Molecular basis for Rift Valley fever phlebovirus NSs protein function
Lead Research Organisation:
University of St Andrews
Department Name: Biology
Abstract
Rift Valley fever is a dangerous disease caused by a virus that is transmitted by mosquitos in most parts of Africa and the Arabian Peninsula. It causes large economic losses inducing a very high rate of abortions ("abortion storms") in infected livestock herds. The virus is also frequently transmitted to humans through contact with infected animals, or through mosquito bites. In most cases of human disease symptoms are similar to those seen during flu or meningitis. However, in about 10% of cases serious diseases develop, including encephalitis (brain inflammation) and haemorrhagic fever (similar to Ebola) that are often fatal. Worryingly, Rift Valley fever has also been linked to high rates of abortions in humans, raising the possibility of another widespread virus that causes severe damage to unborn life, reminiscent of Zika. Animal vaccines are available for preventing outbreaks of Rift Valley fever, but these are not completely safe and/or fully effective. No human vaccines or treatments are available. Rift Valley fever virus has a high potential to emerge in new regions in the world, for instance because global warming helps mosquitos capable of transmitting the virus spread from their original habitats into temperate zones. Because of its high potential for widespread epidemics, and because no treatments or safe vaccines are available, Rift Vally fever is one of eight diseases that according to the WHO are an urgent research priority.
Vaccine and drug development for Rift Valley fever requires a detailed understanding of how the virus causes disease. Like other viruses, Rift Valley fever virus has to suppress the immune response of infected animals and humans in order to multiply and spread. It achieves this mainly by using one molecule, the NSs protein. This operates akin to a molecular Swiss army knife - a number of different biological activities are carried out by different parts of the protein. How NSs works in detail is poorly understood, mainly because up to now the three-dimensional structure of the protein was unknown. While the complete structure is still lacking, we obtained the structure of the central part of NSs, which now will enable us to dissect the biological functions of NSs. One of the intriguing properties of NSs is the formation of distinct elongated structures (filaments) in infected cells, specifically in the cell nucleus where genetic information is stored and accessed. These filaments may be important for the virus to cause disease. They disrupt the machinery required to read out the genetic information. They may also be linked to the abortions observed in Rift Valley fever cases. Our molecular structure revealed how the NSs protein can form thread-like assemblies, but it is unclear how these would come together to form filaments in cell nuclei.
We now would like to use our molecular insights and powerful new imaging methods, mainly cryo-electron microscopy, to see at very high magnification what NSs filaments look like in infected cells. This information will help us understand the Swiss army knife activities of NSs. For instance we will be able to see if NSs in cell nuclei interacts with other molecules. We will also equip the NSs protein with molecular handles that will allow us to isolate the protein together with other proteins that it binds to. The same molecular handles can be used to make NSs glow under a microscope. This will also allow us to find out how NSs travels into the cell nucleus. We will use our knowledge gained so far to design variants and fragments of NSs that will allow us to obtain the missing structural information. We expect that dissecting structure and functions of NSs will help us understand how several dangerous viruses cause disease, and will pave the way to making drugs that would disrupt NSs filaments and may be used to treat Rift Valley fever.
Vaccine and drug development for Rift Valley fever requires a detailed understanding of how the virus causes disease. Like other viruses, Rift Valley fever virus has to suppress the immune response of infected animals and humans in order to multiply and spread. It achieves this mainly by using one molecule, the NSs protein. This operates akin to a molecular Swiss army knife - a number of different biological activities are carried out by different parts of the protein. How NSs works in detail is poorly understood, mainly because up to now the three-dimensional structure of the protein was unknown. While the complete structure is still lacking, we obtained the structure of the central part of NSs, which now will enable us to dissect the biological functions of NSs. One of the intriguing properties of NSs is the formation of distinct elongated structures (filaments) in infected cells, specifically in the cell nucleus where genetic information is stored and accessed. These filaments may be important for the virus to cause disease. They disrupt the machinery required to read out the genetic information. They may also be linked to the abortions observed in Rift Valley fever cases. Our molecular structure revealed how the NSs protein can form thread-like assemblies, but it is unclear how these would come together to form filaments in cell nuclei.
We now would like to use our molecular insights and powerful new imaging methods, mainly cryo-electron microscopy, to see at very high magnification what NSs filaments look like in infected cells. This information will help us understand the Swiss army knife activities of NSs. For instance we will be able to see if NSs in cell nuclei interacts with other molecules. We will also equip the NSs protein with molecular handles that will allow us to isolate the protein together with other proteins that it binds to. The same molecular handles can be used to make NSs glow under a microscope. This will also allow us to find out how NSs travels into the cell nucleus. We will use our knowledge gained so far to design variants and fragments of NSs that will allow us to obtain the missing structural information. We expect that dissecting structure and functions of NSs will help us understand how several dangerous viruses cause disease, and will pave the way to making drugs that would disrupt NSs filaments and may be used to treat Rift Valley fever.
Technical Summary
Rift Valley fever phlebovirus (RVFV) is one of the 8 priority pathogens identified by the WHO that urgently require strategies for the prevention of epidemics and treatment of the disease caused. RVFV relies on its non-structural NSs protein for pathogenicity. NSs is imported into the cell nucleus where it forms characteristic filaments and suppresses the interferon response by interfering with the TFIIH complex, shutting down global transcription, and by specifically inhibiting IFN expression.
The modes of action of NSs are poorly understood. The first structure for NSs provided a molecular basis for filament formation and will now inform dissection of NSs functions and interactions. We will use cryo-EM and X-ray microscopy to resolve NSs filament structures. We will use correlative fluorescence and EM to home in on filaments, and various sectioning approaches to obtain medium to near-atomic resolution structures. Crystallography and NMR will be used to obtain the missing structure of the N-terminal domain of NSs, which has been shown to be functionally important. Comparative structural analyses of NSs proteins of two other human-pathogenic phleboviruses will provide insights into the molecular determinants of NSs localisation and polymerisation. Structural work will be complemented by functional analyses using a recombinant viruses, and transient expression systems. We will use a variant of NSs that lacks the ability to form filaments to assess their role in interferon suppression. Using a covalent tagging system for pull-downs we will identify NSs binding partners in cytosol and nuclei that might be required for filament formation and import into the nucleus. Our data show a direct interaction of a soluble NSs variant with the TFIIH complex, which we will characterise using structural biology and functional assays.
This project will generate fundamental insights into molecular mechanisms underpinning pathogenesis of RVFV and other phleboviruses.
The modes of action of NSs are poorly understood. The first structure for NSs provided a molecular basis for filament formation and will now inform dissection of NSs functions and interactions. We will use cryo-EM and X-ray microscopy to resolve NSs filament structures. We will use correlative fluorescence and EM to home in on filaments, and various sectioning approaches to obtain medium to near-atomic resolution structures. Crystallography and NMR will be used to obtain the missing structure of the N-terminal domain of NSs, which has been shown to be functionally important. Comparative structural analyses of NSs proteins of two other human-pathogenic phleboviruses will provide insights into the molecular determinants of NSs localisation and polymerisation. Structural work will be complemented by functional analyses using a recombinant viruses, and transient expression systems. We will use a variant of NSs that lacks the ability to form filaments to assess their role in interferon suppression. Using a covalent tagging system for pull-downs we will identify NSs binding partners in cytosol and nuclei that might be required for filament formation and import into the nucleus. Our data show a direct interaction of a soluble NSs variant with the TFIIH complex, which we will characterise using structural biology and functional assays.
This project will generate fundamental insights into molecular mechanisms underpinning pathogenesis of RVFV and other phleboviruses.
