2014 REU Poster: Quantifying Lipid Contents In Liposomes with Enveloped Plasmonic Nanoparticles

Poster presentation at REU Summer's End Research Symposium, 2014, by REU participant Beatrix Seytono, Calhoun Community College - Bjoern Reinhard group, Amin Feizpour lab mentor.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. We learn the binding infinity of PS and GM1 lipid by using gold nanoparticle (GNP) since it is stable and conductive. Besides, changing concentration of lipid on the virus can control how the virus infective. The performed studies can use identify unknown concentration of lipid.NSF-RE

Growth of Pseudotypes of Vesicular Stomatitis Virus with N-Tropic Murine Leukemia Virus Coats in Cells Resistant to N-Tropic Viruses

Formation of pseudotypes between murine RNA tumor viruses and vesicular stomatitis virus (VSV) has been confirmed. Pseudotypes of VSV genomes coated by the surface envelope from an N-tropic tumor virus grew equally well in cells homozygous for either the Fv-1n or Fv-1b alleles. Therefore, the product of the Fv-1 locus, which restricts growth of murine RNA tumor viruses, must act on an intracellular aspect of tumor virus replication, a step after attachment and penetration

Modeling Virus Transport and Removal during Storage and Recovery in Heterogeneous Aquifers

A quantitative understanding of virus removal during aquifer storage and recovery (ASR) in physically and geochemically heterogeneous aquifers is needed to accurately assess human health risks from viral infections. A two-dimensional axisymmetric numerical model incorporating processes of virus attachment, detachment, and inactivation in aqueous and solid phases was developed to systematically evaluate the virus removal performance of ASR schemes. Physical heterogeneity was considered as either layered or randomly distributed hydraulic conductivities (with selected variance and horizontal correlation length). Geochemical heterogeneity in the aquifer was accounted for using Colloid Filtration Theory to predict the spatial distribution of attachment rate coefficient. Simulation results demonstrate that the combined effects of aquifer physical heterogeneity and spatial variability of attachment rate resulted in higher virus concentrations in the recovered water at the ASR well (i.e. reduced virus removal). While the sticking efficiency of viruses to aquifer sediments was found to significantly influence virus concentration in the recovered water, the solid phase inactivation under realistic field conditions combined with the duration of storage phase had a predominant influence on the overall virus removal. The relative importance of physical heterogeneity increased under physicochemical conditions that reduced virus removal (e.g. lower value of sticking efficiency or solid phase inactivation rate). This study provides valuable insight on site selection of ASR projects and an approach to optimize ASR operational parameters (e.g. storage time) for virus removal and to minimize costs associated with post-recovery treatment

Use of a lambda gt11 expression library to localize a neutralizing antibody-binding site in glycoprotein E2 of Sindbis virus

The Sindbis virus envelope contains two species of integral membrane glycoproteins, E1 and E2. These proteins form heterodimers, and three dimeric units assemble to form spikes incorporated into the viral surface which play an important role in the specific attachment of Sindbis virus to host cells. To map the neutralization epitopes on the surface of the virus, we constructed a lambda gt11 expression library with cDNA inserts 100 to 300 nucleotides long obtained from randomly primed synthesis on Sindbis virus genomic RNA. This library was screened with five different neutralizing monoclonal antibodies (MAbs) specific for E2 (MAbs 50, 51, 49, 18, and 23) and with one neutralizing MAb specific for E1 (MAb 33). When 10(6) lambda gt11 plaques were screened with each antibody, four positive clones that reacted with E2-specific MAb 23 were found. These four clones contained overlapping inserts from glycoprotein E2; the domain from residues 173 to 220 of glycoprotein E2 was present in all inserts, and we concluded that this region contains the neutralization epitope recognized by the antibody. No clones that reacted with the other antibodies examined were found, and we concluded that these antibodies probably recognize conformational epitopes not present in the lambda gt11 library. We suggest that the E2 domain from residues 173 to 220 is a major antigenic determinant of Sindbis virus and that this domain is important for virus attachment to cells

On the Spread of Viruses on the Internet

We analyze the contact process on random graphs generated according to the preferential attachment scheme as a model for the spread of viruses in the Internet. We show that any virus with a positive rate of spread from a node to its neighbors has a non-vanishing chance of becoming epidemic. Quantitatively, we discover an interesting dichotomy: for it virus with effective spread rate λ, if the infection starts at a typical vertex, then it develops into an epidemic with probability λ^Θ ((log (1/ λ)/log log (1/ λ))), but on average the epidemic probability is λ^(Θ (1))

Cloning of hemagglutinin (HA) protein of influenza A virus - Potential for sialic acid linkage discrimination

The initial step in infection of a cell by influenza A virus is the attachment of a virus particle to the target cell. This is accomplished by interaction of a glycoprotein, hemagglutinin (HA), found on the surface of the viral lipid membrane with cell-surface oligosaccharides containing sialic acids. All influenza virus attachment requires terminal sialic acid residues and two major linkages between sialic acid (Neu5Ac) and the penultimate galactose (Gal) residues of carbohydrate side chains are found in nature, Neu5Ac(α2,3)-Gal and Neu5Ac(α2,6)-Gal. The HA’s of different subtypes of influenza A virus exhibit different recognition specificities for these linkages and these linkage specificities have been correlated with host range specificity. The ability of the HA protein to differentiate sialic acid linkages makes it an interesting candidate for use in the characterization of glycoprotein's potentially facilitating the discrimination of alternate glycoforms of biopharmaceutical therapeutics and their subsequent purification

Quantification and a Molecular Dynamics Study of Viral Membrane Lipids through Plasmon Coupling Microscopy

Phosphatidylserine (PS) and monosialotetrahexosylganglioside (G_M1) 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 G_M1 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. In addition, the mechanical properties of the viral membrane have been found to be contributing to the efficient reproduction cycle of the virus. Membrane fluidity which is a function of temperature and membrane composition is one of the crucial factors in viral activity. We have used temporally-resolved microscopy on silver NPs to track these molecular dynamics

Monomeric ephrinB2 binding induces allosteric changes in Nipah virus G that precede its full activation.

Nipah virus is an emergent paramyxovirus that causes deadly encephalitis and respiratory infections in humans. Two glycoproteins coordinate the infection of host cells, an attachment protein (G), which binds to cell surface receptors, and a fusion (F) protein, which carries out the process of virus-cell membrane fusion. The G protein binds to ephrin B2/3 receptors, inducing G conformational changes that trigger F protein refolding. Using an optical approach based on second harmonic generation, we show that monomeric and dimeric receptors activate distinct conformational changes in G. The monomeric receptor-induced changes are not detected by conformation-sensitive monoclonal antibodies or through electron microscopy analysis of G:ephrinB2 complexes. However, hydrogen/deuterium exchange experiments confirm the second harmonic generation observations and reveal allosteric changes in the G receptor binding and F-activating stalk domains, providing insights into the pathway of receptor-activated virus entry.Nipah virus causes encephalitis in humans. Here the authors use a multidisciplinary approach to study the binding of the viral attachment protein G to its host receptor ephrinB2 and show that monomeric and dimeric receptors activate distinct conformational changes in G and discuss implications for receptor-activated virus entry

Heparan sulfate proteoglycan is an important attachment factor for cell entry of Akabane and Schmallenberg viruses

Akabane (AKAV) and Schmallenberg (SBV) viruses are Orthobunyavirus transmitted by arthropod vectors with a broad cellular tropism in vitro as well as in vivo Both AKAV and SBV cause arthrogryposis-hydranencephaly syndrome in ruminants. The main cellular receptor and attachment factor for entry of these orthobunyaviruses are unknown. Here, we found that AKAV and SBV infections were inhibited by the addition of heparin or enzymatic removal of cell surface heparan sulfates. To confirm this finding, we prepared heparan sulfate proteoglycan (HSPG)-knockout (KO) cells by using a CRISPR/Cas9 system and measured the binding quantities of these viruses to cell surfaces. We observed a substantial reduction in AKAV and SBV binding to cells, limiting the infections by these viruses. These data demonstrate that HSPGs are important cellular attachment factors for AKAV and SBV, at least in vitro, to promote virus replication in susceptive cells. Importance: AKAV and SBV are the etiological agents of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic losses in the livestock industry. Here, we identified heparan sulfate proteoglycan as a major cellular attachment factor for the entry of AKAV and SBV. Moreover, we found that heparin is a strong inhibitor of AKAV and SBV infections. Revealing the molecular mechanisms of virus-host interactions is critical in order to understand virus biology and develop novel live attenuated vaccines

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 × 106 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.NIH grants RO1CA138509 (B.M.R.), RO1A1064099 (S. G., and 1R56Al104393 (B.M.R. and S. G.; Ethan Edmonds support (CHE 1156666