Joyce A. Feller

Changes in transcription within the CA1 field of the hippocampus are associated with age-related spatial learning impairments.

Aged rats display a broad range of behavioral performance in spatial learning. The aim of this study was to identify candidate genes that are associated with learning and memory impairments. We first categorized aged-superior learners and age learning-impaired rats based on their performance in the Morris water maze (MWM) and then isolated messenger RNA from the CA1 hippocampal region of each animal to interrogate Affymetrix microarrays. Microarray analysis identified a set of 50 genes that was transcribed differently in aged-superior learners that had successfully learned the spatial strategy in the MWM compared to aged learning-impaired animals that were unable to learn and a variety of groups designed to control for all non-learning aspects of exposure to the water maze paradigm. A detailed analysis of the navigation patterns of the different groups of animals during acquisition and probe trials of the MWM task was performed. Young animals used predominantly an allocentric (spatial) search strategy and aged-superior learners appeared to use a combination of allocentric and egocentric (response) strategies, whereas aged-learning impaired animals displayed thigmotactic behavior. The significant 50 genes that we identified were tentatively classified into four groups based on their putative role in learning: transcription, synaptic morphology, ion conductivity and protein modification. Thus, this study has potentially identified a set of genes that are responsible for the learning impairments in aged rats. The role of these genes in the learning impairments associated with aging will ultimately have to be validated by manipulating gene expression in aged rats. Finally, these 50 genes were functioning in the context of an aging CA1 region where over 200 genes was found to be differentially expressed compared to a young CA1.

FRAGILE X MENTAL RETARDATION PROTEIN REPLACEMENT RESTORES HIPPOCAMPAL SYNAPTIC FUNCTION IN A MOUSE MODEL OF FRAGILE X SYNDROME

Fragile X syndrome (FXS) is caused by a mutation that silences the fragile X mental retardation gene (FMR1), which encodes the fragile X mental retardation protein (FMRP). To determine whether FMRP replacement can rescue phenotypic deficits in a fmr1-knockout (KO) mouse model of FXS, we constructed an adeno-associated virus-based viral vector that expresses the major central nervous system (CNS) isoform of FMRP. Using this vector, we tested whether FMRP replacement could rescue the fmr1-KO phenotype of enhanced long-term depression (LTD), a form of synaptic plasticity that may be linked to cognitive impairments associated with FXS. Extracellular excitatory postsynaptic field potentials were recorded from CA3–CA1 synaptic contacts in hippocampal slices from wild-type (WT) and fmr1-KO mice in the presence of AP-5 and anisomycin. Paired-pulse low-frequency stimulation (PP-LFS)-induced LTD is enhanced in slices obtained from fmr1 KO compared with WT mice. Analyses of hippocampal synaptic function in fmr1-KO mice that received hippocampal injections of vector showed that the PP-LFS-induced LTD was restored to WT levels. These results indicate that expression of the major CNS isoform of FMRP alone is sufficient to rescue this phenotype and suggest that post-developmental protein replacement may have the potential to improve cognitive function in FXS.

Effects of Hydrogen Bonding within a Damaged Base Pair on the Activity of Wild Type and DNA-intercalating Mutants of Human Alkyladenine DNA Glycosylase

Human alkyladenine DNA glycosylase “flips” damaged DNA bases into its active site where excision occurs. Tyrosine 162 is inserted into the DNA helix in place of the damaged base and may assist in nucleotide flipping by “pushing” it. Mutating this DNA-intercalating Tyr to Ser reduces the DNA binding and base excision activities of alkyladenine DNA glycosylase to undetectable levels demonstrating that Tyr-162 is critical for both activities. Mutation of Tyr-162 to Phe reduces the single turnover excision rate of hypoxanthine by a factor of 4 when paired with thymine. Interestingly, when the base pairing partner for hypoxanthine is changed to difluorotoluene, which cannot hydrogen bond to hypoxanthine, single turnover excision rates increase by a factor of 2 for the wild type enzyme and about 3 to 4 for the Phe mutant. In assays with DNA substrates containing1,N 6-ethenoadenine, which does not form hydrogen bonds with either thymine or difluorotoluene, base excision rates for both the wild type and Phe mutant were unaffected. These results are consistent with a role for Tyr-162 in pushing the damaged base to assist in nucleotide flipping and indicate that a nucleotide flipping step may be rate-limiting for excision of hypoxanthine.

The C-Terminal 88 Amino Acids of the Sendai Virus P Protein Have Multiple Functions Separable by Mutation

ABSTRACT The Sendai virus P-L polymerase complex binds the NP-encapsidated nucleocapsid (NC) template through a P-NP interaction. To identify P amino acids responsible for binding we performed site-directed mutagenesis on the C-terminal 88 amino acids in the NC binding domain. The mutant P proteins expressed from plasmids were assayed for viral RNA synthesis and for various protein-protein interactions. All the mutants formed P oligomers and bound to L protein. While two mutants, JT3 and JT8, retained all P functions at or near the levels of wild-type (wt) P, three others—JT4, JT6, and JT9—were completely defective for both transcription and genome replication in vitro. Each of the inactive mutants retained significant NC binding but had a different spectrum of other binding interactions and activities, suggesting that the NC binding domain also affects the catalytic function of the polymerase. NC binding was inhibited by combinations of the inactive mutations. The remaining P mutants were active in transcription but defective in various aspects of genome replication. Some P mutants were defective in NP0 binding and abolished the reconstitution of replication from separate P-L and NP0-P complexes. In some of these cases the coexpression of the wt polymerase with the mutant NP0-P complex could rescue the defect in replication, suggesting an interaction between these complexes. For some P mutants replication occurred in vivo, but not in vitro, suggesting that the intact cell is providing an unknown function that cannot be reproduced in extracts of cells. Thus, the C-terminal region of P is complex and possesses multiple functions besides NC binding that can be separated by mutation.

Isolation and molecular characterization of the swinepox virus thymidine kinase gene

Swinepox virus (SPV), the only member of the Suipoxvirus genus, shows little antigenic relatedness or DNA homology to members of the other poxvirus genera. A SPV thymidine kinase (TK) gene was detected and mapped to the left end of the HindIII G fragment using degenerate oligonucleotide probes. Cloning and sequencing of a 1.8-kb HindIII-BamHI fragment containing the SPV TK gene revealed an open reading frame (ORF) of 181 amino acids yielding a predicted polypeptide of Mr 20.6 kDa with significant homology to both poxvirus and vertebrate thymidine kinases. Comparison with other TK protein sequences showed that the SPV thymidine kinase was closely related to the TK genes of avipoxviruses (52.0%) and vertebrates (57.1-59.7%). The TK gene from African swine fever virus (ASF) showed little homology (30.5%) to the SPV TK gene suggesting that these two viruses are not closely related though they share many biochemical features and infect a single, common mammalian host (swine). The SPV TK gene, like that of other poxviruses, is transcribed early, and when cloned into a TK- strain of vaccinia converted the virus to a TK+ phenotype. BUdRR mutants of SPV contained frameshift, deletion, and missense mutations in the TK ORF.

Replication-Competent Controlled Herpes Simplex Virus

ABSTRACT We present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. IMPORTANCE The alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate, stringent regulation of multiple replication-essential genes. By directing activating heat to the region of virus administration, replication is strictly confined to infected cells within this region. The requirement for antiprogestin provides an additional level of safety, ensuring that virus replication cannot be triggered inadvertently. Replication-competent controlled vectors such as HSV-GS3 may have the potential to be superior to conventional attenuated HSV vaccine and oncolytic vectors without sacrificing safety.

Development of Recombinant HSV-Based Vaccine Vectors

Herpes simplex virus (HSV) causes significant morbidity on the human population through such clinical syndromes as cold sores, genital herpes, herpes stromal keratitis, and encephalitis. Attempts to generate efficacious vaccines to date have failed. We have recently described the use of a conditionally replication-competent HSV-1 vector to immunize mice against a lethal challenge of HSV-1. The unique feature of this vaccine vector is that its replication is tightly controlled and can only occur in the presence of local heat and the presence of a small molecule inducer (an antiprogestin). This gives it the safety advantage of a replication-defective vaccine vector as well as the advantage of a replication-competent vector in that it is able to stimulate innate and adaptive aspects of the immune response in a natural context that a replication-defective vector cannot. In this chapter we provide a brief overview of HSV vaccines followed by the methodology used to propagate and utilize replication-conditional HSV vectors as vaccines.

Construction of Recombinant Vaccinia Virus Expressing HCV Genes

Hepatitis C virus (HCV) remains the leading cause of non-A, non-B hepatitis, and a major indicator for orthotopic liver transplantation. To date, finding a cure or even a commonly effective therapy for infection with HCV has proven to be an elusive goal. One major problem that has hampered attempts to develop antiviral agents is the ongoing inability to grow the virus in tissue culture. Without an efficient in vitro system for the propagation of HCV, it is difficult to generate the large amounts of viral protein needed for this sort of study; nor can the effects of known antiviral agents be assessed on the replication of HCV. Recently, some laboratories have reported preliminary evidence of HCV replication in cultured cells (1,2), but even the best of these systems does not produce significant amounts of progeny virus. Although these methods may be applicable in the future, at present they are not yet practical.