Optimization of translation enhancing element use to increase protein expression in a vaccinia virus system.

Since the successful use of vaccinia virus (VACV) in the immunization strategies to eliminate smallpox, research has been focused on the development of recombinant VACV strains expressing proteins from various pathogens. Attempts at decreasing the side effects associated with exposure to recombinant, wild-type viral strains have led to the development of attenuated viruses. Yet while these attenuated VACV's have improved safety profiles compared to unmodified strains, their clinical use has been hindered due to efficacy issues in stimulating a host immune response.

Exploring the Role of AUG Triplets in Human Cap-Independent Translation Enhancing Elements.

Cap-independent translation is believed to play an important role in eukaryotic protein synthesis, but the mechanisms of ribosomal recruitment and translation initiation remain largely unknown. Messenger RNA display was previously used to profile the human genome for RNA leader sequences that can enhance cap-independent translation. Surprisingly, many of the isolated sequences contain AUG triplets, suggesting a possible functional role for these motifs during translation initiation.

Altertoxins with potent anti-HIV activity from Alternaria tenuissima QUE1Se, a fungal endophyte of Quercus emoryi.

Screening of a small library of natural product extracts derived from endophytic fungi of the Sonoran desert plants in a cell-based anti-HIV assay involving T-cells infected with the HIV-1 virus identified the EtOAc extract of a fermentation broth of Alternaria tenuissima QUE1Se inhabiting the stem tissue of Quercus emoryi as a promising candidate for further investigation. Bioactivity-guided fractionation of this extract led to the isolation and identification of two new metabolites, altertoxins V (1) and VI (2) together with the known compounds, altertoxins I (3), II (4), and III (5). The structures of 1 and 2 were determined by detailed spectroscopic analysis and those of 3-5 were established by comparison with reported data.

A leader sequence capable of enhancing RNA expression and protein synthesis in mammalian cells.

Many applications in biotechnology require human proteins generated from human cells. Stable cell lines commonly used for this purpose are difficult to develop, and scaling to large numbers of proteins can be problematic. Transient expression can circumvent this problem, but protein yields are generally too low for most applications.

Genome-wide profiling of human cap-independent translation-enhancing elements.

We report an in vitro selection strategy to identify RNA sequences that mediate cap-independent initiation of translation. This method entails mRNA display of trillions of genomic fragments, selection for initiation of translation and high-throughput deep sequencing. We identified >12,000 translation-enhancing elements (TEEs) in the human genome, generated a high-resolution map of human TEE-bearing regions (TBRs), and validated the function of a subset of sequences in vitro and in cultured cells.

Differential HIV-1 integration targets more actively transcribed host genes in neonatal than adult blood mononuclear cells.

We have recently shown an increased HIV-1 replication and gene expression in neonatal (cord) blood mononuclear cells compared with adult cells, which could be due to HIV-1 integration as it targets active host genes. Here we have characterized 468 HIV-1 integration sites within cord and adult blood T-lymphocytes and monocyte-derived macrophages (MDM) from five donors. Several functional classes of genes were identified by gene ontology to be over represented, including genes for cellular components, maintenance of intracellular environment, enzyme regulation, cellular metabolism, catalytic activity and cation transport.

Molecular characterization of the HIV-1 gag nucleocapsid gene associated with vertical transmission.

Background: The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) plays a pivotal role in the viral lifecycle: including encapsulating the viral genome, aiding in strand transfer during reverse transcription, and packaging two copies of the viral genome into progeny virions. Another gag gene product, p6, plays an integral role in successful viral budding from the plasma membrane and inclusion of the accessory protein Vpr within newly budding virions. In this study, we have characterized the gag NC and p6 genes from six mother-infant pairs following vertical transmission by performing phylogenetic analysis and by analyzing the degree of genetic diversity, evolutionary dynamics, and conservation of functional domains.