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Dengue Project

PTDC/QUI/69937/2006 - O papel das membranas lipidicas na infecção celular pelo vírus de Dengue. (The role of lipid membranes in Dengue virus cell infection)


PTDC/QUI-BIQ/112929/2009 - O papel de membranas lipídicas hepáticas e corpúsculos lipídicos na encapsidação do vírus da Dengue. Uma via para novas terapias. (The role of hepatic lipid membranes and lipid droplets in Dengue virus encapsidation. A gateway to new therapies.


Resumo
A febre hemorrágica de Dengue é causada por diferentes vírus, sendo uma ameaça muito séria para a saúde pública. De todos os agentes causadores, a infecção pelo vírus da Dengue (DV) é a mais frequente. Cerca de dois terços da população mundial vive em regiões onde a febre hemorrágica de Dengue é endémica e cerca de 80 milhões de pessoas são infectadas todos os anos, levando a um total estimado em 20000 mortos anuais. De acordo com a Organização Mundial de Saúde, o número de casos de febre de Dengue só no Brasil, por exemplo, triplicou nos últimos cinco anos, com cerca de 700 mortes no mesmo período.
Uma vez que não existe disponível qualquer tratamento ou vacina, é essencial conhecer os mecanismos moleculares envolvidos no ciclo de vida do vírus para que se possa num futuro próximo desenvolver tratamentos anti-virais. O presente projecto é uma contribuição para a elucidação do papel das membranas lipídicas na infecção viral. A importância das membranas é muitas vezes menorizada, se não mesmo esquecida, mas o resultado final deste projecto abrirá novas oportunidades terapêuticas ao permitir o desenvolvimento de novos fármacos que tenham como alvo a fusão do vírus e/ou a formação da cápside, processos que envolvem directamente a interacção de proteínas com membranas.
O projecto reúne especialistas de três equipas diferentes, cada uma delas com um papel distinto, mas complementares entre si. A equipa da Universidade Federal do Rio de Janeiro/Instituto de Bioquímica Médica (Brasil) é perita em biologia molecular de vírus, com ênfase para o DV, entre outros. As equipas do instituto de Medicina Molecular (Faculdade de Medicina de Lisboa) são peritas na aplicação de técnicas espectroscópicas de dispersão de luz e Microscopia de Forças Atómicas a estudos biomédicos e na aplicação de espectroscopias ópticas a estudos estruturais de sistemas modelo de membranas biológicas. Daí resulta que o projecto consiste numa reunião ecléctica de “know-how” para estudar a interacção das proteínas E e C do DV com membranas de lípidos em condições experimentais de relevância biológica. A proteína E é responsável pela fusão do vírus com a membrana da célula-alvo e a proteína C é a proteína da cápside, que interage com membranas e tem um segmento activo em membranas na sua sequência. Descobrir a forma como estas moléculas interagem entre si e se organizam quando inseridas ou adsorvidas em bicamadas lípidicas é o primeiro passo para o desenvolvimento de estratégias para o seu controlo em projectos subsequentes.
A equipa brasileira domina as metodologias de biologia molecular necessárias para obter as proteínas C e E, que serão usadas nos estudos de Microscopia de Força Atómica acoplada a microscopia de fluorescência, espectroscopia de força, dispersão de luz e espectroscopias ópticas (fluorescência, dicroísmo circular e FT-IR). As outras duas equipas desenvolveram recentemente metodologias para estudar a interacção entre bicamadas lípidicas e péptidos. O projecto tira partido da combinação das especificidades das equipas envolvidas


Abstract

2006 - Viral hemorrhagic fever is a clinical syndrome caused by different viruses, posing a serious global health threat. Among the causative agents, dengue virus (DV) infection has the highest incidence rate and is the major cause of viral hemorrhagic fever in the world. About two-thirds of the world population lives in regions where dengue fever is endemic, and roughly 80 million people are infected with the virus every year, leading to an estimated 20 thousand deaths annually. According to the World Health Organization (WHO), the number of dengue fever cases in Brazil, for instance, increased three times in the last five years, with about 700 deaths in the same period. Since no treatment or commercial vaccine against DV-caused diseases are available, a detailed understanding of the molecular mechanisms involved in the life cycle of the virus is essential to the future development of anti-viral treatments and new therapies. This project is a contribution towards the elucidation of the role of lipidic membranes in viral infection. The importance of membranes is frequently underestimated or even overlooked but this project opens a gateway to the future development of fusion and/or capsid assembly-targeted drugs and therapies. The project gathers the expertise of three different teams, each having a very distinct, yet synergistic, role. The team from the Federal University of Rio de Janeiro / Medical Biochemistry Institute (Brazil) has expertise in molecular biology of viruses, with emphasis on DV, among others. The teams from the Institute of Molecular Medicine (Medical School, Lisbon) have expertise in the application of light scattering spectroscopy and Atomic Force Microscopy to biomedical studies, as well as expertise in the application of optical spectroscopies to structural studies involving lipid bilayers. Therefore, this project consists of an eclectic gathering of expertise to study the interaction of the E and C proteins of DV with lipid membranes in experimental conditions of biological relevance. The E protein is responsible for virus fusion with the target cell membrane and the C protein is the capsid protein, which has a membrane-active segment in its sequence. Finding the way these molecules interact and organize upon interaction in membranes is the first step towards the development of strategies for its control. The Brazilian team masters the molecular biology methodologies needed to obtain the E and C proteins that will be studied later using a combination of Atomic Force microscopy coupled to fluorescence imaging, force spectroscopy, light scattering and optical spectroscopies (fluorescence, circular dichroism and FT-IR). The other teams have developed in the recent years methodologies to study the interaction between lipid bilayers and peptides. The project takes advantage from the combination of the specificities of the teams involved.


2009 - Viral hemorrhagic fever is a serious health threat mostly caused by the Dengue virus (DV), a member of the Flavivirus genus, Flaviviridae family. DV infection occurs via Aedes spp. mosquitoes. This vector, previously confined to tropical and subtropical regions, is now spreading, due to global warming and increased movement of people and goods, being now present in Portugal and other European countries. In spite of the increasing global reach and the resulting media coverage, Dengue is still a seriously neglected disease, for which after infection occurs, no specific and effective treatment is currently available. One of the reasons is the lack of detailed knowledge on the viral life cycle, namely relating the molecular mechanisms of viral assembly. One of the key steps of this process involves the Dengue virus capsid protein (DVCP) interaction with RNA. Lipids have recently been proposed to have an important role in the mechanism of DV assembly and encapsidation, although their contribution for the process is still not fully understood. The most accepted pathways for dengue virus expression and assembly involves the endoplasmic reticulum (ER). DVCP interaction with lipid droplets was also recently documented. Lipid droplets are ER derived organelles that consist on storage depots for neutral lipids. This project aims to fill that gap in the understanding of the DV life cycle by studying DVCP in the context of interaction with RNA and lipid membranes. DVCP interaction with lipid systems will be fully characterized in biophysical terms, using lipid bilayers mimicking ER membranes and lipid droplets directly isolated from hepatic cell lines. In order to achieve this purpose, an eclectic gathering of complementary expertise and know-how of the participants in the research effort proposed here has been assembled. In this project we will use fluorescence spectroscopy and atomic force microscopy (AFM) to determine the affinity of DVCP, in the presence and absence RNA, towards lipid membranes and droplets. Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies will determine the structural features of the protein complexed with lipids and/or RNA. Information about DVCP oligomerization and aggregation will be given by light scattering and zeta-potential studies. The results obtained will allow us to establish the preferential site of viral assembly, ER or lipid droplets, and the specific structural features of DVCP governing lipid and RNA binding. This will provide a ground for the future development of drugs and/or therapies aimed at the DVCP-mediated mechanism of viral assembly.

 



 

Av. Professor Egas Moniz
Edifício Egas Moniz,
1649-028 Lisboa
Portugal
 

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