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Background The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization. Results By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123). Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIV MAC239 Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface. Conclusions We have demonstrated significant levels of intracellular Nef oligomers by immunoprecipitation from cellular extracts. However, our results are contrary to the identification of salt bridges between R105 and D123 as necessary for self-association. Importantly, binding between HIV-1Nef and SIVNef demonstrates evolutionary conservation and therefore significant function(s) for oligomerization. Based on modeling studies of Nef self-association, we propose a new dimerization interface. Finally, our findings support a stochastic model of Nef function with a dispersed intracellular distribution of Nef oligomers.

Dendritic cells (DCs) transfected with total amplified tumor cell RNA have the potential to induce broad antitumor immune responses. However, analytical methods required for quantitatively assessing the integrity, fidelity, and functionality of the amplified RNA are lacking. We have developed a series of assays including gel electrophoresis, northern blot, capping efficiency, and microarray analysis to determine integrity and fidelity and a model system to assess functionality after transfection into human DCs. We employed these tools to demonstrate that modifications to our previously reported total cellular RNA amplification process including the use of the Fast Start High Fidelity (FSHF) PCR enzyme, T7 Powerswitch primer, post-transcriptional capping and incorporation of a type 1 cap result in amplification of longer transcripts, greater translational competence, and a higher fidelity representation of the starting total RNA population. To study the properties of amplified RNA after transfection into human DCs, we measured protein expression levels of defined antigens coamplified with the starting total RNA populations and measured antigen-specific T cell expansion in autologous DC-T cell co-cultured in vitro. We conclude from these analyses that the improved RNA amplification process results in superior protein expression levels and a greater capacity of the transfected DCs to induce multifunctional antigen-specific memory T cells.

Upstream open reading frames (uORFs) are cis-acting elements that influence translation of the major cistron, sometimes in a regulated manner. For instance, the uORF in the S-adenosylmethionine decarboxylase (AdoMetDC) mRNA confers feedback control by cellular polyamines on translation of AdoMetDC. In one model of regulation, polyamines are thought to modulate an interaction of the peptide product of the AdoMetDC uORF with some component in the translational apparatus. A step in translation, such as chain termination or peptide release, would be inhibited through this interaction, causing the ribosome to stall over the termination codon. Ribosome stalling would create a blockade to the scanning of additional ribosomes, and suppress translation of both the uORF and the downstream cistron. An integral part of this model is that low polyamine levels lead to relief of this blockade, allowing translation. In this dissertation, aspects of the mechanism of AdoMetDC regulation are presented which support the above model. In chapter 2, the first direct evidence of peptide synthesis from the AdoMetDC uORF is demonstrated. Synthesis of the wild-type peptide was more sensitive to elevated polyamines than that of altered peptides. In contrast, synthesis of wild-type and altered peptides was comparable at low levels of polyamines, and at all concentrations of magnesium tested. In chapter 3, a ribosome was detected paused over the termination codon of the AdoMetDC uORF. This paused ribosome was stabilized by elevated polyamines, and was associated with decreased detection of ribosomes at a downstream AUG codon. In chapter 4, an intermediate in the uORF termination process was detected and identified to be the complete nascent peptide linked to the tRNA decoding the final codon (MAGDIS-tRNASer). This intermediate was stabilized by elevated polyamines and this stabilization required the wild-type peptide sequence. Altogether, these studies suggest that polyamines modulate translation of the AdoMetDC cistron by regulating translation termination of the uORF. Furthermore, the exact step in uORF termination regulated by polyamines must occur after formation of MAGDIS-tRNASer , but before its hydrolysis.

The phonology of Chichewa. By Laura J. Downing and Al Mtenje. Oxford: Oxford University Press, 2017. Pp. xvi, 294. ISBN 9780198724742. $90 (Hb).