Using viruses to study kinesin-1 recruitment, regulation and function

Cells need to sort and transport cargo to distinct parts of the cell and this is fundamental for cell survival and for determining cell structure and function. To do this cells possess a skeleton (cytoskeleton) that is composed of microtubules. Microtubules are associated with motors that bind and transport cargo in a highly specific and regulated manner. One type of motor, called kinesin-1, is responsible for transporting cargos to specific locations within the cell. Defects in kinesin-1 transport cause human diseases, ranging from age-related macular degeneration and cataracts, to invasive cancers and neurological disorders. Kinesin-1 transport is very important for moving cargos longer distances along nerve axons and defects in kinesin-1 cause several neurodegenerative disorders, including Parkinson's, Huntington's, Alzheimer's and inherited spastic paraplegia. How kinesin-1 binds to and moves along microtubules is quite well studied. In contrast, how kinesin-1 recruits specific cargos and how kinesin-1 activity and target destination are regulated remain poorly understood.

Viruses replicate inside cells and have evolved mechanisms to hijack cellular machinery to enhance their replication and spread. Several viruses exploit kinesin-1 during their replication cycle, for instance to help export newly formed virus particles out of the cell. An example is vaccinia virus (VACV), the prototype poxvirus and the vaccine used to eradicate smallpox. Our lab uses the recruitment of kinesin-1 by VACV as a model to study the mechanisms that control the binding of cargo to kinesin-1 and activation of the motor.

The kinesin-1 complex is made up of two kinesin heavy chains (KHC) and two kinesin light chains (KLC). Its activity is regulated by autoinhibition. Activation involves multiple binding events by cargo proteins that induce a structural change in the complex enabling association with microtubules. Mammalian cells express multiple kinesin-1 types (isoforms), each with slightly different biochemical properties that may bind different cargos or move to different destinations.

Three VACV proteins, A36 and the F12/E2 complex, are involved in recruiting kinesin-1 to transport virus particles to the cell surface. To understand the role of each of these proteins, their interaction with each other and with components of the kinesin-1 complex will be characterised biochemically and using structural biology approaches. We have shown that F12/E2 and A36 associate with different domains of KLC and preferentially bind to different KLC isoforms. Gene knock-down using RNA interference and gene knock-out using CRISPR/Cas9-mediated gene editing will be used to determine the importance of different KLC isoforms for virus transport and for normal cellular function.

The roles of various KLC isoforms in cell function remain largely unknown. Cells in which individual KLCs or combinations of KLC have been knocked-out will be characterised by microscopy. Additionally, the cellular functions of individual KLCs will be determined by identifying and comparing the proteins that associate with them. Proteins that are also found to associate with the viral transport complex will be studied to determine if they are required for virus export. The contribution of viral proteins, individual KLC isoforms and cellular proteins to the activation of kinesin-1, and their possible role in its modulation will be measured using a combination of in vitro and in cell assays.

The proposed research project aims to investigate the mechanisms used by VACV to recruit and activate kinesin-1 by characterising the virus and cellular proteins involved. This research will also help our understanding of the normal cellular processes and how disease ensues if these dysfunction. Structural information about the interaction of the virus with kinesin-1 will aid the development of compounds that inhibit this process and prevent disease caused by poxviruses.

The microtubule network and associated kinesin-1 motor mediate the transport of cargo to specific places within cells and this is critical for normal cell function. Defects in kinesin-1 function or regulation can lead to disease, such as Parkinson's, Huntington's and Alzheimer's. How kinesin-1 binds to and moves along microtubules is well known, but how it binds specific cargos and how its activity and target destination are regulated are less well known.

Several viruses exploit kinesin-1 to transport newly formed virus particles out of the cell. An example is vaccinia virus (VACV), the vaccine used to eradicate smallpox. Our lab uses the recruitment of kinesin-1 by VACV as a model to study the mechanisms that control the binding of cargo to kinesin-1 and activation of the motor. This is the subject of this application.

The kinesin-1 complex contains 2 kinesin heavy chains (KHC) and 2 kinesin light chains (KLC). Its activity is regulated by autoinhibition. Activation involves binding by cargo proteins that induce a structural change in the complex enabling association with microtubules. Mammalian cells express multiple kinesin-1 types (isoforms), each with slightly different biochemical properties that may bind different cargos or move to different destinations.

This project will study the mechanisms by which VACV recruits and activates kinesin-1 to transport virus particles out of infected cells, to determine the structure of the virus proteins involved, and to investigate the expression and functions of the different KLC isoforms and splice variants for cell biology and which ones are hijacked by the virus.

The information gained will help understanding of the recruitment and regulation of kinesin-1 activity is in normal cells and how dysregulation can lead to disease. Another objective of the project is to study the properties and functions of different kinesin light chain (KLC) isoforms and splice variants to understand their roles in cell biology.

The impact of this research proposal will be to provide an increased understanding of how vaccinia virus (VACV) interacts with the host cell and hijacks the cellular transport system composed of microtubules and the kinesin-1 motor to mediate the export of newly synthesised virus particles from the infected cell. The information gained from this study will also increase understanding of how the kinesin-1 motor recruits cargo and is activated in normal (uninfected) cells and how its dysregulation can lead to disease. The research will also study fundamental properties and roles of kinesin light chains (of which there are several isoforms and many splice variants) in cell biology and increase our understanding of the functions of these cellular proteins.

Vaccinia virus is not itself a cause of disease in man, accept for rare complications following its use as a vaccine, but it is famous as the vaccine that was used to eradicate smallpox and is closely related to other orthopoxviruses that are a concern, such as monkeypox virus in parts of central and west Africa, cowpox virus in Europe, and variola virus the cause of smallpox. Although the disease smallpox has been eradicated, variola virus remains in high security laboratories in USA and Russia and there are concerns that the virus might re-merge naturally, be re-created by synthetic biology, or be released deliberately in an act of bioterrorism. This concern has prompted research to develop drugs to combat infection by variola virus and orthopoxviruses in general. The information gained from the research programme described in this application, specifically the structure of the virus proteins that bind to the kinesin-1 motor, may also lead to the development of antiviral drugs that prevent the interaction of the newly synthesised orthopoxvirus virions with the kinesin-1 motor and so prevent spread of the virus. Without the ability to spread efficiently from the infected cell orthopoxviruses are avirulent, and so blocking spread is an excellent way of preventing disease. Indeed the lead drug (ST-246) developed against smallpox and that has been purchased by the US Government for its national stockpile, acts by blocking virus egress from the cell, rather than affecting virus entry or replication.

It is now 40 years since the last naturally occurring case of smallpox, but research with VACV continues. In part this is because the virus is being engineered as a vaccine against other infectious diseases and cancers, and developed as an oncolytic agent, but it is also largely because VACV is an excellent tool with which to study fundamental aspects of immunology, cell biology and virus-host interactions. This application is an example of this. It will increase understanding of the mechanism by which VACV hijacks kinesin-1 and activates this motor to transport virus particles to the cell surface, but also increase our understanding of the regulation of kinesin-1 in uninfected cells and how specific cargos are selected and transported to different parts of the cell. An increased understanding of these processes will, in due course, lead to understanding of how dysregulation of kinesin-1 occurs and leads to disease, of which there are several examples, notably in neurological disease. Understanding how disease is caused is the first step to intervention.

So those who might benefit from this research are those also studying how kinesin-1 functions and how this motor is hijacked by pathogens to achieve export from the infected cell. Others are those studying diseases linked to dysfunction of kinesin-1, such as Parkinson's, Huntington's, Alzheimer's and inherited spastic paraplegia. And a third group of beneficiaries are those seeking to develop drugs to treat orthopoxvirus infections.