Rapid concentration and purification of retrovirus by flocculation with Polybrene

Landázuri, Natalia; Gupta, Monique; Doux, Joseph M. Le

doi:10.1016/j.jbiotec.2006.03.026

Cited by 14 publications

(11 citation statements)

References 22 publications

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“…If viral particles aggregate together, the inefficient diffusionlimited capture process may be bypassed in favor of much more rapid sedimentation of the virus to the cell surface by convective processes (17). This process has been shown to be effective in promoting retrovirus infection in vitro, where directed mass transfer of the virus to plated cells significantly enhances infection rates (11,17,(51)(52)(53). The effect of virus aggregation in the in vivo situation, where cell densities are much higher and target cells are in a three-dimensional matrix, is much less certain; however, the influence of PAP 248-286 /SEVI on virus aggregation and convective transport warrants further investigation (54,55).…”

Section: Discussionmentioning

confidence: 99%

Helical Conformation of the SEVI Precursor Peptide PAP248-286, a Dramatic Enhancer of HIV Infectivity, Promotes Lipid Aggregation and Fusion

Brender

Hartman

Gottler

et al. 2009

Biophysical Journal

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In previous in vivo studies, amyloid fibers formed from a peptide ubiquitous in human seminal fluid (semen-derived enhancer of viral infection (SEVI)) were found to dramatically enhance the infectivity of the HIV virus (3-5 orders of magnitude by some measures). To complement those studies, we performed in vitro assays of PAP(248-286), the most active precursor to SEVI, and other polycationic polymers to investigate the physical mechanisms by which the PAP(248-286) promotes the interaction with lipid bilayers. At acidic (but not at neutral) pH, freshly dissolved PAP(248-286) catalyzes the formation of large lipid flocculates in a variety of membrane compositions, which may be linked to the promotion of convective transport in the vaginal environment rather than transport by a random Brownian motion. Furthermore, PAP(248-286) is itself fusiogenic and weakens the integrity of the membrane in such a way that may promote fusion by the HIV gp41 protein. An alpha-helical conformation of PAP(248-286), lying parallel to the membrane surface, is implicated in promoting bridging interactions between membranes by the screening of the electrostatic repulsion that occurs when two membranes are brought into close contact. This suggests that nonspecific binding of monomeric or small oligomeric forms of SEVI in a helical conformation to lipid membranes may be an additional mechanism by which SEVI enhances the infectivity of the HIV virus.

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Section: Discussionmentioning

confidence: 99%

Helical Conformation of the SEVI Precursor Peptide PAP248-286, a Dramatic Enhancer of HIV Infectivity, Promotes Lipid Aggregation and Fusion

Brender

Hartman

Gottler

et al. 2009

Biophysical Journal

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“…The viral supernatant was concentrated by ultracentrifugation in a Beckman Optima XL‐100 centrifuge (Beckman Coulter Inc., Fullerton, CA, USA): 100 000 g for 1.5 h at 4 °C using a Beckman 45Ti rotor 8, 17. The pelleted virus was resuspended in storage buffer [Tris (Calbiochem, Darmstadt, Germany), pH 7.2, 10 m M MgCl 2 (Merck) and 0.01% Tween 80 (Merck)] and purified by centrifugation on a 20% (w/v) sucrose (Fluka, Steinheim, Germany) solution at 200 000 g for 2 h at 4 °C using a Beckman 90Ti rotor 8, 18. The final virus pellet was resuspended in storage buffer or in storage buffer with a stabilizing agent.…”

Section: Methodsmentioning

confidence: 99%

Stabilization of gammaretroviral and lentiviral vectors: from production to gene transfer

Carmo

Alves

Rodrigues

et al. 2009

The Journal of Gene Medicine

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The presented study reveals that it is possible to increase the half-life of purified gammaretroviral and lentiviral vectors at 37 degrees C and at 4 degrees C, but the two vectors have different stabilization requirements: for retroviral vectors, the addition of rHSA is enough and, for lentiviral vectors, it is necessary to add both lipoproteins and rHSA. The increase of the stability of the reverse transcription process was shown to have a high impact with respect to the increase of the stability of infectivity.

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“…Transfections were with the calcium phosphate coprecipitation method. Forty-eight hours after transfection, virus supernatant was harvested and concentrated by precipitation with chondroitin 6-sulfate (CSC) and polybrene (both Sigma) and centrifuged [33].…”

Section: Cells and Cell Culturementioning

confidence: 99%

The Polycomb Protein Ezh2 Impacts on Induced Pluripotent Stem Cell Generation

DingXiaolei

WangXiaoying

SontagStephanie

et al. 2014

Stem Cells and Development

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Rapid concentration and purification of retrovirus by flocculation with Polybrene

Cited by 14 publications

References 22 publications

Helical Conformation of the SEVI Precursor Peptide PAP248-286, a Dramatic Enhancer of HIV Infectivity, Promotes Lipid Aggregation and Fusion

Helical Conformation of the SEVI Precursor Peptide PAP248-286, a Dramatic Enhancer of HIV Infectivity, Promotes Lipid Aggregation and Fusion

Stabilization of gammaretroviral and lentiviral vectors: from production to gene transfer

The Polycomb Protein Ezh2 Impacts on Induced Pluripotent Stem Cell Generation

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