Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy

<p>Abstract</p> <p>Background</p> <p>Unlike CD4+ T cells, HIV-1 infected macrophages exhibit extended life span even upon stress, consistent with their <it>in vivo </it>role as long-lived HIV-1 reservoirs.</p> <p>Results</p> <p>Here, we demonstrate that PI3K/Akt inhibitors, including clinically available Miltefosine, dramatically reduced HIV-1 production from long-living virus-infected macrophages. These PI3K/Akt inhibitors hyper-sensitize infected macrophages to extracellular stresses that they are normally exposed to, and eventually lead to cell death of infected macrophages without harming uninfected cells. Based on the data from these Akt inhibitors, we were able to further investigate how HIV-1 infection utilizes the PI3K/Akt pathway to establish the cytoprotective effect of HIV-1 infection, which extends the lifespan of infected macrophages, a key viral reservoir. First, we found that HIV-1 infection activates the well characterized pro-survival PI3K/Akt pathway in primary human macrophages, as reflected by decreased PTEN protein expression and increased Akt kinase activity. Interestingly, the expression of HIV-1 or SIV Tat is sufficient to mediate this cytoprotective effect, which is dependent on the basic domain of Tat – a region that has previously been shown to bind p53. Next, we observed that this interaction appears to contribute to the downregulation of PTEN expression, since HIV-1 Tat was found to compete with PTEN for p53 binding; this is known to result in p53 destabilization, with a consequent reduction in PTEN protein production.</p> <p>Conclusion</p> <p>Since HIV-1 infected macrophages display highly elevated Akt activity, our results collectively show that PI3K/Akt inhibitors may be a novel therapy for interfering with the establishment of long-living HIV-1 infected reservoirs.</p

Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly.

Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein

Species difference in ANP32A underlies influenza A virus polymerase host restriction.

Influenza pandemics occur unpredictably when zoonotic influenza viruses with novel antigenicity acquire the ability to transmit amongst humans. Host range breaches are limited by incompatibilities between avian virus components and the human host. Barriers include receptor preference, virion stability and poor activity of the avian virus RNA-dependent RNA polymerase in human cells. Mutants of the heterotrimeric viral polymerase components, particularly PB2 protein, are selected during mammalian adaptation, but their mode of action is unknown. We show that a species-specific difference in host protein ANP32A accounts for the suboptimal function of avian virus polymerase in mammalian cells. Avian ANP32A possesses an additional 33 amino acids between the leucine-rich repeats and carboxy-terminal low-complexity acidic region domains. In mammalian cells, avian ANP32A rescued the suboptimal function of avian virus polymerase to levels similar to mammalian-adapted polymerase. Deletion of the avian-specific sequence from chicken ANP32A abrogated this activity, whereas its insertion into human ANP32A, or closely related ANP32B, supported avian virus polymerase function. Substitutions, such as PB2(E627K), were rapidly selected upon infection of humans with avian H5N1 or H7N9 influenza viruses, adapting the viral polymerase for the shorter mammalian ANP32A. Thus ANP32A represents an essential host partner co-opted to support influenza virus replication and is a candidate host target for novel antivirals

Modeling Host Genetic Regulation of Influenza Pathogenesis in the Collaborative Cross

Genetic variation contributes to host responses and outcomes following infection by influenza A virus or other viral infections. Yet narrow windows of disease symptoms and confounding environmental factors have made it difficult to identify polymorphic genes that contribute to differential disease outcomes in human populations. Therefore, to control for these confounding environmental variables in a system that models the levels of genetic diversity found in outbred populations such as humans, we used incipient lines of the highly genetically diverse Collaborative Cross (CC) recombinant inbred (RI) panel (the pre-CC population) to study how genetic variation impacts influenza associated disease across a genetically diverse population. A wide range of variation in influenza disease related phenotypes including virus replication, virus-induced inflammation, and weight loss was observed. Many of the disease associated phenotypes were correlated, with viral replication and virus-induced inflammation being predictors of virus-induced weight loss. Despite these correlations, pre-CC mice with unique and novel disease phenotype combinations were observed. We also identified sets of transcripts (modules) that were correlated with aspects of disease. In order to identify how host genetic polymorphisms contribute to the observed variation in disease, we conducted quantitative trait loci (QTL) mapping. We identified several QTL contributing to specific aspects of the host response including virus-induced weight loss, titer, pulmonary edema, neutrophil recruitment to the airways, and transcriptional expression. Existing whole-genome sequence data was applied to identify high priority candidate genes within QTL regions. A key host response QTL was located at the site of the known anti-influenza Mx1 gene. We sequenced the coding regions of Mx1 in the eight CC founder strains, and identified a novel Mx1 allele that showed reduced ability to inhibit viral replication, while maintaining protection from weight loss

MicroRNA Regulation of Human Protease Genes Essential for Influenza Virus Replication

Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies

N-Glycans on the Nipah virus attachment glycoprotein modulate fusion and viral entry as they protect against antibody neutralization

Nipah virus (NiV) is the deadliest known paramyxovirus. Membrane fusion is essential for NiV entry into host cells and for the virus' pathological induction of cell-cell fusion (syncytia). The mechanism by which the attachment glycoprotein (G), upon binding to the cell receptors ephrinB2 or ephrinB3, triggers the fusion glycoprotein (F) to execute membrane fusion is largely unknown. N-glycans on paramyxovirus glycoproteins are generally required for proper protein conformational integrity, transport, and sometimes biological functions. We made conservative mutations (Asn to Gln) at the seven potential N-glycosylation sites in the NiV G ectodomain (G1 to G7) individually or in combination. Six of the seven N-glycosylation sites were found to be glycosylated. Moreover, pseudotyped virions carrying these N-glycan mutants had increased antibody neutralization sensitivities. Interestingly, our results revealed hyperfusogenic and hypofusogenic phenotypes for mutants that bound ephrinB2 at wild-type levels, and the mutant's cell-cell fusion phenotypes generally correlated to viral entry levels. In addition, when removing multiple N-glycans simultaneously, we observed synergistic or dominant-negative membrane fusion phenotypes. Interestingly, our data indicated that 4- to 6-fold increases in fusogenicity resulted from multiple mechanisms, including but not restricted to the increase of F triggering. Altogether, our results suggest that NiV-G N-glycans play a role in shielding virions against antibody neutralization, while modulating cell-cell fusion and viral entry via multiple mechanisms

Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy-4

SIV mac239 and SIV PBJ. Numbers indicate residues on the first amino acids of the shown sequences. Colored residues in HIV-1 Tat were mutated in this study. CHME5 cells were cotransfected with a plasmid encoding GFP and constructs expressing the first exon of HIV-1 Tat (psvTat72), SIV-PBJ Tat, or with an empty plasmid (pcDNA3.1) using Lipofectamine. Cells were then treated with LPS/CHX and analyzed for cell death. Bright fields (BF) and merged (red+green) fields are shown. Transfected cells are GFP+ cells (green), dead cells (red). The percentage of cell death induced in GFP+, EthD+ cells is shown with the SD from three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy"</p><p>http://www.retrovirology.com/content/5/1/11</p><p>Retrovirology 2008;5():11-11.</p><p>Published online 31 Jan 2008</p><p>PMCID:PMC2265748.</p><p></p

Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy-3

Rease cell survival by preventing PTEN from binding to p53. Binding of HIV-1 Tat to p53 may result in reduced levels of both p53 by destabilization and PTEN by downregulation of PTEN expression. In vitro binding assay: Lysates containing p53-FLAG were incubated with BSA (control) or HIV-1 Tat protein and then mixed with lysate containing PTEN V5-tag. Proteins bound to p53 were immunoprecipitated using anti-FLAG immobilized antibody and analyzed for PTEN-V5 tag levels by Western blotting. Ratios of PTEN-V5 levels normalized by p53-FLAG levels are shown.<p><b>Copyright information:</b></p><p>Taken from "Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy"</p><p>http://www.retrovirology.com/content/5/1/11</p><p>Retrovirology 2008;5():11-11.</p><p>Published online 31 Jan 2008</p><p>PMCID:PMC2265748.</p><p></p

Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy-1

Roteins and EGFP (+) or heat-inactivated vector (-). Levels of PTEN protein in YU-2 infected macrophage and HIV vector-transduced macrophages as determined by Western blotting. Ratios of PTEN normalized by β-tubulin levels are shown. Reverse transcriptase PCR analysis of PTEN mRNA levels following transduction of macrophage with HIV vector (+) or treatment with heat-inactivated vector (-). M: 100 bp size marker. β-tubulin and β-Actin were used for loading controls in the Western analysis and RT-PCR, respectively.<p><b>Copyright information:</b></p><p>Taken from "Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy"</p><p>http://www.retrovirology.com/content/5/1/11</p><p>Retrovirology 2008;5():11-11.</p><p>Published online 31 Jan 2008</p><p>PMCID:PMC2265748.</p><p></p

Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy-0

Reated (media only) or were treated with one of four different PI3K/Akt kinase inhibitors in the presence or absence of stress (SNP, 1 mM): the PI3K inhibitor wortmannin (100 nM), Akt inhibitor IV (200 nM), Akt inhibitor VIII (105 nM) or Miltefosine 5 μM. Viral supernatants were collected every 3 days for 12 days and supernatants were analyzed using the HIV-1 p24 EIA. Asterisks denote undetectable p24 levels. On day 12, YU2-infected macrophages were analyzed for cell viability using the live/dead assay. Viable cells are green; dead cells are red. Results are representative of 5 independent, triplicate experiments using cells obtained from multiple blood donors. BF: Bright field. Merge: overlay of red and green fluorescence. The average ± SD percentage of dead cells is also shown.<p><b>Copyright information:</b></p><p>Taken from "Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy"</p><p>http://www.retrovirology.com/content/5/1/11</p><p>Retrovirology 2008;5():11-11.</p><p>Published online 31 Jan 2008</p><p>PMCID:PMC2265748.</p><p></p