Alphavirus Entry and Membrane Fusion

The study of enveloped animal viruses has greatly advanced our understanding of the general properties of membrane fusion and of the specific pathways that viruses use to infect the host cell. The membrane fusion proteins of the alphaviruses and flaviviruses have many similarities in structure and function. As reviewed here, alphaviruses use receptor-mediated endocytic uptake and low pH-triggered membrane fusion to deliver their RNA genomes into the cytoplasm. Recent advances in understanding the biochemistry and structure of the alphavirus membrane fusion protein provide a clearer picture of this fusion reaction, including the protein’s conformational changes during fusion and the identification of key domains. These insights into the alphavirus fusion mechanism suggest new areas for experimental investigation and potential inhibitor strategies for anti-viral therapy

Functional Characterization of Circulating Tumor Cells with a Prostate-Cancer-Specific Microfluidic Device

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45− cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2–400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents

A Conserved Histidine in the ij Loop of the Semliki Forest Virus E1 Protein Plays an Important Role in Membrane Fusion

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered membrane fusion reaction mediated by the E1 protein. E1 is a class II fusion protein that contains the hydrophobic fusion peptide loop and converts to a stable homotrimer during the fusion reaction. Intriguingly, the fusion loop is closely associated with a loop connecting the i and j β-strands. This ij loop plays a role in the cholesterol dependence of membrane fusion and is specifically susceptible to proteolysis in the protease-resistant E1 homotrimer. The SFV ij loop contains a histidine residue at position 230. Sequence comparisons revealed that an analogous histidine is completely conserved in all alphavirus and flavivirus fusion proteins. An E1 H230A mutant was constructed using the SFV infectious clone. Although cells infected with H230A RNA produced virus particles, these virions were completely noninfectious and were blocked in both cell-cell fusion and lipid mixing assays. The H230A virions efficiently bound to cell surface receptors and responded to low pH by undergoing acid-dependent conformational changes including dissociation of the E1/E2 dimer, exposure of the fusion loop, association with target liposomes, exposure of acid-conformation-specific epitopes, and formation of the stable E1 homotrimer. Studies with a soluble fragment of E1 showed that the mutant protein was defective in lipid-dependent conformational changes. Our results indicate that the E1 ij loop and the conserved H230 residue play a critical role in alphavirus-membrane fusion and suggest the presence of a previously undescribed late intermediate in the fusion reaction

Second-Site Revertants of a Semliki Forest Virus Fusion-Block Mutation Reveal the Dynamics of a Class II Membrane Fusion Protein

The alphavirus Semliki Forest virus (SFV) infects cells through low-pH-induced membrane fusion mediated by the E1 protein, a class II virus membrane fusion protein. During fusion, E1 inserts into target membranes via its hydrophobic fusion loop and refolds to form a stable E1 homotrimer. Mutation of a highly conserved histidine (the H230A mutation) within a loop adjacent to the fusion loop was previously shown to block SFV fusion and infection, although the mutant E1 protein still inserts into target membranes and forms a homotrimer. Here we report on second-site mutations in E1 that rescue the H230A mutant. These mutations were located in a cluster within the hinge region, at the membrane-interacting tip, and within the groove where the E1 stem is believed to pack. Together the revertants reveal specific and interconnected aspects of the fusion protein refolding reaction

On-chip assessment of effective drug-target engagement on viable GEDI-captured CTCs.

<p>Tubulin response to taxane treatment can be assessed in GEDI-captured cells. (A) C4-2 prostate cancer cells were spiked at 200 cells/ml into healthy-donor whole blood. One ml of spiked blood was then flowed in each of three GEDI devices. Captured cells were incubated on each device at 37°C for 24 hr with either DMSO control (upper panel) or DTX 100 nM (middle panel) or 1 µM (lower panel). Following drug treatment, cells were fixed and processed for immunofluorescence staining using antibodies against tubulin and CD45. DAPI was used as a DNA counterstain to evaluate nuclear integrity. Note the fine and intricate microtubule network in the DMSO control (top panel) and the distinct microtubule bundles in the DTX treated devices (arrows, middle and bottom panels). Apoptotic nuclei were observed at higher DTX concentrations (arrowhead, bottom panel). (B) GEDI-captured CTCs from the blood of a CRPC patient were treated <i>ex vivo</i> on the GEDI device with 100 nM DTX (top panel) or the addition of 100 nM PTX (bottom panel) at 37°C for 24 hr. Following drug treatment the PSMA-captured cells were fixed and processed for immunofluorescence staining as in (A) with the addition of cytokeratin-18 as an alternative epithelial marker. In this patient, the presence of an unperturbed microtubule network following DTX treatment (top panel) indicates lack of efficient drug-target engagement. In contrast, addition of PTX resulted in microtubule bundling (bottom panel).</p

CTC enumeration using the GEDI and comparison to capture by Cell Search.

<p>(A) Representative images of circulating tumor cells captured with the GEDI device from 1 mL of blood from prostate cancer patients. CTCs are imaged on the device and are identified following immunostaining with DAPI, PSMA, CD45, and EpCAM. Intact, nucleated cells that are PSMA+/CD45− are identified as CTCs. Leucocytes are identified as DAPI+/PSMA−/CD45+ (bottom row, arrow). Note the heterogeneity of EpCAM expression in the PSMA+ cell population (top and bottom rows, EpCAM-; middle row, EpCAM+). Scale bar: 10 µm. (B) Disease-specific GEDI capture of CTCs. CTC enumeration (CTCs/ml) was performed using blood from healthy donors (median = 3) and CRPC patients (median = 54). (p<.001; Wilcoxon) (C) Comparison of CTC enumeration between GEDI-based- and CellSearch®-based capture. This comparison was performed using same-day blood draws from 25 individual CRPC patients. * indicates that CellSearch-based enumeration was performed 1 week before GEDI-based enumeration; ∉ indicates same patient whose blood was drawn on two separate time points three months apart (blood draw no 14 occurred 3 months after blood draw no 19); # indicates same patient whose blood was drawn on two separate time points 1 year apart (blood draw no 22 occurred 1 year before blood draw no 23).</p