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Dr Kirsten Spann

 
 
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OVERVIEW
 

The Respiratory Virus Research Unit (RVRU) at SASVRC was established in February of 2006 when Dr. Kirsten Spann joined SASVRC/CMVC from the National Institute of Allergy and Infectious Diseases, NIH, USA. The long-term vision of the unit is to develop antiviral treatments for paediatric respiratory viruses for which vaccines are not yet available. The unit will focus on Respiratory Syncytial Virus (RSV) for the first few years. In collaboration with Prof. Jeff Gorman at QIMR, the RVRU will identify the host-viral and viral-viral protein interactions which result in suppression of the cellular immune response during RSV infection. Ms Claire Straub was recruited to the unit as research assistant in May 2006. Start-up grants from RCH and UQ have assisted greatly in establishing the RVRU.
RSV is the most important cause of severe lower respiratory tract disease in infants and young children, and can also cause serious disease in immunocompromised adults and the elderly. Clinical manifestations vary from rhinitis and otitis media, to bronchiolitis and pneumonia. The global annual RSV infection rate is estimated to be 64 million, with approximately 160,000 cases resulting in death. The hospitalization burden due to RSV infection in Australia is considerable. Various research efforts are underway to develop vaccines to combat RSV. However none have been licensed. Monoclonal antibody prophylaxis and treatments have been moderately successful, but are expensive. There is a need to explore alternate antiviral treatments for RSV.
Synthetic compounds which block essential virus functions have been developed as treatments for other viral infections, such as HIV, and have proven to be effective in speeding recovery from disease. Compounds which inhibit immunosuppression by viruses would be ideal antiviral treatments as they would allow patients to mount an appropriate cellular immune response to infection. All viruses produce specific proteins which inhibit the cellular immune response, namely the induction and/or signaling of type I interferon. This inhibition limits the production of antiviral chemokines and cytokines and therefore the induction of an antiviral state in infected cells.
The non-structural proteins (NS1 and NS2) of RSV block type I interferon induction and signaling during RSV infection. We aim to characterize the mechanism by which this occurs, identify the host-viral and viral-viral protein interactions involved, and identify the domains of NS1 and NS2 involved in this response. Ultimately we aim to design synthetic peptides which block these interactions and boost cellular immune responses to RSV infection.
 

Research Projects:
 

The effect of NS1 and NS2 on host immunoregulatory proteins.
Kirsten Spann, Claire Straub in collaboration with Jeff Gorman (QIMR).
We are investigating the effect of NS1 and NS2 on the activation of key immunoregulatory proteins involved in type I interferon induction and signaling. NS1 and NS2 block the activation of IRF3, one of the principal transcription factors involved in type I interferon induction. They also affect the JAK-STAT pathway responsible for interferon signaling. We are investigating the exact mechanism(s) by which this occurs by tracking the activation of key immunoregulatory proteins in the presence and absence of NS1/NS2. We have treated cells with Poly I:C, a synthetic dsRNA polymer, and universal type I interferon to induce the activation of IRF3 and STAT2 respectively. In this way, we have established a time-line of activation by tracking nuclear translocation using immunofluorescence. This time-line will be used to compare activation in RSV-infected cells. We will also investigate the activation of other key factors in the interferon induction and JAK-STAT pathways in the presence and absence of RSV NS1/NS2.
Once we have identified the immunoregulatory proteins that are affected by NS1 and NS2, we will investigate the post-translational modifications of these proteins. We believe NS1 and NS2 block type I interferon induction and signaling by blocking essential post-translational modifications of the immunoregulatory proteins, such as phosphorylation.

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Immunoprecipitation of host-viral and viral-viral protein complexes using FLAG affinity tag.
Kirsten Spann, Claire Straub, in collaboration with Jeff Gorman, Tristan Wallis (QIMR) and Peter Collins (NIAID, USA).
We have demonstrated previously that NS1 and NS2 act co-operatively to suppress type I interferon induction and signaling. It is therefore likely that these two viral proteins bind in complex with each other and host proteins during infection. Recombinant RSVs expressing either FLAG and/or HexaHis tags on either NS1 and/or NS2 were genetically engineered by Kirsten Spann and Peter Collins at NIAID. These tags will be used to purify proteins in complex with NS1 and NS2 from RSV-infected cells. These viruses have been imported and high titre stocks successfully grown and stored at SASVRC. Western blot analysis has confirmed the expression of the FLAG tags.

Immunoprecipitation protocols using the FLAG and HexaHIS tags are presently being developed. We have purified FLAG-tagged NS1 from infected cells in complex with several other proteins. This complex will be analysed by mass spectrometry for the presence of NS2 and host immunoregulatory proteins. The NS1 protein will also be characterized for post-translational modifications such as phosphorylation and N-terminal acetylation. Understanding the modifications of NS1 and NS2 is essential to the identification of mechanisms by which these proteins interact with each other and host proteins.


NS1 immunoprecipitated using FLAG tag.

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