Final Report Summary - VIRUS ENTRY (Molecular Mechanisms of Cell Entry of Enveloped Viruses)
Virus infections are a major cause of diseases and death among men and animals. To combat virus infection and propagation, systematic and comprehensive studies both on viral components mediating virus-cell interactions, and on the cell biology behind virus entry are necessary. The network Virus Entry (https://www2.hu-berlin.de/virusentry/) comprising groups with complementary interdisciplinary and inter-sector expertise, was aimed at undertaking a joined effort towards systematic understanding molecular mechanisms behind virus entry by using techniques of molecular biology, structural biology, biochemistry, proteomics, experimental and theoretical biophysics and applying them to human and animal pathogen enveloped viruses. The participating members of the network were Ineke Brakmann and Peter Rottier (both The Netherlands), Felix Rey and Yves Gaudin (both France), Ari Helenius (Switzerland), Misha Kozlov (Israel), Henrik Garoff (Sweden), Michael Veit and Andreas Herrmann (both Germany)the two companies Capsulution (Germany) and Vironova (Sweden) .
The network has made highly relevant studies on the interaction of viruses with cells and various steps in the entry process for a number of different viruses using technologies ranging from high-through put screening to imaging of virus movements in live cells. The studies resulted in many new insights especially regarding the role of host cell factors in entry and virus fusion and uncoating after endocytosis. Novel technologies for studying the earliest phases of virus infection, i.e. receptor binding, virus uptake and membrane fusion were established. New insights about the host cell requirements for successful cell entry, for example for coronavirus about the processing events that trigger the conformational rearrangements of the viral fusion protein, and about the fusion-active form of this protein were generated. The information will be essential for developing infection prevention and treatment methods. Complementary approaches with molecular resolution reveal a variety of binding pathways that indicate a highly dynamic interaction between the influenza virus glycoprotein hemagglutinin and its receptor allowing to rationalize the binding of influenza virus to host cells quantitatively at molecular level and transmission to other hosts, i.e. from avian to humans. Apart from adaptation of the receptor binding site of the host also an adaptation of the fusion pH has to occur in order to allow the virus to circulate in a specific host.
For enveloped viruses being focus of the network an essential step of infection is the fusion between the viral envelope and the target membrane of the host cell releasing the viral genome into the cell. This is triggered by a conformational change of virus spike proteins. Several groups could characterize the structure of those proteins and their conformational dynamics for several viruses. For the first time, a detailed molecular scenario of the conformational changes and respective intermediates of a viral fusion protein was identified and described for the homotrimeric G glycoprotein of rhabodviruses. The major conclusion is that along the conformational change the G protein adopts an intermediate monomeric organization and forms subsequently a crystalline like network on the target membrane. The conformational changes of the trimeric HIV-1 Env spike protein turned out to be a cooperative process. It is suggested that this model of an obligatory coordinated spike protomer activation is also applicable to oligomeric viral fusion proteins of other viruses. This observation provides new therapeutical approaches to inhibit the fusion triggering conformational change and thus infection.
An interesting target for infection inhibition is synthesis and folding of virus spike proteins becoming integrated into new virions. The network revealed the inhibitory mechanism of co-translational and the triggering mechanism of post-translational signal peptide cleavage of HIV-1 Env and its importance for HIV-1, and novel and known chaperones as factors affecting spike protein folding. This can now be used for rational design of compounds or peptides blocking spike protein maturation and, hence, formation of new infectious viruses.
Understanding essential steps of virus infection as membrane fusion and membrane bending and budding of newly forming viruses require detailed molecular analysis and modeling. For example, membrane shaping consists of two consecutive geometrical transformations: generation of curvature and remodeling of curvature by fusion or fission. In a joint experimental and theoretical approach it was demonstrated her that insertion of hydrophobic protein domains into the membrane matrix can drive membrane fission in addition to bending while protein scaffolds favor membrane fusion in addition to the curvature generation.
Viral strategies for application in nanobiotechnology and –medicine have been exploited. Recent research has indicated the potential of virus protein decorated multicoloured Layer-by-Layer-technology (LbL)- particles as a diagnostic tool for detecting virus specific antibodies. In projects are planned to develop and optimise enveloping of LbL-particles with lipid bilayers containing reconstituted virus proteins.
An approach has been developed using immunoliposomes that can bind HIV-1 virus-like particles (HIV-VLPs) while being specifically phagocytosed by macrophages, thus allowing the co-internalization of HIV-VLPs. This could be a potential approach for efficient removal of HIV-1 and other viruses.
The network has made highly relevant studies on the interaction of viruses with cells and various steps in the entry process for a number of different viruses using technologies ranging from high-through put screening to imaging of virus movements in live cells. The studies resulted in many new insights especially regarding the role of host cell factors in entry and virus fusion and uncoating after endocytosis. Novel technologies for studying the earliest phases of virus infection, i.e. receptor binding, virus uptake and membrane fusion were established. New insights about the host cell requirements for successful cell entry, for example for coronavirus about the processing events that trigger the conformational rearrangements of the viral fusion protein, and about the fusion-active form of this protein were generated. The information will be essential for developing infection prevention and treatment methods. Complementary approaches with molecular resolution reveal a variety of binding pathways that indicate a highly dynamic interaction between the influenza virus glycoprotein hemagglutinin and its receptor allowing to rationalize the binding of influenza virus to host cells quantitatively at molecular level and transmission to other hosts, i.e. from avian to humans. Apart from adaptation of the receptor binding site of the host also an adaptation of the fusion pH has to occur in order to allow the virus to circulate in a specific host.
For enveloped viruses being focus of the network an essential step of infection is the fusion between the viral envelope and the target membrane of the host cell releasing the viral genome into the cell. This is triggered by a conformational change of virus spike proteins. Several groups could characterize the structure of those proteins and their conformational dynamics for several viruses. For the first time, a detailed molecular scenario of the conformational changes and respective intermediates of a viral fusion protein was identified and described for the homotrimeric G glycoprotein of rhabodviruses. The major conclusion is that along the conformational change the G protein adopts an intermediate monomeric organization and forms subsequently a crystalline like network on the target membrane. The conformational changes of the trimeric HIV-1 Env spike protein turned out to be a cooperative process. It is suggested that this model of an obligatory coordinated spike protomer activation is also applicable to oligomeric viral fusion proteins of other viruses. This observation provides new therapeutical approaches to inhibit the fusion triggering conformational change and thus infection.
An interesting target for infection inhibition is synthesis and folding of virus spike proteins becoming integrated into new virions. The network revealed the inhibitory mechanism of co-translational and the triggering mechanism of post-translational signal peptide cleavage of HIV-1 Env and its importance for HIV-1, and novel and known chaperones as factors affecting spike protein folding. This can now be used for rational design of compounds or peptides blocking spike protein maturation and, hence, formation of new infectious viruses.
Understanding essential steps of virus infection as membrane fusion and membrane bending and budding of newly forming viruses require detailed molecular analysis and modeling. For example, membrane shaping consists of two consecutive geometrical transformations: generation of curvature and remodeling of curvature by fusion or fission. In a joint experimental and theoretical approach it was demonstrated her that insertion of hydrophobic protein domains into the membrane matrix can drive membrane fission in addition to bending while protein scaffolds favor membrane fusion in addition to the curvature generation.
Viral strategies for application in nanobiotechnology and –medicine have been exploited. Recent research has indicated the potential of virus protein decorated multicoloured Layer-by-Layer-technology (LbL)- particles as a diagnostic tool for detecting virus specific antibodies. In projects are planned to develop and optimise enveloping of LbL-particles with lipid bilayers containing reconstituted virus proteins.
An approach has been developed using immunoliposomes that can bind HIV-1 virus-like particles (HIV-VLPs) while being specifically phagocytosed by macrophages, thus allowing the co-internalization of HIV-VLPs. This could be a potential approach for efficient removal of HIV-1 and other viruses.