1 Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh EH9 3JF, UK
2 Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, UK
3 Sir Alastair Currie Cancer Research UK Laboratories, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Edinburgh, UK
Correspondence
Rona M. Barron
rona.barron{at}bbsrc.ac.uk
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ABSTRACT |
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Published online ahead of print on 5 January 2005 as DOI 10.1099/vir.0.80525-0.
A supplementary figure showing ME7 and 301V incubation times in mice expressing codon 108 and 189 polymorphisms is available in JGV Online.
Present address: Department for Environment, Food and Rural Affairs (DEFRA), Veterinary Research Division, TSE Research Unit, Dean Stanley Street, London, UK.
Present address: Department of Genetics and Genomics, Roslin Institute, Edinburgh, UK.
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INTRODUCTION |
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The host PrP gene (Prnp) has a major influence over the outcome of TSE disease. PrP polymorphisms have been shown to alter incubation time and TSE susceptibility in mice (Moore et al., 1998), sheep (Goldmann et al., 1994
) and man (Palmer et al., 1991
; Goldfarb et al., 1992
). Classical genetic analysis of the control of TSE incubation time in mice identified the presence of a single gene (Sinc) encoding two alleles (s7 and p7), which programmed short and long incubation times, respectively, for the ME7 strain of scrapie (Dickinson et al., 1968
). Similar differences in incubation time have been found between NZW/LacJ and Iln/J mouse strains when infected with the Chandler isolate, identifying a single locus, Prni, that controlled incubation time, which was linked closely to Prnp (Carlson et al., 1986
). Following isolation and characterization of the gene encoding PrP in mice (Prnp) (Basler et al., 1986
; Locht et al., 1986
), mouse strains carrying Sinc s7 were shown to have a Prnp gene encoding 108L_189T (Prnpa) and those carrying Sinc p7 had a Prnp gene encoding 108F_189V (Prnpb) (Westaway et al., 1987
). However, due to the different genetic backgrounds of these lines of inbred mice, it was difficult to confirm that the Prnp, Sinc and Prni genes were congruent and that the polymorphisms in Prnp were responsible for the control of scrapie incubation time in mice.
The introduction of 108F and 189T into the murine Prnpa gene by gene targeting produced a line of transgenic mice (FV/FV) that differed from the 129/Ola parental line by only the targeted polymorphisms. Inoculation of these mice with TSE infectivity produced incubation times similar to those in Prnpb mice, demonstrating that Sinc, Prni and Prnp were indeed the same gene and that the codon 108 and/or 189 polymorphisms were the major factors controlling TSE incubation time in mice (Moore et al., 1998; Barron et al., 2003
; Barron & Manson, 2004
).
In order to determine the individual involvement of the codon 108 and 189 polymorphisms in disease and the mechanism by which they control TSE incubation time in mice, 108F and 189V have been introduced separately into the murine Prnpa gene by gene targeting. These new alleles are designated Prnpa[108L_189V] (PrnpLV) and Prnpa[108F_189T] (PrnpFT). Inoculation of mice that were homozygous for Prnpa, Prnpb, PrnpLV or PrnpFT and their heterozygous crosses with mouse scrapie have demonstrated the involvement of both polymorphisms in the control of incubation time.
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METHODS |
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Cre deletion of the HPRT minigene.
The LoxP-flanked PGK/HPRT module was excised from targeted alleles by transient transfection of ES cells with the Cre recombinase expression construct pMCCreN (Gu et al., 1993). Clones were transfected with 150 µg pMCCreN (10x106 cells, 900 V, 3 µF) and selected by 6-thioguanine. Drug-resistant clones were screened for deletion of the LoxP-flanked HPRT minigene by PCR with primers W7878 (5'-AGTCAGGGAGGAGTAACACAGAAGG-3') and W6991 [94 °C (60 s), 66 °C (60 s), 72 °C (95 s); 35 cycles]. This generated a 1·61 kb product from the wild-type allele and a slightly larger band of 1·66 kb from the HPRT-deletant allele. This size difference arises from the retention of a single 38 bp LoxP site and a portion of the pBluescript multiple-cloning site. ES cell clones were examined by Southern analysis for the expected gene structure and the absence of additional recombination events, as described previously (Moore et al., 1995
).
Generation of mice.
Germ-line chimeras were generated as described previously (Thompson et al., 1989). Briefly, HM-1 ES cells with the desired alterations were introduced into BALB/c 3·5-day-old blastocysts and transferred to pseudopregnant MF1 recipients. Chimeras were crossed directly to 129/Ola stock-generating mice with PrP codon 108 and 189 alterations, which were co-isogenic with the parental strain 129/Ola Hsd (Harlan). Germ-line pups were screened by a combination of PCR and MnlI or BstEII restriction digestion, as described above. 129/Ola littermates heterozygous for the targeted PrP allele that were also wild-type at the deleted HPRT locus in HM-1 cells were crossed to establish the line.
Northern blot analysis.
Total RNA was isolated from terminal brains by using RNAzol B, based on the guanidinium thiocyanate/phenol/chloroform extraction method (Chomczynski & Sacchi, 1987). Total RNA (50 µg) was separated on a 1·0 % agarose/formaldehyde denaturing gel and transferred to Hybond-N (Amersham Biosciences) by capillary transfer overnight. RNA was fixed to the membrane by baking at 80 °C for 2 h before probing. A 936 bp KpnIEcoRI fragment from Prnp exon 3 was used to generate the PrP probe. Membranes were hybridized overnight by using ULTRAhyb (Ambion). As a loading control, membranes were reprobed for 18S rRNA by using a 275 bp PCR-generated murine DNA fragment.
Western blot analysis.
Homogenates of frozen brain tissue [10 % (w/v)] were prepared in NP40 buffer [0·5 % (v/v) NP40, 0·5 % (w/v) sodium deoxycholate, 150 mM NaCl, 50 mM Tris/HCl (pH 7·5)]. Clarified homogenates (10 000 g for 10 min at 10 °C) were incubated with or without PK (20 µg ml1) for 1 h at 37 °C and the reaction was terminated by the addition of PMSF to 1 mM. Samples were prepared at 10 mg ml1 in SDS-PAGE sample buffer (Novex; Invitrogen), incubated at 90 °C for 20 min and separated on 12 or 412 % Novex Tris/glycine acrylamide gels (Invitrogen). Proteins were transferred onto a PVDF membrane by electroblotting and incubated overnight at room temperature with mouse anti-PrP monoclonal antibody 7A12 (Li et al., 2000) at a dilution of 50 ng ml1, or with rat anti-tubulin monoclonal antibody (Abcam) at 250 ng ml1. Proteins were visualized with horseradish peroxidase-conjugated anti-mouse or anti-rat secondary antibody diluted to 200 ng ml1 (Jackson ImmunoResearch) and a chemiluminescence detection kit (Roche Diagnostics). Membranes were exposed to X-ray film for periods ranging from 10 s to 10 min.
Preparation of inoculum.
Inocula were prepared from the brains of C57BL mice with terminal ME7 scrapie and the brains of VM mice with terminal 301V disease. A 1 % homogenate of each sample was prepared in sterile saline prior to use as an inoculum. All mice were inoculated intracerebrally with 20 µl inoculum under anaesthesia. All experimental protocols were submitted to the Local Ethical Review Committee for approval before mice were inoculated. All experiments were performed under licence to and in accordance with the UK Home Office Regulations [Animals (Scientific Procedures) Act 1986].
Scoring of clinical TSE disease.
The presence of clinical TSE disease was assessed as described previously (Dickinson et al., 1968). Animals were scored for clinical disease without reference to the genotype of the mouse. Genotypes were confirmed for each animal by PCR analysis of tail DNA at the end of the experiment. Incubation times were calculated as the interval between inoculation and cull due to terminal TSE disease. Mice were killed by cervical dislocation at the terminal stage of disease, at termination of the experiment (between 600 and 700 days) or for welfare reasons due to intercurrent illness. Half-brains were fixed in 10 % formal saline for 48 h, followed by decontamination in 98 % formic acid for 1 h. The remaining half-brain was frozen at 70 °C for biochemical analysis. Fixed brain tissue was dehydrated in alcohol and impregnated in wax during a 7 h automated processing cycle. Sections were cut coronally at four levels and mounted on Superfrost slides.
Lesion profiles.
Sections were haematoxylin/eosin-stained and scored for vacuolar degeneration on a scale of 05 in nine standard grey-matter areas and three standard white-matter areas, as described previously (Fraser & Dickinson, 1967).
Genotyping of mouse-tail DNA.
A 23 cm portion of tail was removed post-mortem from each mouse. DNA was prepared from a 1 cm piece of tail by digestion overnight at 37 °C in tail lysis buffer [300 mM sodium acetate, 1 % SDS, 10 mM Tris (pH 8), 1 mM EDTA, 200 µg PK ml1)] and subsequent extraction with an equal volume of phenol/chloroform. DNA was precipitated with 2-propanol, washed with 70 % ethanol and resuspended in 100 µl TE buffer [10 mM Tris, 1 mM EDTA (pH 7·4)]. A 605 bp PCR fragment for the Prnp ORF was generated from purified tail DNA with an ORF primer, PrP5'> (5'-GTGGCTGGGGACAACCCCAT-3'), and an exon 3 3'-untranslated region primer, PrP3'< (5'-GCCTAGACCACGAGAATGCG-3'). Cycle conditions were: 35 cycles, 94 °C (30 s), 65 °C (30 s), 72 °C (1 min). The PrnpLV allele was detected by the absence of a BstEII site within this product compared with the wild-type Prnpa allele. The PrnpFT allele was detected by the absence of an MnlI site compared with the wild-type Prnpa allele.
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RESULTS |
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HM-1 ES cell clones bearing the targeted PrnpFT or PrnpLV alleles were injected into 3·5-day-old BALB/c blastocysts to create chimeric mice (Moore et al., 1995), which were crossed with 129/Ola mice to establish the FT/FT and LV/LV transgenic lines co-isogenic with 129/Ola. The identical genetic background of the lines ensured that any observed alterations in incubation time and disease pathology were a direct result of the targeted mutations and were not due to the effects of genes in mice of other genetic backgrounds.
Expression of PrP in the gene-targeted lines
Expression levels of Prnp mRNA and mature PrPC were investigated in FT/FT and LV/LV brain and compared with those in wild-type 129/Ola mice (LT/LT) and the existing gene-targeted transgenic line expressing Prnpa[108F_189V] (FV/FV) (Moore et al., 1998). Northern blot (Fig. 2
) and Western blot (Fig. 3
) analysis of tissues from each line confirmed expression from the PrnpFT and PrnpLV alleles and indicated that the levels of expression in the three homozygous lines (FV/FV, LV/LV and FT/FT) were indistinguishable from those of wild-type 129/Ola mice. Prnp gene expression was therefore not affected by the process of gene targeting, the introduction of a single polymorphism or the presence of the LoxP site 3' of the Prnp gene. Thus, any alteration in incubation time and neuropathology observed on inoculation of these animals with TSE infectivity should be attributable to the specific alterations at codons 108 and 189, and not due to any differences in Prnp gene-expression levels.
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Introducing heterozygosity at codons 108 and 189 revealed that the control of incubation time by these two polymorphisms is not as straightforward as suggested by data from the homozygous lines (Table 1). There is no obvious pattern of allelic combination associated with increasing incubation time (see Supplementary Figure in JGV Online), with the exception that the two shortest incubation times are in mice that are homozygous for 189T (155 and 168 days) and the two longest are in mice that are homozygous for 189V (295 and 325 days), in keeping with identity at codon 189 with the source of the agent (LT/LT). However, homozygosity at codon 189 does not always produce extreme incubation times, as LT/FT and LV/LV mice have intermediate incubation times of 196 and 261 days, respectively (Table 1
).
An association between genotype and incubation time can be identified when the 10 lines are examined by the allelic combinations at either codon 108 or codon 189 (Table 1). When lines are sorted into groups by codon 189 genotype (189TT, 189VV or 189FV), the effect of codon 108 on incubation time is the same in each of the three groups: the shortest incubation times occur when the codon at 108 is homozygous for either L or F, although the shorter of the two is always 108LL, matching that of the source of the inoculum (LT/LT). Therefore, at codon 108, heterozygosity always leads to increased incubation times with respect to the combination at codon 189, despite the inoculum matching one allele at codon 108. Indeed, incubation times in excess of those in Prnpb (FV/FV) mice were observed in FV/LV mice [Table 1
and Supplementary Figure (available in JGV Online)]. However, when sorted by codon 108 genotype (108LL, 108FF or 108LF), a different pattern of incubation time is observed. For each of the three groups, homozygosity for threonine at codon 189 (which matches the source of the inoculum) gives the shortest incubation times and homozygosity for valine (which does not match the inoculum) gives the longest incubation times, whilst the heterozygotes (TV) produce intermediate incubation times. Hence, at codon 108, ME7 incubation times follow the pattern LL<FF<LF, whilst at codon 189, the pattern of incubation time is TT<TV<VV. These results show that the effect on incubation period depends upon the combination of PrP alleles, particularly the presence of homozygosity or heterozygosity at codons 108 and 189 and on identity with the source of the inoculum at these positions in PrP.
Interactions between codon 108 and 189 polymorphisms and the 301V strain
In contrast to ME7 challenge, LT/LT mice inoculated with the Prnpb (FV/FV)-passaged strain 301V have long incubation periods of 240 days. Similarly, FV/FV mice that have long ME7 incubation periods produce short incubation periods of 125 days with 301V. The role of codons 108 and 189 in the control of 301V incubation time was explored by challenge of FT/FT and LV/LV mice. Incubation times of 141 and 202 days were obtained in the LV/LV and FT/FT lines of mice, respectively, compared to 125 days in FV/FV mice and 240 days in LT/LT mice (Table 1). Hence, in agreement with the data obtained from inoculations with ME7, both codons 108 and 189 are responsible for the control of incubation time for the 301V agent strain. Similarly to ME7, the greatest effect was produced by the T to V alteration at codon 189 (
100 days reduction). However the L to F change produced a greater effect on incubation time with 301V (
60 days reduction) than with ME7, again showing that challenge with an agent that shows identity at codon 108 or 189 results in the shortest incubation times (Table 1
). The full range of allelic combinations has not been examined with this strain of TSE agent. In addition, no incubation time is available for the LT/FT line. Six LT/FT mice were culled due to welfare reasons between 327 and 377 days and all showed positive TSE pathology on examination of the brain tissue, suggesting an incubation time in excess of that of the LT/LT line (240 days). However, the combinations that were examined have produced patterns of incubation-time alteration identical to those identified with ME7 [Table 1
and Supplementary Figure (available in JGV Online)], with the exception that the F1 cross LT/FV has an incubation time in excess of those of both parental lines. This overdominance is characteristic of 301V and can be observed with other TSE strains. Thus, the pattern of incubation time for 301V appears to be FF<LL<LF at codon 108 and VV<TV<TT at codon 189. This pattern is the same as that observed for ME7, where heterozygosity results in extended incubation times for 108LF, but intermediate incubation times with 189TV. The shortest incubation times for both strains of agent reveal a preference for identity with the genotype of the donor of infectivity (LT/LT for ME7 and FV/FV for 301V).
Effect of polymorphisms on PrPSc production
Detergent homogenates were prepared from three terminal ME7 brains of each genotype. The homogenates were treated with PK and analysed by immunoblotting to assess the levels of PK-resistant PrP present in each model. Equal levels of PK-resistant PrP were detected between the three mice of each genotype (data not shown). Levels were also similar between the 10 different lines of mice (Fig. 4), suggesting that the codon 108 and 189 polymorphisms have no effect on PrPSc levels in the brains of mice culled with clinical ME7 infection. This was also true for the lines challenged with 301V (data not shown).
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DISCUSSION |
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The control of incubation time is thought to be due to PrP sequence identity between the host and the donor of infectivity. However, our results show that short incubation times are obtained in FT/FT mice inoculated with LT/LT-derived ME7 and in LV/LV mice inoculated with FV/FV-derived 301V, despite the sequence incompatibility between donor and host at codon 108. Hence, although codon 108 polymorphisms can affect scrapie incubation time independently, the major control is due to identity with the source of inoculum at codon 189. The inoculation of mice expressing different combinations of PrnpLT, PrnpFV, PrnpLV and PrnpFT produced a complex series of incubation times (see Supplementary Figure in JGV Online). It was clear that, in addition to identity with the inoculum at codon 189, homozygosity at codon 108 consistently gave rise to shorter incubation times in both 108L/L- and 108F/F-expressing lines. Heterozygosity at codon 108 always resulted in extended incubation times with respect to the genotype at codon 189. Although the series of allelic crosses produced for these experiments are described as homozygous and heterozygous at codons 108 and 189, expression from both Prnp alleles in a heterozygote will result in the presence of two different PrP populations in the cell. It is interactions between these proteins, and not between Prnp alleles, that will determine the incubation time of disease. Hence, the nature or combination of PrP variants in the recipient may be more important than PrP identity with the inoculum, indicating that interactions between amino acids 108 and 189, either on the same or different proteins, is a critical part of disease propagation.
It has been proposed that the production of PrPSc is a two-step process that involves binding of PrP-res (PK-resistant PrP) to PrP-sen (PK-sensitive PrP), followed by the conversion of PrP-sen to PrP-res (Caughey, 2001). However, it is unknown which process requires PrP amino acid identity. Cell-free conversion experiments have shown that mouse PrP-res (108L_111V) will convert mouse PrP-sen expressing the 3F4 epitope (108M_111M), but not hamster PrP-sen (also possessing the 3F4 epitope), even though both proteins were found to bind to mouse PrP-res (Horiuchi et al., 2000
). Deletion mutants lacking residues 34113 (MoPrP
34113) also bound to mouse PrP-res and converted, albeit with reduced efficiency (Lawson et al., 2001
). These results imply that amino acid 108 is not required for either initiation of conversion or binding to PrPSc, but may be involved in the control of the rate of conversion and final conformation of PrP-res (Lawson et al., 2004
). Moreover, MoPrP
34113 was found to bind heterologous PrPSc, but, unlike the full-length molecule, did not prevent the conversion of homologous PrPC, again suggesting that this region is involved in the control of conversion, but not binding (Lawson et al., 2001
). The
34113 truncation of PrPC was also found to affect the solubility of the molecule, as loss of these residues reduced the percentage of PrP-sen that was seen to self-aggregate when incubated under cell-free assay conditions in the absence of PrP-res. This region may therefore be responsible for some degree of multimerization/aggregation of PrP-sen, which may be beneficial for conversion (Lawson et al., 2001
).
The individual effects of codon 108 and 189 polymorphisms in PrP have also been modelled in vitro by expressing recombinant 108F_189T and 108L_189V PrP in Escherichia coli (Brown et al., 2000). These intermediate forms of PrP were found to be less stable than both PrP-A (108L_189T) and PrP-B (108F_189V) and were observed to lose their normal conformation and gain some PK resistance over time. It was proposed that inheritance of either 108F or 189V alone may be a disadvantage in terms of mouse survival, and that mice expressing either of these polymorphisms separately could potentially develop spontaneous disease (Brown et al., 2000
). However, the recently reported existence of a Prnpc (108F_189T)-expressing line (Lloyd et al., 2004
) and the data presented here for transgenic FT/FT and LV/LV lines show that such mice are viable and do not develop any neurological phenotype during their lifespan. PrP expressed in the brains of transgenic LT/LT and FV/FV mice is PK-sensitive and expressed at levels identical to those in wild-type mice of both genotypes. These results demonstrate clearly that in vitro and in vivo studies can produce very different outcomes; these may be due to the difference in PrP post-translational modification and/or the physiological environment of the native protein.
From the data presented here, we predict that: (i) polymorphisms at PrP codon 189 control the initial interaction and binding with the agent, as incubation times reflect a preference for sequence identity between the host and inoculum at codon 189; and (ii) polymorphisms at codon 108 control the rate of conversion of PrPC to PrPSc, where the ability to induce multimerization of PrPC and increase the efficiency of conversion is favoured when all PrP expressed in a cell is homogeneous in the N-terminal region. Alternatively, the preference for codon 108 homozygosity may reflect a more complex interaction between host PrPC and PrPSc that is less efficient in the presence of heterogeneous protein populations. However, the importance of codon 108 homozygosity in murine scrapie transmission may explain the role of PrP codon 129 in human TSE disease, where the majority of CJD cases occur in individuals who are homozygous for methionine or valine at codon 129 (Palmer et al., 1991; Zeidler et al., 1997
; Alperovitch et al., 1999
). There is an under-representation of M/V heterozygotes with CJD when compared to the distribution of genotypes in the normal population (Palmer et al., 1991
), suggesting that a similar mechanism requiring N-terminal homogeneity controls the efficiency of disease transmission in humans.
The results of transmissions to the 108L/F_189T/V lines of transgenic mice have therefore shown that codon 189 polymorphisms exert the major control over scrapie incubation time, but that the efficiency of disease transmission is increased when codon 108 is homozygous, suggesting that a multimer of PrPC may be involved in conversion of PrP during TSE disease. Several other studies have suggested the involvement of PrP dimers in disease (Priola et al., 1995; Warwicker, 1997
, 2000
; Meyer et al., 2000
; Jansen et al., 2001
; Meier et al., 2003
), yet most existing dimer models do not consider dimerization of the N-terminal region of PrP, due to the lack of structural information available for this region. Combined with the in vivo data produced in this study, the observations that amino acids 90121 are retained after PK cleavage of PrPSc and that deletion of this region affects susceptibility to disease (Fischer et al., 1996
; Shmerling et al., 1998
; Flechsig et al., 2000
; Lawson et al., 2001
; Supattapone et al., 2001
) prove that this is a structurally important part of the molecule that is involved in the control of disease incubation time.
Analysis of LV/LV and FT/FT transgenic mice has therefore shown that amino acid polymorphisms at both codons 108 and 189 in murine PrP are involved in the control of scrapie incubation time, and that distinct regions of PrP may play different roles in the disease process. Interactions within a single PrP molecule and between molecules must therefore underlie the mechanism by which incubation times are controlled. Such structural interactions may provide the key to replication of TSE infectivity.
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ACKNOWLEDGEMENTS |
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Received 13 August 2004;
accepted 13 December 2004.
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