Amin Feizpour

Glycosphingolipid-functionalized nanoparticles recapitulate CD169-dependent HIV-1 uptake and trafficking in dendritic cells

Ganglioside GM3, a host-derived glycosphingolipid incorporated in the membrane of HIV-1 viral particles, mediates interactions between HIV-1 and Siglec1/CD169, a protein expressed on dendritic cells (DCs). Such interactions, which seem to be independent of viral envelope glycoprotein gp120, are poorly understood. Here we develop a model system consisting of self-assembled artificial virus nanoparticles (AVNs) that are free of viral glycoproteins or other host-derived glycolipids and glycoproteins. These plasmonic AVNs contain a membrane of defined composition wrapped around a solid metal core. GM3-containing AVNs are captured by CD169-expressing HeLa cells or mature DCs, and are sequestered within non-lysosomal tetraspanin-positive compartments. This distribution is reminiscent of CD169-dependent HIV-1 sequestration in mature DCs. Our results highlight GM3-CD169 binding as a gp120-independent signal for sequestration and preservation of HIV-1 infectivity. They also indicate that plasmonic AVNs offer improved features over liposome-based systems and represent a versatile tool for probing specific virus-cell interactions.

Illuminating the Lateral Organization of Cell-Surface CD24 and CD44 through Plasmon Coupling between Au Nanoparticle Immunolabels

CD44 and CD24 are important cell surface glycoproteins whose relative expression levels are used to identify so-called cancer stem cells (CSCs). While current diagnostic applications of CD44 and CD24 focus primarily on their expression levels, we demonstrate here that noble metal nanoparticle (NP) immunolabeling in combination with plasmon coupling microscopy (PCM) can reveal more subtle differences, such as the spatial organization of these surface species on subdiffraction limit length scales. We quantified both expression and spatial clustering of CD44 and CD24 on MCF7 and SKBR3 breast cancer cells through analysis of the labeling intensity and the electromagnetic coupling of the NP labels, respectively. The labeling intensity was well correlated with the receptor expression, but the inspection of the labeled cell surface in the optical microscope revealed that the NP immunolabels were not homogeneously distributed. Consistent with a heterogeneous spatial distribution of the targeted CD24 and CD44 in the plasma membrane, a significant fraction of the NPs were organized into clusters, which were easily detectable in the optical microscope as discrete spots with colors ranging from green to orange. To further quantify the spatial organization of the targeted proteins, we characterized individual NP clusters through spatially resolved elastic scattering spectroscopy. The statistical analysis of the single cluster spectra revealed a higher clustering affinity for CD24 than for CD44 in the investigated cancer models. This preferential clustering was removed upon lipid raft disruption through cholesterol sequestration. Overall, these observations confirm a preferential enrichment of CD24 in lipid rafts and a more random distribution of CD44 in the plasma membrane as cause for the observed differences in protein clustering.

Plasmonic nanoshell functionalized etched fiber Bragg gratings for highly sensitive refractive index measurements

A novel fiber optical refractive index sensor based on gold nanoshells immobilized on the surface of an etched single-mode fiber including a Bragg grating is demonstrated. The nanoparticle coating induces refractive index dependent waveguide losses, because of the variation of the evanescently guided part of the light. Hence the amplitude of the Bragg reflection is highly sensitive to refractive index changes of the surrounding medium. The nanoshell functionalized fiber optical refractive index sensor works in reflectance mode, is suitable for chemical and biochemical sensing, and shows an intensity dependency of 4400% per refractive index unit in the refractive index range between 1.333 and 1.346. Furthermore, the physical length of the sensor is smaller than 3 mm with a diameter of 6 μm, and therefore offers the possibility of a localized refractive index measurement.

Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins

ABSTRACT Gammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called “glycogag” (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes. IMPORTANCE Many murine leukemia viruses (MLVs) encode a protein called “glycogag.” The function of glycogag is not fully understood, but it can assist HIV-1 replication in the absence of the HIV-1 protein Nef under some circumstances. In turn, Nef counteracts the cellular protein Serinc5. Glycogag enhances the infectivity of MLVs with some but not all MLV Env proteins (which mediate viral entry into the host cell upon binding to cell surface receptors). We now report that glycogag acts by enhancing viral entry and that, like Nef, glycogag antagonizes Serinc5. Surprisingly, the effects of glycogag and Serinc5 upon the entry and infectivity of MLV particles carrying an Ebolavirus glycoprotein are the opposite of those observed with the MLV Env proteins. The unrelated S2 protein of equine infectious anemia virus (EIAV) is functionally analogous to glycogag in our experiments. Thus, three retroviruses (HIV-1, MLV, and EIAV) have independently evolved accessory proteins that counteract Serinc5. Many murine leukemia viruses (MLVs) encode a protein called “glycogag.” The function of glycogag is not fully understood, but it can assist HIV-1 replication in the absence of the HIV-1 protein Nef under some circumstances. In turn, Nef counteracts the cellular protein Serinc5. Glycogag enhances the infectivity of MLVs with some but not all MLV Env proteins (which mediate viral entry into the host cell upon binding to cell surface receptors). We now report that glycogag acts by enhancing viral entry and that, like Nef, glycogag antagonizes Serinc5. Surprisingly, the effects of glycogag and Serinc5 upon the entry and infectivity of MLV particles carrying an Ebolavirus glycoprotein are the opposite of those observed with the MLV Env proteins. The unrelated S2 protein of equine infectious anemia virus (EIAV) is functionally analogous to glycogag in our experiments. Thus, three retroviruses (HIV-1, MLV, and EIAV) have independently evolved accessory proteins that counteract Serinc5.

Nanoconjugation: a materials approach to enhance epidermal growth factor induced apoptosis

Apoptosis evasion is a hallmark of cancer that motivates the development of novel strategies for inducing cell death in a controlled fashion. The size-compatibility of nanoparticles (NPs) with cellular components provides new opportunities for regulating cellular processes, potentially including apoptosis. We investigated the impact of the covalent attachment of epidermal growth factor (EGF) to 40 nm diameter Au NPs on cellular apoptosis levels, quantified as caspase-3 activity, in two in vitro cancer cell lines: A431 and HeLa. Our studies show that nanoconjugation enhances EGF-induced apoptosis in EGF receptor (EGFR) overexpressing A431 and triggers a quantifiable increase in apoptosis in HeLa. The latter has physiological receptor expression levels and does not show apoptosis in response to free EGF. Endocytosis and trafficking are involved in key EGFR regulation processes, most prominently signal termination. Our experimental findings indicate that these processes can be manipulated through nanoconjugation to induce apoptosis.

Quantifying Lipid Contents in Enveloped Virus Particles with Plasmonic Nanoparticles

Phosphatidylserine (PS) and monosialotetrahexosylganglioside (GM1 ) are examples of two host-derived lipids in the membrane of enveloped virus particles that are known to contribute to virus attachment, uptake, and ultimately dissemination. A quantitative characterization of their contribution to the functionality of the virus requires information about their relative concentrations in the viral membrane. Here, a gold nanoparticle (NP) binding assay for probing relative PS and GM1 lipid concentrations in the outer leaflet of different HIV-1 and Ebola virus-like particles (VLPs) using sample sizes of less than 3 × 10(6) particles is introduced. The assay evaluates both scattering intensity and resonance wavelength, and determines relative NP densities through plasmon coupling as a measure for the target lipid concentrations in the NP-labeled VLP membrane. A correlation of the optical observables with absolute lipid contents is achieved by calibration of the plasmon coupling-based methodology with unilamellar liposomes of known PS or GM1 concentration. The performed studies reveal significant differences in the membrane of VLPs that assemble at different intracellular sites and pave the way to an optical quantification of lipid concentration in virus particles at physiological titers.

Membrane Fluidity Sensing on the Single Virus Particle Level with Plasmonic Nanoparticle Transducers

Viral membranes are nanomaterials whose fluidity depends on their composition, in particular, the cholesterol (chol) content. As differences in the membrane composition of individual virus particles can lead to different intracellular fates, biophysical tools capable of sensing the membrane fluidity on the single-virus level are required. In this manuscript, we demonstrate that fluctuations in the polarization of light scattered off gold or silver nanoparticle (NP)-labeled virus-like-particles (VLPs) encode information about the membrane fluidity of individual VLPs. We developed plasmonic polarization fluctuation tracking microscopy (PFTM) which facilitated the investigation of the effect of chol content on the membrane fluidity and its dependence on temperature, for the first time on the single-VLP level. Chol extraction studies with different methyl-β-cyclodextrin (MβCD) concentrations yielded a gradual decrease in polarization fluctuations as a function of time. The rate of chol extraction for individual VLPs showed a broad spread, presumably due to differences in the membrane composition for the individual VLPs, and this heterogeneity increased with decreasing MβCD concentration.

Access of HIV-2 to CD169-dependent dendritic cell-mediated trans infection pathway is attenuated.

The mechanisms behind the low viral loads and lower mortality rates of HIV-2(+) individuals remain unknown. We hypothesized that reduced interaction of HIV-2 with CD169, the primary HIV-1 attachment factor on monocyte-derived dendritic cells (DCs) that targets captured virus particles to the trans infection pathway, contributes to its diminished pathogenic phenotype in vivo. We observed a significant decrease in capture of HIV-2 Gag-eGFP virus-like particles (VLPs) and infectious GFP-containing HIV-2 particles compared to corresponding HIV-1 particles by CD169(+) mature DCs. Interestingly, there was decreased co-localization of HIV-2 with HIV-1 Gag at plasma membrane microdomains in virus producer cells which correlated with reduced incorporation of GM3, the CD169 ligand, in HIV-2 virions, and reduction in mature DC-mediated HIV-2 trans infection compared to HIV-1. We conclude that limited interaction of HIV-2 with CD169 diminishes virus access to the mature DC-mediated trans infection pathway and might result in attenuated HIV-2 dissemination in vivo.

Approximating the Concentration of Lipids on the Surface of Virus-Like Particles through Plasmon Coupling

The superior optical properties of gold nanoparticles were used to estimate the concentration of two crucial lipids on virus surface. This is a novel high-throughput and rapid surface characterization approach for viruses in low-concentration samples.

Probing the nano-bio interface with nanoplasmonic optical probes

Noble metal nanoparticles have large cross-sections in both optical and electron microscopy and plasmon coupling between noble metal nanoparticles facilitate the characterization of subdiffraction limit separations through spectral analysis of the scattered light in Plasmon Coupling Microscopy (PCM). The size compatibility of noble metal nanoparticles together with the ability to encode specific functionality in a rational fashion by control of the nanoparticle surface makes noble metal nanoparticles unique probes for a broad range of biological processes. Recent applications of the technology include i.) characterization of cellular heterogeneity in nanomaterial uptake and processing through macrophages, ii.) testing the role of viral membrane lipids in mediating viral binding and trafficking, and iii.) characterizing the spatial organization of cancer biomarkers in plasma membranes. This paper reviews some of these applications and introduces the physical and material science principles underlying them. We will also introduce the use of membrane wrapped noble metal nanoparticles, which combine the superb photophysical properties of a nanoparticle core with the biological functionality of a membrane, as probes in PCM.