Effects of hydrostatic pressure on the stability and thermostability of poliovirus: a new method for vaccine preservation

Submitted by Repositório Arca ([email protected]) on 2019-03-07T16:42:53Z No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) F90-1-s2.0-S0264410X09009736-main.pdf: 543360 bytes, checksum: 46e5a708f50aedd123f781634f7ca772 (MD5)Approved for entry into archive by monique santos ([email protected]) on 2019-03-11T14:41:43Z (GMT) No. of bitstreams: 2 F90-1-s2.0-S0264410X09009736-main.pdf: 543360 bytes, checksum: 46e5a708f50aedd123f781634f7ca772 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-03-11T14:41:43Z (GMT). No. of bitstreams: 2 F90-1-s2.0-S0264410X09009736-main.pdf: 543360 bytes, checksum: 46e5a708f50aedd123f781634f7ca772 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Programa de Biologia Estrutural. Instituto de Bioquímica Médica e Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas Jiri Jonas. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Programa de Biologia Estrutural. Instituto de Bioquímica Médica e Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas Jiri Jonas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Programa de Biologia Estrutural. Instituto de Bioquímica Médica e Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas Jiri Jonas, Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Viruses are a structurally diverse group of infectious agents that differ widely in their sensitivities to high hydrostatic pressure (HHP). Studies on picornaviruses have demonstrated that these viruses are extremely resistant to HHP treatments, with poliovirus appearing to be the most resistant. Here, the three attenuated poliovirus serotypes were compared with regard to pressure and thermal resistance. We found that HHP does not inactivate any of the three serotypes studied (1–3). Rather, HHP treatment was found to stabilize poliovirus by increasing viral thermal resistance at 37 °C. Identification of new methods that stabilize poliovirus against heat inactivation would aid in the design of a more heat-stable vaccine, circumventing the problems associated with refrigeration during storage and transport of the vaccine prior to use

Fusion of a new world Alphavirus with membrane microdomains involving partially reversible conformational changes in the viral spike proteins

This work was supported by an international grant from International Centre for Genetic Engineering and Biotechnology (ICGEB) and by Brazilian grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Programa de Apoio ao Desenvolvimento Cientıfíco e Tecnológico (PADCT), and Programa de Apoio a Nucleos de Excelência (PRONEX).Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Centro de Ciências da Saúde. Rio de Janeiro, RJ, Brazil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brazil / Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brazil / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brazil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Centro de Ciências da Saúde. Rio de Janeiro, RJ, Brazil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Centro de Ciências da Saúde. Rio de Janeiro, RJ, Brazil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Centro de Ciências da Saúde. Rio de Janeiro, RJ, Brazil.Alphaviruses are enveloped arboviruses mainly proposed to infect host cells by receptor-mediated endocytosis followed by fusion between the viral envelope and the endosomal membrane. The fusion reaction is triggered by low pH and requires the presence of both cholesterol and sphingolipids in the target membrane, suggesting the involvement of lipid rafts in the cell entry mechanism. In this study, we show for the first time the interaction of an enveloped virus with membrane microdomains isolated from living cells. Using Mayaro virus (MAYV), a New World alphavirus, we verified that virus fusion to these domains occurred to a significant extent upon acidification, although its kinetics was quite slow when compared to that of fusion with artificial liposomes demonstrated in a previous work. Surprisingly, when virus was previously exposed to acidic pH, a condition previously shown to inhibit alphavirus binding and fusion to target membranes as well as infectivity, and then reneutralized, its ability to fuse with membrane microdomains at low pH was retained. Interestingly, this observation correlated with a partial reversion of low pH-induced conformational changes in viral proteins and retention of virus infectivity upon reneutralization. Our results suggest that MAYV entry into host cells could alternatively involve internalization via lipid rafts and that the conformational changes triggered by low pH in the viral spike proteins during the entry process are partially reversible

The structural dynamics of the flavivirus fusion peptide-membrane interaction.

Membrane fusion is a crucial step in flavivirus infections and a potential target for antiviral strategies. Lipids and proteins play cooperative roles in the fusion process, which is triggered by the acidic pH inside the endosome. This acidic environment induces many changes in glycoprotein conformation and allows the action of a highly conserved hydrophobic sequence, the fusion peptide (FP). Despite the large volume of information available on the virus-triggered fusion process, little is known regarding the mechanisms behind flavivirus-cell membrane fusion. Here, we evaluated the contribution of a natural single amino acid difference on two flavivirus FPs, FLA(G) ((98)DRGWGNGCGLFGK(110)) and FLA(H) ((98)DRGWGNHCGLFGK(110)), and investigated the role of the charge of the target membrane on the fusion process. We used an in silico approach to simulate the interaction of the FPs with a lipid bilayer in a complementary way and used spectroscopic approaches to collect conformation information. We found that both peptides interact with neutral and anionic micelles, and molecular dynamics (MD) simulations showed the interaction of the FPs with the lipid bilayer. The participation of the indole ring of Trp appeared to be important for the anchoring of both peptides in the membrane model, as indicated by MD simulations and spectroscopic analyses. Mild differences between FLA(G) and FLA(H) were observed according to the pH and the charge of the target membrane model. The MD simulations of the membrane showed that both peptides adopted a bend structure, and an interaction between the aromatic residues was strongly suggested, which was also observed by circular dichroism in the presence of micelles. As the FPs of viral fusion proteins play a key role in the mechanism of viral fusion, understanding the interactions between peptides and membranes is crucial for medical science and biology and may contribute to the design of new antiviral drugs

The secondary structures of FLA_G and FLA_H in the presence of membrane models.

<p>(A and B) The secondary structures of the fusion peptides FLA_G (A) and FLA_H (B) in the presence of POPE membranes at 35°C. (C and D) The circular dichroism spectra of the FLA_G (C) and FLA_H (D) FP in solution (solid line) and in the presence of SDS (dashed line) or n-OGP (dotted line) micelles. The experiments were performed at room temperature at pH 5.5.</p

Fluorescence spectroscopy data of FLA_G and FLA_H in the presence of micelles.

<p>All measurements were performed in triplicate in the same experiment, and the results were obtained from at least six independent experiments. ΔCM and S/S₀ are expressed as the mean ± SD.</p>[1]<p>Blue shift was determined by subtracting emission wavelength from control.</p

The secondary structure of peptides FLA_G and FLA_H in aqueous buffer.

<p>The secondary structure patterns of the fusion peptides FLA_G (A) and FLA_H (B), at 35°C and secondary structure patterns of the FP FLA_G (C) and FLA_H (D) at 85°C in solution. (E and F) The MD simulation of FLA_G (E) and FLA_H (F) in water at 35°C (red line) and at 85°C (black line). (G and H) The minimal distance between FLA_G (G) or FLA_H (H) Trp101 and Phe108 residues at 35°C (red line) and at 85°C (black line). (I and J) The circular dichroism spectra of the FLA_G (I) and FLA_H (J) fusion peptides in solution at 25°C (solid line), at 85°C (dashed line), and the return to 25°C (dotted line). The experiments were performed at room temperature at pH 5.5.</p

Molecular dynamics simulation of the interaction of the fusion peptides with the POPE membrane.

<p>Representative snapshots from simulations of FLA_G (A) and FLA_H (B) interaction with a POPE membrane at 35°C. The peptides are shown in light green, the membranes are in gray and, in each peptide, the side chains of residues Trp101 and Phe 108 are highlighted in dark green and blue, respectively.</p

Molecular dynamics studies of the POPE membrane environment.

<p>(A) The RMSD of FLA_G (black) and FLA_H (red). (B) The distance between the peptide and membrane was determined as follows: the distance between the center of mass of the peptide and the membrane in the axis perpendicular to the membrane surface plane for both FLA_G (black) and FLA_H (red); and the minimal distance between Gly104 (green) or His104 (blue) atoms and the phosphorus atoms of the lipids. (C and D) The number of intramolecular hydrogen bonds (black) and those formed between the fusion peptides FLA_G (C) or FLA_H (D) and the water (green) or the POPE membrane (red). (E and F) The minimal distance between the Trp101 residue and the POPE membrane (red) and between the Phe108 residue and the POPE membrane (black) during MD simulation in the presence of the POPE membrane at 35°C. The intermolecular distance between Trp101 and Phe108 is also presented (green). The results for MD simulation of FLA_G and FLA_H are shown in E and F, respectively.</p

The flavivirus E glycoprotein fusion loop and hydrophobicity plots of the fusion peptides.

<p>(A) The crystallographic structure of West Nile virus E protein (PDB ID 2HG0) and the schematic representation and sequence of the two fusion peptides of flaviviruses studied in this work. FLA_G has a Gly residue (red) in position 104 of glycoprotein E, while FLA_H presents a His residue (blue). Trp101, Gly104 and His104 are indicated in bold, red and blue, respectively. The conserved amino acids are underlined. (B) Hydrophobicity plots for the fusion peptides FLA_G (solid line) and FLA_H (dashed line) were elaborated using the Wimley-White hydrophobicity scale.</p