Investigating the plasticity of viral envelope proteomes

A subset of virus species, e.g. HIV and Influenza, are surrounded by a lipid bi-layer membrane, termed the envelope. Envelopes contain at least one viral glycoprotein and may contain several host-derived membrane proteins in addition. We could show previously that proteins modified post-translationally with a glycosyl- phosphatidyl-inositol (GPI) anchor can insert into the viral envelope of a number of virus species. In this study we hypothesize that this has evolved to happen in a natural context as well and provides benefits for the virus (i.e. stabilization of virus particles, immune evasion, etc.). We intend to test for association of GPI anchored proteins to envelopes, but on a more general level also for association of any proteins to the virus post-exit, i.e. in circulation. The hypothesis that the virus acquires proteins from the surrounding medium seems reasonable and consequently, different proteins should associate to the virus upon a change of the virus`s environment (i.e. by budding into extracellular space, changing compartments within a host, changing hosts or even host species) This should lead to a distinct plasticity of the viral envelope proteome. Studies investigating this have not been undertaken so far and we believe that such mechanisms may play an important role in viral transmission. Three different research fields will be initiated: In the insertion bait approach a protein known to insert into viral envelopes will be used to define conditions permissive for insertion into HIV and Influenza, specifically type and level of protein background, temperature and stability. In association dynamics monitoring, the pre-defined composition of lipid model membranes, including different levels of protein content allows for the investigation of the influence of lipid composition and protein pre-loading on insertion/association events. In the differential proteomics approach the comparison of the proteome of viral envelopes before and after challenge with a complex protein mixture, i.e. serum will allow identifying differential display of proteins thus indicating insertion/association events. The project will be carried out in close scientific collaboration with the Department of Nanobiotechnology of the University of Natural Resources and Life Sciences (BOKU) in Vienna providing a strong background in biophysics/model membranes - and the University of Veterinary Medicine`s technology core facility, providing tools and experience for the proteomics approach. This will ensure the optimal combination of expertise for carrying out the project. Very little is known about how viruses manage to face challenges in the stage between infected cells. We believe that gathering information as described throughout the proposal will help to identify a new aspect of host-pathogen interaction since the mechanisms investigated may very well be of great importance for success of viral transmission. Subsequently, this research could also open new routes for anti-viral intervention, perhaps especially in the context of zoonotic or emerging infections

Some virus species, such as HIV and Influenza, are surrounded by a lipid layer, termed the envelope. Envelopes contain at least one viral protein and may contain several host proteins in addition. We could show previously that a certain type of proteins (i.e. glycosyl- phosphatidyl-inositol or GPI-anchored proteins) can insert into the viral envelope. We believe that this has also evolved in a natural context and provides benefits for the virus such as an increased stability of virus particles or an escape from the host`s immune system. We were testing for association of GPI anchored proteins to envelopes, but on a more general level also for association of any proteins to the virus upon a change of the viruss biochemical environment. To investigate this attachment, we were optimizing a method that would preserve viral particles in a state as close to their natural situation as possible, but still would be compatible with biophysical methods for interaction analysis. This was achieved by using different tethering components to immobilize the virus on sensors. In a first step we were using liposomes, non-viral lipid vesicles of controlled composition and a size similar to viral particles, as a model. Different types of liposomes were tested for immobilization by tethering and then exposed to different proteins or protein mixtures. We could identify different types of interaction: from transient binding to stable binding via mixed intermediates depending on the proteins or protein mixtures used. When in a second step, viral particles were attached to the sensors and analyzed, we found the same patterns. Based upon these results we tried to identify proteins bound to the viral particle. Preliminary data identified a range of proteins with known lipid binding character. Additionally, we could confirm that virus can benefit from the uptake of proteins from the surrounding: the GPI-anchored protein CD59 protects from the complement system, a part of the immune system. In summary, our results suggest strong levels of interaction between virus particles and the surrounding media and a high degree of similarity between liposomes and virus. Taken together with the identification of a range of lipophilic binding partners this underlines the importance of protein-lipid interactions. With this we collected evidence for our original hypothesis: virus can associate with proteins from novel surroundings and gain new beneficial properties as a result. In the future we will work on a direct proof-of concept and identifying biological consequences.