Evaluation of RNA Amplification Methods to Improve DC Immunotherapy Antigen Presentation and Immune Response.

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.Molecular Therapy-Nucleic Acids (2013) 2, e91; doi:10.1038/mtna.2013.18; published online 7 May 2013.

Self-association of the Lentivirus protein, Nef.

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.

Structural constraints on human immunodeficiency virus type 1 Nef function.

HIV-1 Nef is a multifunctional protein that exerts its activities through interactions with multiple cellular partners. Nef uses different domains and mechanisms to exert its functions including cell surface down-modulation of CD4 and MHC-I receptors and activation of the serine/threonine kinase PAK-2. We inserted tags at the C-terminus and proximal to the N-terminus of Nef and the effects on Nef's structure/function relationships were examined. We discovered significant defects in MHC-I down-modulation with the insertion of HA/FLAG tags at either region. We also found impaired PAK-2 activation with a C-terminal fusion with GFP. Interestingly, Nef-GFP and Nef-GH(7) induced MHC-I down-modulation, suggesting that the negative charge of the HA/FLAG tag could contribute to the observed defect. Together, these observations highlight elements of Nef's functional complexity and demonstrate previously unsuspected structural requirements for PAK-2 activation and MHC-1 down-modulation in Nef's flexible N- and C-terminal regions.

Activation of p21-activated kinase 2 by human immunodeficiency virus type 1 Nef induces merlin phosphorylation.

The accessory human immunodeficiency virus type 1 (HIV-1) protein Nef activates the autophosphorylation activity of p21-activated kinase 2 (PAK2). Merlin, a cellular substrate of PAK2, is homologous to the ezrin-radixin-moesin family and plays a critical role in Rac signaling. To assess the possible impact on host cell metabolism of Nef-induced PAK2 activation, we investigated the phosphorylation of merlin in Nef expressing cells. Here we report that Nef induces merlin phosphorylation in multiple cell lines independently of protein kinase A. This intracellular phosphorylation of merlin directly correlates with in vitro assay of the autophosphorylation activity of Nef-activated PAK2. Importantly, merlin phosphorylation induced by Nef was also observed in human primary T cells. The finding that Nef induces phosphorylation of the key signaling molecule merlin suggests several possible roles for PAK2 activation in HIV pathogenesis.

Reconstitution and molecular analysis of an active human immunodeficiency virus type 1 Nef/p21-activated kinase 2 complex.

Human immunodeficiency virus type 1 (HIV-1) Nef activation of p21-activated kinase 2 (PAK-2) was recapitulated in a cell-free system consisting of in vitro-transcribed RNA, rabbit reticulocyte lysate, and microsomal membranes on the basis of the following observations: (i) Nef associated with a kinase endogenous to the rabbit reticulocyte lysate that was identified as PAK-2, (ii) Nef-associated kinase activity was detected with Nefs from HIV-1(SF2), HIV-1(YU2), and SIV(mac239), (iii) kinase activation was not detected with a myristoylation-defective Nef (HIV-1(SF2)NefG2A) or with a Nef defective in PAK-2 activation but fully competent in other Nef functions (HIV-1(SF2)NefF195I), and (iv) Nef-associated kinase activation required activated endogenous p21 GTPases (Rac1 or Cdc42). The cell-free system was used to analyze the mechanism of Nef activation of PAK-2. First, studies suggest that the p21 GTPases may act transiently to enhance Nef activation of PAK-2 in vitro. Second, addition of wortmannin to the cell-free system demonstrated that Nef activation of PAK-2 does not require PI 3-kinase activity. Third, ultracentrifugation analysis revealed that whereas the majority of Nef and PAK-2 partitioned to the supernatant, Nef-associated PAK-2 activity partitioned to the membrane-containing pellet as a low-abundance complex. Lastly, Nef activation of PAK-2 in vitro requires addition of microsomal membranes either during or after translation of the Nef RNA. These results are consistent with a model in which activation of PAK-2 by Nef occurs by recruiting PAK-2 to membranes. As demonstrated herein, the cell-free system is a new and important tool in the investigation of the mechanism of PAK-2 activation by Nef.

Studies with chimeric Mpl/JAK2 receptors indicate that both JAK2 and the membrane-proximal domain of Mpl are required for cellular proliferation.

The thrombopoietin (TPO) receptor c-Mpl, like other members of the cytokine receptor superfamily, requires the association and activation of Janus kinases (JAKs) for normal signal transduction. The membrane-proximal portion of the signaling domain, containing conserved box1 and box2 motifs, is sufficient to support the proliferation of cytokine-dependent cell lines and basal megakaryocytopoiesis in vivo. We hypothesized that activation of the JAK2 kinase alone might be sufficient for proliferative signaling. To test this premise, we constructed chimeric receptors in which the extracellular and transmembrane portions of Mpl were fused to the pseudokinase and kinase domains of murine JAK2 kinase. When expressed in the interleukin-3-dependent cell line Ba/F3, the chimeric receptors were appropriately expressed on the cell surface and were able to initiate tyrosine kinase activity upon exposure to TPO. However, chimeric receptors lacking an intact box2 domain of Mpl were unable to support proliferation at any concentration of TPO. Only chimeric receptors containing both JAK2 kinase activity and the box2 region initiated proliferative signaling. Within the box2 motif, we determined that the sequence Glu(56)-Ile(57)-Leu(58) of the Mpl cytoplasmic domain is critical for proliferation of the chimeric receptors. Furthermore, TPO-dependent induction of c-myc transcription is also dependent on this motif. These results indicate that JAK2 activation alone is not sufficient for TPO-induced proliferation and that one or more essential signaling pathways must arise from the cytoplasmic domain of Mpl that includes box2. Although the nature of the signal transduction pathway is not yet known, this second proliferative event is likely to regulate c-myc expression.

Regulated translation termination at the upstream open reading frame in s-adenosylmethionine decarboxylase mRNA.

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a cis-acting element that confers feedback control by cellular polyamines on translation of this message. Recent studies demonstrated that elevated polyamines inhibit synthesis of the peptide encoded by the uORF by stabilizing a ribosome paused in the vicinity of the termination codon. These studies suggested that polyamines act at the termination step of uORF translation. In this paper, we demonstrate that elevated polyamines stabilize an intermediate in the termination process, the complete nascent peptide linked to the tRNA that decodes the final codon. The peptidyl-tRNA molecule is found associated with the ribosome fraction, and decay of this molecule correlated with release of the paused ribosome from the message. Furthermore, the stability of this complex is influenced by the same parameters that influence regulation by the uORF in vivo, namely the concentration of polyamines and the sequence of the uORF-encoded peptide. These results suggest that the regulated step in uORF translation is after formation of the peptidyl-tRNA molecule but before hydrolysis of the peptidyl-tRNA bond. This regulation may involve an interaction between the peptide, polyamines, and a target in the translational apparatus.