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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.

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.

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.

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.

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.

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.

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.