DNA analogues are currently being intensely investigated owing to their potential as gene-targeted drugs. Furthermore, their properties and interaction with DNA and RNA could provide a better understanding of the structural features of natural DNA that determine its unique chemical, biological and genetic properties. We recently designed a DNA analogue, PNA, in which the backbone is structurally homomorphous with the deoxyribose backbone and consists of N-(2-aminoethyl)glycine units to which the nucleobases are attached. We showed that PNA oligomers containing solely thymine and cytosine can hybridize to complementary oligonucleotides, presumably by forming Watson-Crick-Hoogsteen (PNA)2-DNA triplexes, which are much more stable than the corresponding DNA-DNA duplexes, and bind to double-stranded DNA by strand displacement. We report here that PNA containing all four natural nucleobases hybridizes to complementary oligonucleotides obeying the Watson-Crick base-pairing rules, and thus is a true DNA mimic in terms of base-pair recognition.

Fibroblast growth factor 21 is a member of endocrine FGFs subfamily, along with FGF19 and FGF23. It is emerging as a novel regulator with beneficial effects on a variety of metabolic parameters, including glucose and lipid control. FGF21 activity depends on membrane protein betaKlotho that physically complexes with various FGF receptors, thus conferring them the ability to bind FGF21 and activate downstream signaling pathways. FGF21, like other FGFs, folds to a beta-trefoil-like core region, with disordered N- and C-termini. In order to investigate their role in the activity of FGF21, we have constructed a series of deletion mutants and tested them for their ability to (1) bind betaKlotho, analyzed by surface plasmon resonance spectroscopy (2) signal through MAPK phosphorylation and inhibit apoptosis in 3T3-L1/betaKlotho fibroblasts (3) stimulate GLUT1 mRNA upregulation and glucose uptake in 3T3-L1 adipocytes. Binding studies with betaKlotho revealed that the interaction with the co-receptor involves the C-terminus, as progressive removal of amino acids from the carboxy end decreased affinity for betaKlotho. By contrast, removal of up to 17 amino acids from the N-terminus had no effect on the interaction with betaKlotho. Terminal deletions had greater effect on function, as deletions of six amino acids from the amino-terminus and only four from the carboxy-terminus each significantly impacted activity (10-fold). Of the extreme terminal truncations, with no detectable activity, DeltaN17 acted as competitive antagonist while DeltaC20 did not. Our structure/function studies show that the C-terminus is important for betaKlotho interaction whereas the N-terminus likely interacts directly with FGF receptors.

Alphavirus vectors, derived principally from Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus, are widely used for gene expression studies in vitro and are being developed for both vaccine and gene therapy applications (25). Typically, these vectors are constructed in a format known as a replicon, due to the selfamplifying nature of the vector RNA (30). Replicons contain both the cis and trans alphavirus genetic elements required for RNA replication, as well as heterologous gene expression via the native subgenomic promoter. Upon introduction into cells, replicon RNA is translated to produce four nonstructural proteins (nsPs), which together comprise the alphaviral replicase. Replication proceeds through a minus-strand RNA intermediate and subsequently generates two distinct positive-strand RNA species, corresponding to a genomic-length vector RNA and an abundant subgenomic RNA encoding the heterologous gene (27). The replicon RNA can be packaged into virion-like particles by providing the structural proteins in trans, from in vitro-transcribed defective helper RNA (4, 15-17) or using packaging cell lines (16). Alternatively, the replicon RNA can be introduced directly into cells as plasmid DNA (2,6,8,13).In most mammalian cells, host macromolecular synthesis is inhibited following the introduction of alphavirus replicons, leading to eventual cell death by an apoptotic mechanism (11, 25). Thus, application of these vectors for some gene therapy applications and extended gene expression studies in cultured cells is limited. Given the many other attractive features of the alphavirus replicon system, it would be useful to extend the utility of these vectors to include long-term expression and reduced cytopathogenicity options.Under appropriate conditions, alphaviruses and alphavirusderived vectors can establish persistence in cultured cells (14,26,29) or exhibit delayed onset of cytopathic effects (9). The establishment of SIN replicon persistence in BHK cells has been associated with mutation of the protease domain of nsP2 (7, 10), and studies have suggested that the use of such mutants for long-term expression may be possible (1, 3). It remains to be determined whether mutation of other alphavirus nsPs or nsP2 domains can provide a noncytopathic phenotype by a similar or alternative mechanism.To expand the utility of the noncytopathic replicon and further explore how persistence is established, we isolated additional SIN replicons with this phenotype, as well as SFV replicons with a similar phenotype. Mutations that conferred the establishment of persistent replication were mapped to several regions of nsP2 for both SIN and SFV replicons, in addition to the same residue 726 mutation identified previously (7, 10). These mutations had various effects on the levels of genomic and subgenomic replicon RNA and, in some cases, processing of the nonstructural polyprotein.Selection of replicons that establish persistent replication. To select alphavirus replicon variants capable of...

Alphavirus vectors are being developed for possible human vaccine and gene therapy applications. We have sought to advance this field by devising DNA-based vectors and approaches for the production of recombinant vector particles. In this work, we generated a panel of alphavirus vector packaging cell lines (PCLs). These cell lines were stably transformed with expression cassettes that constitutively produced RNA transcripts encoding the Sindbis virus structural proteins under the regulation of their native subgenomic RNA promoter. As such, translation of the structural proteins was highly inducible and was detected only after synthesis of an authentic subgenomic mRNA by the vectorencoded replicase proteins. Efficient production of biologically active vector particles occurred after introduction of Sindbis virus vectors into the PCLs. In one configuration, the capsid and envelope glycoproteins were separated into distinct cassettes, resulting in vector packaging levels of 10 7 infectious units͞ml, but reducing the generation of contaminating replication-competent virus below the limit of detection. Vector particle seed stocks could be amplified after low multiplicity of infection of PCLs, again without generating replicationcompetent virus, suggesting utility for production of largescale vector preparations. Furthermore, both Sindbis virusbased and Semliki Forest virus-based vectors could be packaged with similar efficiency, indicating the possibility of developing a single PCL for use with multiple alphavirusderived vectors.The use of virus-derived expression vectors for gene therapy and vaccine applications increasingly is being pursued, with a number of diverse virus types and approaches. Alphaviruses are attractive candidates for such applications because of their high levels of replication and gene expression, their ability to infect a variety of diverse cell types, and the ability to manipulate cDNA clones from which infectious viral RNA may be transcribed (for review, see refs. 1 and 2). The alphavirus genome is a single-stranded, positive-sense RNA of approximately 11.7 kb and is encapsidated within an icosahedral capsid protein shell (for review, see ref.3). Nucleocapsids, in turn, are surrounded by a host-derived lipid envelope from which the viral spike glycoproteins E1 and E2 protrude. Cytoplasmic replication of the RNA genome is mediated by four viral-encoded nonstructural proteins and proceeds through a full-length negative-sense intermediate. Subsequent positive-strand RNA synthesis results in both progeny genome RNA and an abundant, internally initiated subgenomic mRNA. The virus structural proteins are translated from the subgenomic mRNA as a polyprotein that is processed into the individual components of the virion.The general strategy for construction of alphavirus-based expression vectors has been to substitute the viral structural protein genes with a heterologous gene, maintaining transcriptional control via the highly active subgenomic RNA promoter (1, 2, 4). As such, these vector r...

The ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55Gag elicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.Dendritic cells (DC) are the most potent antigen-presenting cell population and play a major role in the activation of both memory and naïve T cells. Immature DC capture antigen in the periphery and migrate to the draining lymph nodes, where they undergo maturation. Presentation of acquired antigen by mature DC is critical for induction of antigen-specific immune responses (1, 9, 13, 36) and stimulation of protective T-cell responses (3, 10). Transduction of autologous cultured DC ex vivo with gene delivery vectors encoding a desired antigen, followed by adoptive transfer, has been shown to stimulate antigen-specific T-cell responses in vivo (45,46). Unfortunately, the ability to target the DC cell population in vivo has been quite limited or has been shown to interfere with DC function or development (5,17,20,23,32,39). We rationalized that enhanced delivery of antigen to immature DC may provide an opportunity for improvement of vaccines, particularly for gene-based vaccination approaches.Toward a goal of improving DC-targeting approaches, we have focused on alphavirus-based vectors. The use of alphavirus vectors for vaccine and gene therapy applications is a rapidly emerging field (15, 42, 44). These RNA-based vectors, known as "replicons" because they retain the replicase functions necessary for RNA self-amplification and high-level expression, can be launched in vi...