Inherited prion disease caused by the V210I mutation: transmission to transgenic mice.

OBJECTIVE: To describe the clinical and neuropathologic profile and determine the strain characteristics of familial Creutzfeldt-Jakob disease (fCJD) caused by a point mutation of the PRNP gene at codon 210 that results in a valine-to-isoleucine substitution in the prion protein (PrP). METHODS: The clinicopathologic features of four individuals from the United States who died of fCJD(V210I) were compared. Transgenic (Tg) mice expressing a chimeric human-mouse PrP transgene were inoculated with brain extracts from three fCJD(V210I) cases, sporadic CJD (sCJD), fCJD(E200K), and fatal familial insomnia (FFI), to compare prion strain characteristics. RESULTS: The clinicopathologic profile of fCJD(V210I) was variable among cases but shared similarities with sCJD. The pattern of PrP(Sc) deposition in the brains of Tg mice was similar to that caused by sCJD but different from that associated with fCJD(E200K) or FFI. CONCLUSIONS: Each of these prion diseases is characterized by a rapidly progressive dementia with myoclonus, periodic complexes on EEG, and spongiform change without PrP plaque deposition in the brain. The occurrence of a different PrP(Sc) phenotype with each PRNP mutation argues that each respective amino acid sequence substitution produces a different prion strain.

Transgenic studies of prion diseases.

This chapter reviews studies that involve the manipulation of prion protein (PrP) genes by transgenesis in mice. These consist of two approaches: PrP gene knockout and gene replacement using homologous recombination in embryonic stem cells; and microinjection of transgenes into fertilized embryos. These studies have provided important insights into the pathogenesis of prion diseases including the molecular basis of prion strains and species barriers. Transgenic approaches have also provided important information about the mechanism by which human prion diseases can be both genetic and infectious. Despite these advances, our understanding of these unique pathogens is far from complete. Transgenic approaches will doubtless remain the cornerstone of investigations into the prion diseases, and will be important in the development of therapeutic agents in coming years.

Prion protein genes and prion diseases: studies in transgenic mice.

In the past decade, manipulation of PrP genes by transgenesis in mice has provided important insights into mechanisms of prion propagation and the molecular basis of prion strains and species barriers. Despite these advances, our understanding of these unique pathogens is far from complete. This review focuses on PrP gene knockout and gene replacement studies, PrP structure and function, and transgenic models of human and animal prion diseases. Transgenic approaches will doubtless remain the cornerstone of investigations into the prion diseases in the coming years, which will include mechanistic studies of prion pathogenesis and prion transmission barriers. Transgenic models will also be important tools for the evaluation of potential therapeutic agents for prion diseases.

Heritable disorder resembling neuronal storage disease in mice expressing prion protein with deletion of an alpha-helix.

Mice were constructed carrying prion protein (PrP) transgenes with individual regions of putative secondary structure deleted. Transgenic mice with amino-terminal regions deleted remained healthy at >400 days of age, whereas those with either of carboxy-terminal alpha-helices deleted spontaneously developed fatal CNS illnesses similar to neuronal storage diseases. Deletion of either C-terminal helix resulted in PrP accumulation within cytoplasmic inclusions in enlarged neurons. Deletion of the penultimate C-terminal helix resulted in proliferation of rough endoplasmic reticulum. Mice with the C-terminal helix deleted were affected with nerve cell loss in the hippocampus and proliferation of smooth endoplasmic reticulum. Whether children with the human counterpart of this malady will be found remains to be determined.

N-terminally tagged prion protein supports prion propagation in transgenic mice.

The eight amino acid sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, representing the FLAG peptide, was inserted after codons 22 or 88 of the mouse (Mo) prion protein (PrP) gene. Inclusion of the FLAG sequence at these locations interfered neither with the cellular processing of PrPC nor its conversion into PrPSc. Inclusion of the FLAG epitope at residue 22 but not at residue 88 facilitated immunodetection of tagged PrP by anti-FLAG monoclonal antibodies (mAbs). Inoculation of transgenic (Tg) mice expressing N-terminally tagged MoPrP with Mo prions resulted in abbreviated incubation times, indicating that the FLAG sequence was not deleterious to prion propagation. Immunopurification of FLAG-tagged MoPrPC in the brains of Tg mice was achieved using the calcium-dependent anti-FLAG M1 mAb and non-denaturing procedures. Although the function of PrPC remains unknown, our studies demonstrate that some modifications of PrPC do not inhibit the one biological activity that can be measured, i.e., conversion into PrPSc. Tagged PrP molecules may prove useful in the development of improved assays for prions as well as structural studies of the PrP isoforms.

Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity.

The fundamental event in prion diseases seems to be a conformational change in cellular prion protein (PrPC) whereby it is converted into the pathologic isoform PrPSc. In fatal familial insomnia (FFI), the protease-resistant fragment of PrPSc after deglycosylation has a size of 19 kilodaltons, whereas that from other inherited and sporadic prion diseases is 21 kilodaltons. Extracts from the brains of FFI patients transmitted disease to transgenic mice expressing a chimeric human-mouse PrP gene about 200 days after inoculation and induced formation of the 19-kilodalton PrPSc fragment, whereas extracts from the brains of familial and sporadic Creutzfeldt-Jakob disease patients produced the 21-kilodalton PrPSc fragment in these mice. The results presented indicate that the conformation of PrPSc functions as a template in directing the formation of nascent PrPSc and suggest a mechanism to explain strains of prions where diversity is encrypted in the conformation of PrPSc.

Interactions between wild-type and mutant prion proteins modulate neurodegeneration in transgenic mice.

Transgenic mice overexpressing approximately eightfold the mouse (Mo) prion protein (PrP) gene carrying the P102L mutation of GSS developed neurodegeneration between 150 and 300 days of age, while controls expressing the wild-type MoPrP-A transgene at the same level remained healthy. Mice overexpressing the wild-type MoPrP-A transgene were highly susceptible to inoculated mouse prions, exhibiting abbreviated scrapie incubation times of 45 days. After crossing the mutant transgene onto a null (Prnp 0/0) background, the resulting Tg(MoPrP-P101L)Prnp 0/0 mice displayed a highly synchronous onset of illness at 145 days of age, which was shortened to 85 days upon breeding to homozygosity for the transgene array. Besides occasional PrP plaques and modest spongiform degeneration, Tg(MoPrP-P101L) mice suffered from a myopathy and a peripheral neuropathy. Disruption of the wild-type MoPrP gene increased the number of PrP plaques and the severity of spongiform degeneration. Brain extracts prepared from spontaneously ill transgenic mice transmitted disease to Tg196/Prnp 0/0 mice, expressing low levels of the mutant transgene. Our results demonstrate that the presence of wild-type PrP genes, the level of PrP transgene expression, and the sequence of the transgene can profoundly modify experimental prion disease.

Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein.

Transgenic (Tg) mice expressing human (Hu) and chimeric prion protein (PrP) genes were inoculated with brain extracts from humans with inherited or sporadic prion disease to investigate the mechanism by which PrPC is transformed into PrPSc. Although Tg(HuPrP) mice expressed high levels of HuPrPC, they were resistant to human prions. They became susceptible to human prions upon ablation of the mouse (Mo) PrP gene. In contrast, mice expressing low levels of the chimeric transgene were susceptible to human prions and registered only a modest decrease in incubation times upon MoPrP gene disruption. These and other findings argue that a species-specific macromolecule, provisionally designated protein X, participates in prion formation. While the results demonstrate that PrPSc binds to PrPC in a region delimited by codons 96 to 167, they also suggest that PrPC binds protein X through residues near the C-terminus. Protein X might function as a molecular chaperone in the formation of PrPSc.

Transmission of Creutzfeldt-Jakob disease from humans to transgenic mice expressing chimeric human-mouse prion protein.

Transgenic (Tg) mice were constructed that express a chimeric prion protein (PrP) in which a segment of mouse (Mo) PrP was replaced with the corresponding human (Hu) PrP sequence. The chimeric PrP, designated MHu2MPrP, differs from MoPrP by 9 amino acids between residues 96 and 167. All of the Tg(MHu2M) mice developed neurologic disease approximately 200 days after inoculation with brain homogenates from three patients dying of Creutzfeldt-Jakob disease (CJD). Inoculation of Tg(MHu2M) mice with CJD prions produced MHu2MPrPSc (where PrPSc is the scrapie isoform of PrP); inoculation with Mo prions produced Mo-PrPSc. The patterns of MHu2MPrPSc and MoPrPSc accumulation in the brains of Tg(MHu2M) mice were different. About 10% of Tg(HuPrP) mice expressing HuPrP and non-Tg mice developed neurologic disease > 500 days after inoculation with CJD prions. The different susceptibilities of Tg(HuPrP) and Tg(MHu2M) mice to Hu prions indicate that additional species-specific factors are involved in prion replication. Diagnosis, prevention, and treatment of Hu prion diseases should be facilitated by Tg(MHu2M) mice.

Constructing chimeric type 12/type 5 adenovirus E1A genes and using them to identify an oncogenic determinant of adenovirus type 12.

The E1A gene of highly oncogenic type 12 adenovirus (Ad12) possesses a segment unique to this serotype and comprising 60 base pairs contiguous with and separating conserved regions 2 and 3 in the gene. A similar but slightly longer segment is also present in the E1A gene of highly oncogenic simian adenovirus type 7 (D. Kimelman, J. S. Miller, D. Porter, and B. E. Roberts, J. Virol. 53:399-409, 1985). This segment is missing entirely from the E1A gene of type 5 adenovirus, which is nononcogenic. To test the hypothesis that this unique separating or "spacer" region influences the oncogenicity of Ad12, we constructed ClaI and SmaI restriction sites on either side of it, which allowed reciprocal exchange between this and the equivalent cassette from type 5 adenovirus E1A, bounded by the same restriction sites intrinsic to that gene. The resultant Ad12-based chimeric viruses, ch702 and ch704, in which the spacer region is replaced with (in-frame) type 5 sequence, grow normally on human A549 cells and display wild-type transformation frequencies on baby rat and mouse kidney cells. In contrast, the oncogenic capacity of these chimeric viruses, as measured by tumor induction following virus inoculation in Hooded Lister rats, is greatly reduced. Likewise, cells transformed by ch702 and ch704 display reduced tumorigenicity compared with wild-type transformants in syngeneic rats. These results, coupled with recent preliminary tests using a mutant with a point mutation in this region, support the view that the unique spacer region of type 12 is an oncogenic determinant of this virus.

Absence of an essential regulatory influence of the adenovirus E1B 19-kilodalton protein on viral growth and early gene expression in human diploid WI38, HeLa, and A549 cells.

Mutations in the gene encoding the adenovirus (Ad) early region 1B 19-kDa protein (the 19K gene) result in multiple phenotypic effects upon infection of permissive human cells. It has been reported, for example, that Ad type 2 (Ad2) and Ad5 with mutations in the 19K gene (19K-defective mutants) have a marked growth advantage compared with wild-type virus in human diploid WI38 cells (E. White, B. Faha, and B. Stillman, Mol. Cell. Biol. 6:3763-3773, 1986), and it was proposed that this host range phenotype stems from the large increase in viral early gene expression reported to occur in the mutant-infected cells. These observations gave rise to the hypothesis that the 19-kDa protein (the 19K protein) normally functions as a negative regulator of Ad early gene expression and growth. We have tested this hypothesis and find that Ad5 and Ad12 wild-type viruses grow as efficiently as their respective 19K-defective mutants, in1 and dl337 and pm700 and in700, in WI38 and other human cell types. Neither the accumulation of E1A cytoplasmic mRNAs nor the synthesis of E1A and other viral early proteins in these cells is altered as a result of these mutations in the 19K gene, and we conclude that the 19K protein does not play an essential role in regulating viral early gene expression or viral growth in human cells.

The E1B 19-kilodalton protein is not essential for transformation of rodent cells in vitro by adenovirus type 5.

The newly constructed adenovirus type 5 mutant in1 carries a single AT base pair insertion immediately after nucleotide position 1715 in the E1B gene sequence which destroys the proximal AUG normally present in E1B messages and prevents production of intact E1B 19-kDa protein in infected cells. We have used in1, variants of in1 containing mutant alleles of viral genes known to enhance transformation frequency, and adenovirus type 5 mutant dl337 (S. Pilder, J. Logan, and T. Shenk, J. Virol. 52:664-671, 1984), in which the sequence between nucleotides 1770 and 1916 within the 19-kDa reading frame is deleted, to test the generally accepted hypothesis that this E1B protein is essential for the transformation of rodent cells and maintenance of the transformed phenotype. We find that these mutants transform rat embryo cells, rat kidney and mouse kidney primary cells, and cells of the 3Y1 rat line with decreased frequencies only when virus is added to these various cells at high input multiplicities of infection. In contrast, when lower doses of virus are used, the mutants transform with wild-type frequencies. Cells infected with higher doses of mutant virus show increased levels of DNA degradation and cell killing compared with those of cells infected with the same levels of wild-type virus, and these effects most likely contribute to the decreased transformation frequencies observed. On the basis of these results and the results of phenotypic analyses of numerous transformants, we propose that the E1B 19-kDa protein is not required for induction and/or maintenance of transformed-cell characteristics in rodent cells infected with adenovirus type 5.

The adenovirus L4 100-kilodalton protein is necessary for efficient translation of viral late mRNA species.

When screening a number of adenovirus type 5 (Ad5) temperature-sensitive mutants for defects in viral gene expression, we observed that H5ts1-infected 293 cells accumulated reduced levels of newly synthesized viral late proteins. Pulse-labeling and pulse-chase experiments were used to establish that the late proteins synthesized in H5ts1-infected cells under nonpermissive conditions were as stable as those made in Ad5-infected cells. H5ts1-infected cells contained normal levels of viral late mRNAs. Because these observations implied that translation of viral mRNA species was defective in mutant virus-infected cells, the association of viral late mRNAs with polyribosomes was examined during the late phase of infection at a nonpermissive temperature. In Ad5-infected cells, the majority of the viral L2, L3, L4, pIX, and IVa2 late mRNA species were polyribosome bound. By contrast, these same mRNA species were recovered from H5ts1-infected cells in fractions nearer the top of polyribosome gradients, suggesting that initiation of translation was impaired. During the late phase of infection, neither the polyribosome association nor the translation of most viral early mRNA species was affected by the H5ts1 mutation. This lesion, mapped by marker rescue to the L4 100-kilodalton (kDa) nonstructural protein, has been identified as a single base pair substitution that replaces Ser-466 of the Ad5 100-kDa protein with Pro. A set of temperature-independent revertants of H5ts1 was isolated and characterized. Either true reversion of the H5ts1 mutation or second-site mutation of Pro-466 of the H5ts1 100-kDa protein to Thre, Leu, or His restored both temperature-independent growth and the efficient synthesis of viral late proteins. We therefore conclude that the Ad5 L4 100-kDa protein is necessary for efficient initiation of translation of viral late mRNA species during the late phase of infection.