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. This deficiency has largely been attributed to decreased production of the target protein for immunization. Efforts to increase protein production from attenuated VACV strains has largely centered around modulation of viral factors, while manipulation of the translation of viral mRNAs has been largely unexplored. In this study we evaluate the use of translation enhancing element hTEE-658 to increase recombinant protein production in an attenuated VACV system. Optimization of the use of this motif is also attempted by combining it with strategies that have demonstrated effectiveness in previous research. We show that extension of the 5' leader sequence containing hTEE-658 does not improve motif function, nor does the combination with other known translation enhancing elements. However, the sole use of hTEE-658 in an attenuated VACV system is shown to increase protein expression levels beyond those of a standard viral promoter when used with a wild-type virus. Taken together these results highlight the potential for hTEE-658 to improve the effectiveness of attenuated VACV vaccine candidates and give insights into the optimal sequence context for its use in vaccine design.

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. Herein, we examine the sequence determinants of AUG triplets within a set of human translation enhancing elements (TEEs). Functional analyses performed in vitro and in cultured cells indicate that AUGs have the capacity to modulate mRNA translation either by serving as part of a larger ribosomal recruitment site or by directing the ribosome to defined initiation sites. These observations help constrain the functional role of AUG triplets in human TEEs and advance our understanding of this specific mechanism of cap-independent 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. When tested in our cell-based assay at concentrations insignificantly toxic to T-cells, altertoxins V (1), I (3), II (4), and III (5) completely inhibited replication of the HIV-1 virus at concentrations of 0.50, 2.20, 0.30, and 1.50 µM, respectively. Our findings suggest that the epoxyperylene structural scaffold in altertoxins may be manipulated to produce potent anti-HIV therapeutics.

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. Here we report a novel 37-nucleotide leader sequence that promotes rapid and high transgene expression in mammalian cells. This sequence was identified by in vitro selection and functions in a transient vaccinia-based cytoplasmic expression system. Vectors containing this sequence produce microgram levels of protein in just 6 h from a small-scale expression in 10(6) cells. This level of protein synthesis is ideal for high throughput production of human proteins, and could be scaled to generate milligram quantities of protein. The technology is compatible with a broad range of cell lines, accepts plasmid and linear DNA, and functions with viruses that are approved for use under BSL1 conditions. We suggest that these advantages provide a powerful method for generating human protein in mammalian cells.

Inhibition of HIV-1 Replication by Secondary Metabolites From Endophytic Fungi of Desert Plants.

Most antiretroviral drugs currently in use to treat an HIV-1 infection are chemically synthesized and lead to the development of viral resistance, as well as cause severe toxicities. However, a largely unexplored source for HIV-1 drug discovery is endophytic fungi that live in a symbiotic relationship with plants. These fungi produce biologically active secondary metabolites, which are natural products that are beneficial to the host. We prepared several hundred extracts from endophytic fungi of desert plants and evaluated the inhibitory effects on HIV-1 replication of those extracts that showed less than 30% cytotoxicity in T-lymphocytes. Those extracts that inhibited viral replication were fractionated in order to isolate the compounds responsible for activity. Multiple rounds of fractionation and antiviral evaluation lead to the identification of four compounds, which almost completely impede HIV-1 replication. These studies demonstrate that metabolites from endophytic fungi of desert plants can serve as a viable source for identifying potent inhibitors of HIV-1 replication.

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. Numerous potential transcription factor binding sites at the sites of integration were identified. Furthermore, the genes at the site of integration, transcription factors which potentially bind upstream of the HIV-1 promoter and factors that assist HIV-1 integration were found to be expressed at higher levels in cord than adult cells. Taken together, these results suggest HIV-1 integration occurred in a more actively transcribed genes in neonatal cells compared with adult cells, which may help explain a higher level of HIV-1 gene expression and replication in neonatal compared with adult cells.

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. RESULTS: Phylogenetic analysis of 168 gag NC and p6 genes sequences revealed six separate subtrees that corresponded to each mother-infant pair, suggesting that epidemiologically linked individuals were closer to each other than epidemiologically unlinked individuals. A high frequency (92.8%) of intact open reading frames of NC and p6 with patient and pair specific sequence motifs were conserved in mother-infant pairs' sequences. Nucleotide and amino acid distances showed a lower degree of viral heterogeneity, and a low degree of estimates of genetic diversity was also found in NC and p6 sequences. The NC and p6 sequences from both mothers and infants were found to be under positive selection pressure. The two important functional motifs within NC, the zinc-finger motifs, were highly conserved in most of the sequences, as were the gag p6 Vpr binding, AIP1 and late binding domains. Several CTL recognition epitopes identified within the NC and p6 genes were found to be mostly conserved in 6 mother-infant pairs' sequences. CONCLUSION: These data suggest that the gag NC and p6 open reading frames and functional domains were conserved in mother-infant pairs' sequences following vertical transmission, which confirms the critical role of these gene products in the viral lifecycle.