</td></tr> <tr> <td>10.1007/s10528-006-9045-y</td></tr></tbody></table><!--Begin Abstract--> A Naturally Occurring Variant of Porcine Mx1 Associated with Increased Susceptibility to Influenza Virus In Vitro
Emiko Nakajima¹, Takeya Morozumi², Kenji Tsukamoto³, Tomomasa Watanabe⁴, Graham Plastow⁵ and Tadayoshi Mitsuhashi¹
<table> <tbody> <tr valign="top"> <td>(1) </td> <td>Animal Gene Function Laboratory, Department of Physiology and Genetic Regulation, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki 305-0901, Japan</td></tr></tbody></table> <table> <tbody> <tr valign="top"> <td>(2) </td> <td>Second Research Division, STAFF Institute, 446-1 Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 305-0854, Japan</td></tr></tbody></table> <table> <tbody> <tr valign="top"> <td>(3) </td> <td>Department of Virology, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-0856, Japan</td></tr></tbody></table> <table> <tbody> <tr valign="top"> <td>(4) </td> <td>Laboratory of Animal Breeding and Reproduction, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan</td></tr></tbody></table> <table> <tbody> <tr valign="top"> <td>(5) </td> <td>Sygen International, 2 Kingston Business Park, Kingston Bagpuize, Oxon, OX13 5FE, UK</td></tr></tbody></table>
<table class="Contact"> <tbody> <tr> <td valign="top"></td> <td>Tadayoshi Mitsuhashi
Email: tad@affrc.go.jp</td></tr></tbody></table> Published online: 4 January 2007
Mx1 has been implicated in resistance to the influenza virus. We have now identified four alleles of the Mxl gene in domesticated breeds of pigs. Two of the alleles encode deletion variants (a 3-bp deletion in exon 13 and an 11-bp deletion in exon 14), which might be expected to interfere with Mx activity. The porcine Mxl genes corresponding to wild type, the 3-bp deletion mutant, and the 11-bp deletion mutant were cloned and expressed in NIH3T3 cells, and the antiviral activity for influenza virus was assayed. Virus yield was observed to be 10?100-fold greater with the 11-bp deletion allele than that for wild type and the 3-bp deletion alleles. The results suggest that the 11-bp deletion type is lacking antiviral activity able to contribute to the interference of influenza virus replication.
KEY WORDS porcine Mx1 - influenza virus - deletion allele
<hr> INTRODUCTION
Influenza virus infections are common and occasionally cause epidemic disease as a result of antigenic drift and genetic shift mutations not only in humans but also in a variety of domestic animals, such as the chicken (fowl plague), the horse (equine influenza), and the pig (swine influenza) (Sereda, <cite>1974</cite>).
Influenza virus was the first mammalian virus for which replication could be studied biochemically (Hirst, <cite>1941</cite>). The genome and transcriptome of this RNA virus have now been well characterized (Enami, <cite>2002</cite>). Little is known, however, about the host mechanisms of resistance and susceptibility to influenza virus infection except for the Mx1 locus in mouse. The mouse influenza virus resistance locus Mx1 was first identified in 1964 (Lindenmann, <cite>1964</cite>), and the Mx protein was subsequently identified (Horisberger et al., <cite>1983</cite>) in the A2G mouse strain. The mouse Mx1 protein is induced by interferon (IFN) α/β (Haller et al., <cite>1980</cite>) as well as by dsRNA or several other viral infections (Pestka et al., <cite>1987</cite>), and it has an intrinsic antiviral activity for protection against influenza virus infection (Arnheiter et al., <cite>1991</cite>; Noteborn et al., <cite>1987</cite>; Staeheli et al., <cite>1986</cite>).
Mx-related proteins have been described in humans (Aebi et al., <cite>1989</cite>; Horisberger and Hochkeppel, <cite>1987</cite>; Staeheli and Haller, <cite>1985</cite>), cattle (Horisberger et al., <cite>1988</cite>), rats (Meier et al., <cite>1990</cite>), fish (Staeheli et al., <cite>1989</cite>), and even in yeast (Rothman et al., <cite>1990</cite>). The cDNA clone encoding the porcine Mx1 has been isolated and sequenced (M?ller et al., <cite>1992</cite>). A genetic approach was used to identify the role of Mx1 protein in protection against virus infection in the mouse, but little is known about the function of Mx proteins in farm animals and humans, as Mx-defective mutants have not been found in these species.
We previously reported polymorphisms (an 11-bp deletion) in exon 14 in the porcine (Landrace) Mx1 gene (Morozumi et al., <cite>2001</cite>), and more recently, we have identified an additional mutation of the porcine (Meishan) Mx1 in exon 13. The percentage of individuals that were homozygous for the 11-bp deletion was relatively low. We suspected therefore that the deletion might have a considerable effect on the susceptibility of pig to some diseases originating from RNA viruses. In order to investigate the antiviral potential of these porcine Mx1 proteins, we have established stable clones of transfected 3T3 cells that express normal or mutant types of Mx1. In this report, we show that the normal porcine Mx1 confers a degree of resistance to influenza A virus infection in vitro, but an 11-bp deletion variant appears to be completely defective in any antiviral activity for influenza A virus infection.

<hr> MATERIALS AND METHODS
cDNA Cloning of the Porcine Mx1
DNAs were extracted by DNAZol reagent (Invitrogen) from the porcine primary mononuclear cells of Landrace and Meishan breeds according to the manufacturer?s instructions. Exon 13 fragments from the Meishan breed (194 bp) were amplified with 20 pmol of each primer (forward primer 5′-AAGAAGCTGAGACGTCGATCCGGCT-3′ and reverse primer 5′-CTGAAAGATCTCGGCTATGGAGGG-3′). The PCR method consisted of an initial denaturation at 94?C for 5 min followed by 35 cycles of 94?C denaturation for 30 s, 64?C annealing for 30 s, and 72?C extension for 1 min, with a final 5 min extension at 72?C. Polymorphism in exon 13 (a 3-bp deletion) was detected by the direct sequencing of these PCR fragments. The 11-bp deletion in exon 14 (Landrace) was detected by restriction fragment length polymorphism (RFLP) as described previously (Morozumi et al., <cite>2001</cite>).
To induce Mx1 cDNA, the porcine mononuclear cells were treated for 3 h with serum-free RPMI 1640 medium containing 500 U/mL of the recombinant human IFN α (3?10⁸ U/mg) (Pepro Tech EC). Total RNAs were prepared using ISOGEN (Nippon Gene), and poly (-A) RNAs were purified using a mRNA Purification Kit (GE Healthcare). Each cDNA of Mx1 was amplified with the primers of attB1MxF (5′-GGGGACAAGTTTGTACCAAAAAAGCAGGCTGTCACAGCG AAGAAAAGGAAG-3′) and attB2Mx-R (5′-GGGGACCACTTTGTACAAGAA AGCTGGGTCCTTCTATGATGCTATGCGG-3′) or attB2Rdel (5′-GGGGACC ACTTTGTACAAGAAAGCTGGGTCGTCTGTTAGGGAAGGAGG-3′) by Superscript one-step RT-PCR with Platinum Taq (Invitrogen). The RT-PCR method consisted of 30 min incubation at 50?C and an initial denaturation at 94?C for 2 min followed by 35 cycles of 94?C denaturation for 15 s, 64?C annealing for 30 s, and 72?C extension for 3 min, with a final 5 min extension at 72?C. These PCR fragments were cloned into pDONR201 (Invitrogen) by the BP reaction according to the instructions for the Gateway cloning procedure (Invitrogen). Recombinant plasmids were purified by alkaline lysis with SDS (Sambrook and Russell, <cite>2001</cite>). These plasmids were checked for inserts by colony PCR and direct sequencing of the PCR products (accession no. AB164037). To construct expression clones, recombinant plasmid DNAs were mixed with pDEST26 (Invitrogen) and treated with LR Clonase according to the instructions for the Gateway cloning technology. Each recombinant plasmid DNA was checked by sequencing and purified. The following Mx1 expression vectors were constructed: for the normal Mx1, pExMx1; for the 3-bp deletion variant, pExMx1-3; for the 11-bp deletion variant, pExMx1-11.

Construction of the Transfectants Expressing the Recombinant Porcine Mx1
NIH3T3 cells (4 ? 10⁶/0.4 mL) were transfected with 5 μg of each expression plasmid by electroporation using an ECM 60 Electroporation system (Btx Inc.) at 13 ohms of resistance, 100 microfarads, and a 200 V setting in cuvettes with 2-mm chamber gaps (1.0 KV/cm). The transfected cells were cultured for 4 days in DMEM containing 5% FBS and then selected in DMEM (Gibco) supplemented with 500 μg/mL G418 (Geneticin, Invitrogen) and 5% FBS. The G418-resistant cell clones were examined for Mx1 expression by RT-PCR. Positive clones were subcloned by limiting dilution, and stable cell clones expressing the different Mx1 variants were established as follows: vector only (pEx), 3T3-vec; by pExMx1, 3T3; by pExMx1-3, 3T3-3; by pExMx1-11, 3T3-11.

Influenza Virus Preparation and Challenge Experiments
Influenza virus A/Aichi (H3N2) was grown in the allantoic cavities of 11-day-old embryonated chicken eggs. The titer of stock virus was 1 ? 10^7.5 the 50% egg-infective dose (EID₅₀) /0.2 m.
Challenge experiments were performed with two different concentrations of the virus, at a multiplicity of infection (MOI) of 1 and MOI of 10, in triplicate. Four cell classes were used for each series; 3T3-vec, 3T3, 3T3-3, and 3T3-11 (5 ? 10⁴/well), each grown in 24-well plates for 48 h in DMEM containing 5% FBS and 25 mM HEPES (Invitrogen) (total 108 wells per MOI series). Confluent cell monolayers were infected for 1 h with the diluted virus in DMEM containing 25 mM HEPES. Unadsorbed virus was removed by washing twice with PBS, and the cells were incubated in DMEM containing 0.2% FBS and 25 mM HEPES. Samples of the culture supernatants were harvested (from the triplicate samples) at nine different time points, 0, 6, 12, 18, 24, 30, 36, 48, and 54 h post-infection. The triplicate samples were pooled and centrifuged (880 ?g) for 5 min at 4?C. These supernatants were stored at −80?C. The samples were subjected to tenfold serial dilution, and virus titers were determined by the EID₅₀ method. The challenge experiment (for both MOI 1 and 10) was conducted three times in total.

Inhibition Tests of the IFN α/β Inducible by Influenza Virus Infection
Confluent cell monolayers (3T3-vec, 3T3, 3T3-3, and 3T3-3) in triplicate were incubated with or without anti-mouse IFN α/β (2 ? 10⁴ neut. units/0.101 mL) (Biosource) for 14 days. At 14 days, the cells incubated with anti-mouse IFN α/β were infected with the virus diluted at MOI 10 in DMEM containing 25 mM HEPES and anti-mouse IFN α/β for 1 h. The cells incubated without anti-mouse IFN α/β were infected similarly but with virus diluted without anti-mouse IFN α/β. Unadsorbed virus was removed by twice washing with PBS, and the cells were incubated with or without anti-mouse IFN α/β in DMEM containing 0.2% FBS and 25 mM HEPES.
Samples of the culture supernatants were harvested (from the triplicate samples) at 36 h post-infection. The triplicate samples were pooled and centrifuged (880 ? g) for 5 min at 4?C. These supernatants were stored at −80?C. The samples were subjected to tenfold serial dilutions and virus infectivity was titrated by the EID₅₀ method. Challenge experiments with or without anti-mouse IFN α/β were conducted three times in total.

Quantitative Analysis of Each Recombinant Mx1 Expression
For measurement of the Mx1 mRNA levels in each transfected cell culture, total RNA from 10⁶ cells was prepared using ISOGEN (Nippon Gene), and poly (-A) RNA was purified using the mRNA Purification Kit (GE Healthcare) in accordance with the manufacturer?s instructions. Each Mx1 variant was amplified with the primers of attB1MxF and attB2Mx-R or attB2Rdel by Superscript one-step RT-PCR with Platinum Taq (Invitrogen). The RT-PCR method consisted of 30 min incubation at 50?C and an initial denaturation at 94?C for 2 min followed by 35 cycles of 94?C denaturation for 15 s, 64?C annealing for 30 s, and 72?C extension for 3 min, with a final 5 min extension at 72?C. The identity of the fragment was confirmed by sequencing as described before (Morozumi et al., <cite>2001</cite>). Relative quantity of Mx1 expression was measured by image analyzer (Multi Gauge Fuji Film) as quantum levels of DNA bands emerged in the electrophoresis gel. Expression of mouse G3PDH was used as an internal standard.
Amplification of the mouse glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was performed as an endogenous control with the mouse G3PDH RT-PCR primer set (Toyobo).

Detection of Mx1 Protein by Western Blot Analysis and Immunofluorescence
For determination of Mx1 protein expression, the following Mx1 expression vectors were constructed with pEF5/FRT/V5-DEST (Invitrogen) using the Gateway cloning technology: for normal Mx1, pExMx1V5; for the 11-bp deletion variant, pExMx1V5-11. The transfectants expressing Mx1 recombinant V5-fusion protein were constructed by electroporation using the Flp-In system (Invitrogen) with Flp-In 3T3 cells, each Mx1 expression plasmid, and pOG44 according to the manufacturer?s instructions. Cells were cotransfected with a ratio of 9:1 (w/w) pOG44: Mx1 expression plasmid. Flp-In 3T3 cells had been cultured in TMEM (Wako) containing 2% FBS, 2 mM glutamine, and 100 mM/mL Zeocin (Invitrogen). The transfected cells had been cultured in TMEM (Wako) containing 2% FBS, 2 mM glutamine, and 100 mM/mL Hygromycin B (Invitrogen). Stable cell clones expressing the normal or mutation Mx1 were established as pEx-V53T3-vec, pExMx1V5-V53T3, and pExMx1V5-11-V53T3-11.
Detection of the Mx1 recombinant V5-fusion proteins was performed using supernatants of cell extracts as described for the Western Breeze Chromogenic Immunodetection Protocol (Invitrogen). Each supernatant of a cell extract was electrophoresed in an SDS-polyacrylamide gradient gel (10?20%). Separated proteins were transferred to a PVDF membrane and detected with the alkaline phosphatase (AP)-conjugated mouse monoclonal antibody to V5 (Anti-V5-AP, Invitrogen) at 1:2000. Primary antibodies were detected with chromogenic substrate (Western Breeze, Invitrogen).
To detect the Mx1 V5-fusion proteins by immunofluorescence, each transfectant (8 ? 10⁴ cells/well) in triplicate was cultured for 24 h on Lab-Tek chamber slides (Nalge Nunc International). Cultured cells were subjected to immunofluorescence analysis with the fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal antibody to V5 at 1:200 (Anti-V5-FITC, Invitrogen) according to the manufacturer?s instructions. The hybridization signals were captured with a cooled CCD camera (Hamamatsu Photonics) and the nuclear images obtained by counterstaining using DAPI solution (Wako) were superimposed on the image of hybridization signals.


<hr> RESULTS
Nucleotide Sequence of Exon 13 in the Meishan Breed
The 3-bp deletion at position 1778?1810 is shown in Fig. 1. Comparison of the translated amino acid sequence of exon 13 with the published sequence of the protein (Gene Bank) revealed that exon 13 of Mx1 in the Meishan breed has lost the serine residue at amino acid 565. The 11-bp deletion found in exon 14 of Landrace breeds (Fig. 1) leads to a frameshift with 8 amino acid substitutions and a 23 amino acid extension in the carboxyl terminal region of the Mx1 protein (Morozumi et al., <cite>2001</cite>).
Fig. 1. Comparison of nucleotide and deduced amino acid sequences of exon13 and 14 in porcine Mx1 in ACC M65087 and conventional pigs. The asterisks mark the coincident sequence. The dashes highlight the 3-bp deletion in exon 13 of Meishan (a) and the 11-bp deletion in exon 14 of Landrace (b)
<hr>
Fig. 2. Inhibition of virus multiplication by Mx1. The 3T3-vec (open circle), 3T3 (open box), 3T3-3 (solid box), and 3T3-11 (solid circle) cells were infected with influenza A virus at MOI 1 (a) and MOI 10 (b). The data, derived from three independent experiments, are shown as mean ? standard deviation. Bars show standard deviations.
<hr>


Influenza Virus Growth in Mx1 Expressing Cells
MOI 1

The general shape of the growth curve in 3T3-vec and 3T3-11 is similar to that observed in 3T3 and 3T3-3 (Fig. 2). The initial rise in infectivity was observed at 18 h after infection in 3T3-vec and 3T3-11 but 36 h after infection in 3T3 and 3T3-3. Maximal titers were reached by 36 h post-infection in the four transfectants; the infectivity end points were 1.0 log₁₀ EID₅₀/0.2 mL for 3T3, 3T3-3, but 2.8?3.0 log₁₀ EID₅₀/0.2 mL for 3T3-vec, 3T3-11. Moreover, in 3T3-vec and 3T3-11, infectious virus could be detected at 54 h after infection, even though there were no infectious virus in 3T3 or 3T3-3.

MOI 10

In 3T3-vec and 3T3-11, an infectivity of 0.3-0.5 log₁₀ EID₅₀/0.2 mL was observed at 12 h post-infection and the maximum level (4.5?4.7 log₁₀ EID₅₀/0.2 mL) was reached at 36 h after infection (Fig. 2). The infectious virus titer had dropped at 48?54 h after infection. While the general course of viral increase in 3T3-vec and 3T3-11 was similar to that observed in 3T3 and 3T3-3, infectious virus was detected 12 h earlier, and the maximum level of 4.5?4.6 log₁₀ EID₅₀/0.2 mL was reached at 36 h, or 12 h earlier than for 3T3 and 3T3-3. The maximum virus titers in 3T3-vec and 3T3-11 were 100 times higher than those in 3T3 and 3T3-3.


Influenza Virus Growth in Mx1 Expressing Cells Pretreated with Anti-IFN α/β
As the influenza virus is an IFN-inducing virus, the influenza virus-infected cell is capable of releasing IFN α/β, which may induce an antiviral state in neighboring cells. To eliminate this possibility, we treated 3T3-vec and three transfectants with anti-mouse IFN α/β (Biosource) for 14 days before and during virus infection (MOI 10). The results were essentially the same as for untreated cells irrespective of the porcine Mx1 genotype (Fig. 3).
Fig. 3. Influenza virus growth in the Mx1 expressing cells pretreated with anti-IFN α/β. The Mx1 expressing cells had been treated with (open bars) or without anti-mouse IFN α/β (solid bars) for 14 days before and during influenza virus infection (MOI 10). This experiment was repeated in triplicate, and data are shown as mean ? standard deviation. Bars show standard deviations.
<hr>


Expression of Recombinant Mx1
To standardize the amount of sample RNA from each transfected cell, RT-PCR was performed with 10⁶ cells having the same passage history as that of the cells used for virus challenge experiments. There was no expression of porcine Mx1 in control 3T3 cells. All transfectants expressed the porcine Mx1 gene. It appears, however, that the amount of Mx1 expressed in 3T3-11 cells was greater than that expressed in 3T3 and 3T3-3 cells (Fig. 4 and Table I) based on the similar expression levels observed for mouse G3PDH (used as an internal control).
Fig. 4. Expression of recombinant Mx1 and Western blot analysis of Mx1 protein. (a) For measurement of Mx1 mRNA level in each transfected cell (3T3-vec, 3T3-WT, 3T3-11, and 3T3-3), RT-PCR was performed with mRNA purified from 10⁶ cells. Amplification of G3PDH was performed as an endogenous control. M, Markers; lane 1, 3T3-Vec; lane 2, 3T3-WT; lane 3, 3T3-11; lane 4, 3T3-3. (b) Western blot analysis of the Flp-In 3T3 cells transfected with the porcine Mx1. Each cell (V53T3-vec, V53T3-WT, and V53T3-11) extract was electrophoresed in an SDS-polyacrylamide gradient gel (10?20%). Separated proteins were transferred to PVDF membrane and detected with the Anti-V5-AP. M, Markers; lane 1, V53T3-vec; lane 2, V53T3-WT; lane 3, V53T3-11.
<hr>


Detection of Mx1 Protein
Expression of the porcine Mx1 protein in transfected cells was analyzed by Western blotting with a mouse monoclonal antibody, which allows detection of recombinant fusion proteins containing a V5 epitope tag. Figure 4 shows that both V53T3 (normal Mx1) and V53T3-11 (11 bp deletion) cells synthesized proteins of approximately 77 kDa recognized by anti-V5-AP antibody. Untransfected V53T3-vec cells did not synthesize a protein recognized by anti-V5-AP antibody. Table I. Relative Expression of the Pig Mx1 Genes Transfected into Mouse 3T3 Cells
<table border="1"> <colgroup> <col align="left"> <col align="left"> <col align="left"> <col align="left"></colgroup> <thead> <tr class="header"> <th align="left"> </th> <th colspan="3" align="left"> Quantum level
</th></tr> <tr class="header"> <th align="left"> Type of cell
</th> <th align="left"> Porcine Mx
</th> <th align="left"> Mouse G3PDH
</th> <th align="left"> Mx/G3PDH
</th></tr></thead> <tbody> <tr class="noclass"> <td align="left"> 3T3-vec
</td> <td align="left"> 0
</td> <td align="left"> 16372
</td> <td align="left"> 0
</td></tr> <tr class="noclass"> <td align="left"> 3T3-WT
</td> <td align="left"> 42877
</td> <td align="left"> 18965
</td> <td align="left"> 2.2
</td></tr> <tr class="noclass"> <td align="left"> 3T3-11
</td> <td align="left"> 124574
</td> <td align="left"> 18580
</td> <td align="left"> 6.7
</td></tr> <tr class="noclass"> <td align="left"> 3T3-3
</td> <td align="left"> 42794
</td> <td align="left"> 15920
</td> <td align="left"> 2.7
</td></tr></tbody></table> Note. Relative expression of Mx was measured by quantum level of electrophoresis picture of DNA obtained by RT-PCR of mRNA. Expression of mouse G3PDH was used as internal standard. 3T3-vec: Only vector was transfected. 3T3-WT: Wild-type Mx1 was transfected. 3T3-11: Mx1 of 11-bp deletion was transfected. 3T3-3: Mx1 of 3-bp deletion was transfected.





Cytoplasmic Distribution of the Porcine Mx1 Protein
To determine the intracellular localization of the porcine Mx1 protein, immunofluorescence experiments were performed. The FITC-conjugated mouse monoclonal antibody to the V5-porcine Mx1 fusion protein stained cytoplasmic structures similarly in both V53T3 and V53T3-11 (Fig. 5). The V53T3-vec cells that had been transfected with pEF5/FRT/V5 and pOG44 did not contain the porcine Mx1 protein.
Fig. 5. Cytoplasmic distribution of the porcine Mx protein. Each transfectant (8 ? 10⁴ cells/well) in triplicate was cultured for 24 h and subjected to immunofluorescence analysis with Anti-V5-FITC at 1:200. The nuclear images were obtained by counterstaining using DAPI solution. (a) V53T3-vec. (b) V53T3. (c) V3T3-11.
<hr>



<hr> DISCUSSION
It is well established that the mouse Mx1 gene is necessary and sufficient to establish resistance to influenza virus infection in this species. We demonstrated that the 3T3 cells transfected with the porcine full-length Mx1 cDNA were more resistant to influenza virus infection than untransfected cells. Mx1 is normally not expressed in these cells, and it appears that reduced influenza virus replication takes place in 3T3 cells expressing the porcine Mx1. Expression of Mx1 has been shown to be induced by IFN α/β treatment or in viral infection through the action of IFN α/β (Haller et al., <cite>1980</cite>). However, 3T3 cells expressing the porcine Mx1 retained antiviral activity even when the cells were treated with anti-IFN α/β antibody. This suggests that the inhibition of replication of influenza virus by the porcine Mx1 expressing cells was dependent on the function of the Mx1 protein and was not dependent on induction of Mx1 by IFN α/β. Moreover, it seems likely that the other proteins specifically induced by IFN are not essential for the inhibition observed here. It would be interesting to test whether increasing the level of expression of the Mx1 protein in 3T3 cells would result in complete inhibition of influenza virus replication.
The 3T3 cells transfected with Mx1 cDNA of the 3-bp deletion variant expressed Mx1 protein at a level similar to that of normal Mx1 cDNA transformed cells and also demonstrated reduced replication of influenza virus. It seems likely that the 3-bp deletion of the porcine Mx1 cDNA does not affect the function of Mx1 protein.
On the other hand, the cells transfected with Mx1 cDNA of the 11-bp deletion variant appear to lack the capability to suppress influenza virus multiplication. Similar levels of Mx1 transcript were found in 3T3, 3T3-3, and 3T3-11 (Fig. 4 and Table I, with similar levels of G3PDH expression), although expression of the 11-bp deletion variant may actually be increased, suggesting that normal expression may be self-regulated. Western blotting analysis suggests that the molecular weight (approximately 77 kDa) and amount of expressed Mx1 protein were similar in cells transfected with the normal or the 11-bp deletion variant of the porcine Mx1 gene. The size of the product, which is similar to human Mx1 protein, suggests that a full-length copy of Mx1 is expressed in these cells. Asano et al. <cite>(2002)</cite> reported that VSV replication was inhibited not only in the 3T3 cells expressing wild-type porcine Mx1 mRNA but also in the 3T3 cells expressing the 11-bp deletion variant Mx1 mRNA. The evidence that the 11-bp deletion variant Mx1 lost antiviral activity to influenza virus but not to VSV will be defined in experiments based on the viewpoint of which domain or domains of Mx1 protein is correlated with each virus multiplication.
The 3-bp deletion in exon 13 has been observed only in the Meishan breed to date, where the frequency is approximately 21% with the genotype classes being in line with Hardy?Weinberg. However, although an allele frequency of around 30% was observed for the 11-bp deletion in several herds of Landrace and other breeds of pigs, the frequency of individuals homozygous for the 11-bp deletion was relatively low (3.7%). We suspect therefore that pigs homozygous for the 11-bp deletion might be susceptible to some RNA virus infectious diseases.
Evidence of interspecies influenza virus transmission has been reported, for example, the finding of avian H1N1 viruses in swine, human H3N2 viruses in swine, swine H1N1 viruses in humans, and other avian viruses in seals, whales, and mink (Gorman et al, <cite>1990</cite>, <cite>1991</cite>; Kida et al., <cite>1988</cite>; Scholtissek et al., <cite>1983</cite>). This indicates that pigs may serve as significant reservoirs of H1N1 and H3N2 influenza viruses and are frequently involved in interspecies transmission of influenza viruses. Influenza virus maintenance in pigs and the frequent introduction of new viruses from other species may be important in the generation of pandemic strains of human influenza virus. Early production of or increased expression of an active Mx1 protein may yield pigs with increased resistance to influenza virus. The existence of variation in the porcine Mx1 gene suggests that it may be possible to select pigs less susceptible to influenza virus, which may also have a positive impact on the frequency of the generation of pandemic strains of the virus.

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Re: A Naturally Occurring Variant of Porcine Mx1 Associated with Increased Susceptibility

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