Synthetic within-cell flavivirus sensors
Lead Research Organisation:
University of York
Department Name: Biology
Abstract
The aim of the proposed study is to develop synthetic-biology sensors that can sit in living cells, essentially inert until the cell is infected by a virus, and then activate and respond. For this proposal, we focus on flaviviruses, a major group of viruses that includes mosquito-transmitted viruses dengue, Zika and yellow fever viruses, as well as some viruses transmitted by ticks, for example.
When viruses infect a cell, they use a lot of the cell's own machinery for their replication, but they also have some proteins of their own, encoded in their own small genomes, that an uninfected cell does not have. These provide the basis for specific detection - the cell does not have these proteins and enzymes unless it is infected. Unlike the virus' sequence, which is very specific to individual virus types, or even strains of a single virus, these enzymatic activities are quite similar between different viruses of the same general group - e.g. flaviviruses, or mosquito-transmitted flaviviruses, so we expect to develop sensors that will respond to any of a range of viruses, perhaps to all mosquito-transmitted flaviviruses, rather than to just, for example Zika virus, or one strain of Zika virus.
Such systems have several potential uses. One is in diagnostics. Sequence-based detection systems, such as PCR, need some prior knowledge of the virus sequence, and are very sensitive to variations in this sequence (i.e. may fail to detect a variant). More comprehensive approaches exist, such as complete sequencing, but these are currently relatively slow and expensive. Another standard approach involves applying the putative virus sample to cultured cells and looking for disruption of those cells (cytopathic effect, CPE), but this is a little slow, and not all viruses cause obvious pathology. We have developed prototype within-cell systems that appear to have superior characteristics, at least for some purposes. We will develop these and characterise them in detail, with project partners, to provide a set of tools complementary to current approaches.
We will use the same approach in mosquitoes, to develop mosquitoes that respond in specific ways to infection by Zika virus and other flaviviruses. Our main aim here is for that response to reduce the ability of the mosquito to transmit the virus. Unlike humans, mosquitoes are not severely affected by these viruses, indeed it is important for the virus not to harm the mosquito much since it depends on her (always "her": only female mosquitoes bite) for transmission. If we can arrange that infected mosquitoes are unusually sensitive to infection, e.g. are killed by the virus, this will greatly reduce transmission. Such systems would additionally need to be spread into a target mosquito population in the wild. That is not part of this project, which is entirely lab-based, but potentially a future development if this project is successful.
When viruses infect a cell, they use a lot of the cell's own machinery for their replication, but they also have some proteins of their own, encoded in their own small genomes, that an uninfected cell does not have. These provide the basis for specific detection - the cell does not have these proteins and enzymes unless it is infected. Unlike the virus' sequence, which is very specific to individual virus types, or even strains of a single virus, these enzymatic activities are quite similar between different viruses of the same general group - e.g. flaviviruses, or mosquito-transmitted flaviviruses, so we expect to develop sensors that will respond to any of a range of viruses, perhaps to all mosquito-transmitted flaviviruses, rather than to just, for example Zika virus, or one strain of Zika virus.
Such systems have several potential uses. One is in diagnostics. Sequence-based detection systems, such as PCR, need some prior knowledge of the virus sequence, and are very sensitive to variations in this sequence (i.e. may fail to detect a variant). More comprehensive approaches exist, such as complete sequencing, but these are currently relatively slow and expensive. Another standard approach involves applying the putative virus sample to cultured cells and looking for disruption of those cells (cytopathic effect, CPE), but this is a little slow, and not all viruses cause obvious pathology. We have developed prototype within-cell systems that appear to have superior characteristics, at least for some purposes. We will develop these and characterise them in detail, with project partners, to provide a set of tools complementary to current approaches.
We will use the same approach in mosquitoes, to develop mosquitoes that respond in specific ways to infection by Zika virus and other flaviviruses. Our main aim here is for that response to reduce the ability of the mosquito to transmit the virus. Unlike humans, mosquitoes are not severely affected by these viruses, indeed it is important for the virus not to harm the mosquito much since it depends on her (always "her": only female mosquitoes bite) for transmission. If we can arrange that infected mosquitoes are unusually sensitive to infection, e.g. are killed by the virus, this will greatly reduce transmission. Such systems would additionally need to be spread into a target mosquito population in the wild. That is not part of this project, which is entirely lab-based, but potentially a future development if this project is successful.
Technical Summary
The aim of this project is to develop synbio virus sensors capable of detecting flaviviruses. We will develop such genetically encoded sensors towards two potential applications, both based on substantial preliminary data indicating feasibility. These are cell-culture systems for diagnostics and basic science, and mosquito-based systems primarily aimed at engineering mosquitoes with reduced ability to transmit any of a range of flaviviruses. Flaviviruses express a single polyprotein which is processed by a combination of cellular proteases and viral NS2B/3 protease into the mature viral proteins. NS2B/3 protease is a specific, essential enzymatic activity absent from non-infected cells. We have developed prototype pro-enzyme sensors, in which Cre recombinase - which recombines DNA targets present in the nucleus - is expressed "tethered" to a transmembrane domain that keeps it out of the nucleus, with the linker between the two comprising an NS2B/3 cleavage site. This "tethered-Cre" is activated by NS2B/3 protease only in infected cells. We will refine this system, using variant linkers to optimise response to a range of flaviviruses, in cells and in transgenic mosquitoes. We will additionally explore using this principle to develop mosquitoes less able to transmit such flaviviruses. Since flaviviruses such as Zika and dengue viruses establish persistent, lifelong infections in mosquitoes the virus depends on ongoing mosquito activity for its transmission. In this mosquito-based version, we will arrange that activation of the sensor leads to death of the mosquito. While this might appear to have a severe fitness cost, it is designed to affect only infected mosquitoes. These are quite rare, e.g. typically <2% of females (only females bite) carry dengue virus even in severe dengue outbreaks, so the overall fitness cost may be fairly modest - and even lower if the intervention successfully reduces transmission, so fewer mosquitoes become infected.
