Periodic Report Summary - DENGUE VIRUS CAPSID (The role of lipid membranes in dengue virus assembly)
Abstract
Viral hemorrhagic fever is a serious health threat mostly caused by the Dengue virus (DV), which infects 50 to 100 million people worldwide yearly. Aedes spp. mosquitoes, the disease vectors endemic to tropical and subtropical regions, are spreading to temperate regions, being now found in several European countries. DV infection is thus a potential clinical problem for Europe, as stated in the EU FP7 Emerging Epidemics program. Once infected, no specific treatment is available, partially due to the lack of detailed information on the molecular mechanisms of viral assembly. The project intends to fill that gap by studying the viral assembly in biologically relevant conditions, using the capsid protein and lipid bilayers mimicking biological membranes and intracellular lipid droplets isolated from hepatic cell lines. Following this approach, we expect to open a gateway to the future development of capsid assembly-targeted therapies.
Project objectives
The overall endeavour of the proposed research remains the same, i.e. to contribute to the increase in knowledge on the Dengue virus capsid protein (DVCP) and its mechanism of action at the molecular level, as originally detailed in the application. Briefly, several lines of research point to a mechanism where lipids, especially lipid droplets (which are intracellular lipid deposits), play a significant role in dengue virus encapsidation and assembly. In the closely related Hepatitis C virus, the capsid protein interacts specifically with intracellular lipid droplets in the host cell, affecting viral replication and infection and being essential for viral assembly and release. These findings clearly suggest a role for lipids in DVCP mediated viral RNA assembly and encapsidation, possibly via interaction with intracellular lipid droplets. As such, and in agreement with the original proposal, the characterisation of the molecular details of DVCP interaction with lipid droplets has been performed, as further detailed below.
Description of work performed
Up to now, the work has been focused on the characterisation of the molecular details of the interaction between the DVCP capsid protein and lipids, using lipid droplets directly isolated from hepatic cell lines. Biophysical techniques were employed, namely Circular dichroism (CD), Nuclear magnetic resonance (NMR) and zeta potential analysis, complemented with simulations. CD data is collected on a Chirascan plus spectrometer (Applied Photophysics, UK). NMR employs uniformly labelled 15N DVCP protein on a Bruker Avance III 800 (Rheinstetten, Germany) spectrometer operating at 800 MHz. Zeta potential analysis is performed on a Malvern Zetasizer Nano ZS (Malvern, UK). Membrane integration is simulated via the MAPAS server (http://cancer-tools.sdsc.edu/MAPAS/pro2.html).
Main present public results
DVCP interacts with lipid droplets with a minor decrease in alpha-helical content observed by CD, consistent with interactions in alpha-helical regions, as suggested by NMR. NMR also shows that the interaction occurs via specific amino acids. The interaction is likely initiated at the N-terminal and/or the alpha2-alpha2' hydrophobic interface according to the simulation of binding of the DVCP protein NMR structure to model membranes. Following, conformational rearrangements may occur, exposing the positively charged alpha4-alpha4' interface C-terminal region, as suggested by zeta potential analysis. This later rearrangement may prompt the viral RNA to bind the protein, leading the dengue virus assembly and encapsidation process further.
Expected final results and potential impact and use
The DVCP lipid droplets interaction sites are putative targets for drug/vaccine development. It is anticipated that by blocking this interaction, the viral assembly and encapsidation process can be disrupted, diminishing the viral load in vivo.
Viral hemorrhagic fever is a serious health threat mostly caused by the Dengue virus (DV), which infects 50 to 100 million people worldwide yearly. Aedes spp. mosquitoes, the disease vectors endemic to tropical and subtropical regions, are spreading to temperate regions, being now found in several European countries. DV infection is thus a potential clinical problem for Europe, as stated in the EU FP7 Emerging Epidemics program. Once infected, no specific treatment is available, partially due to the lack of detailed information on the molecular mechanisms of viral assembly. The project intends to fill that gap by studying the viral assembly in biologically relevant conditions, using the capsid protein and lipid bilayers mimicking biological membranes and intracellular lipid droplets isolated from hepatic cell lines. Following this approach, we expect to open a gateway to the future development of capsid assembly-targeted therapies.
Project objectives
The overall endeavour of the proposed research remains the same, i.e. to contribute to the increase in knowledge on the Dengue virus capsid protein (DVCP) and its mechanism of action at the molecular level, as originally detailed in the application. Briefly, several lines of research point to a mechanism where lipids, especially lipid droplets (which are intracellular lipid deposits), play a significant role in dengue virus encapsidation and assembly. In the closely related Hepatitis C virus, the capsid protein interacts specifically with intracellular lipid droplets in the host cell, affecting viral replication and infection and being essential for viral assembly and release. These findings clearly suggest a role for lipids in DVCP mediated viral RNA assembly and encapsidation, possibly via interaction with intracellular lipid droplets. As such, and in agreement with the original proposal, the characterisation of the molecular details of DVCP interaction with lipid droplets has been performed, as further detailed below.
Description of work performed
Up to now, the work has been focused on the characterisation of the molecular details of the interaction between the DVCP capsid protein and lipids, using lipid droplets directly isolated from hepatic cell lines. Biophysical techniques were employed, namely Circular dichroism (CD), Nuclear magnetic resonance (NMR) and zeta potential analysis, complemented with simulations. CD data is collected on a Chirascan plus spectrometer (Applied Photophysics, UK). NMR employs uniformly labelled 15N DVCP protein on a Bruker Avance III 800 (Rheinstetten, Germany) spectrometer operating at 800 MHz. Zeta potential analysis is performed on a Malvern Zetasizer Nano ZS (Malvern, UK). Membrane integration is simulated via the MAPAS server (http://cancer-tools.sdsc.edu/MAPAS/pro2.html).
Main present public results
DVCP interacts with lipid droplets with a minor decrease in alpha-helical content observed by CD, consistent with interactions in alpha-helical regions, as suggested by NMR. NMR also shows that the interaction occurs via specific amino acids. The interaction is likely initiated at the N-terminal and/or the alpha2-alpha2' hydrophobic interface according to the simulation of binding of the DVCP protein NMR structure to model membranes. Following, conformational rearrangements may occur, exposing the positively charged alpha4-alpha4' interface C-terminal region, as suggested by zeta potential analysis. This later rearrangement may prompt the viral RNA to bind the protein, leading the dengue virus assembly and encapsidation process further.
Expected final results and potential impact and use
The DVCP lipid droplets interaction sites are putative targets for drug/vaccine development. It is anticipated that by blocking this interaction, the viral assembly and encapsidation process can be disrupted, diminishing the viral load in vivo.