Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.797645
Title: The mechanisms by which ANP32 proteins support the influenza polymerase
Author: Mistry, Bhakti D.
ISNI:       0000 0004 8504 6971
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2019
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Abstract:
Influenza A naturally resides asymptomatically in wild aquatic birds. However, cross over into humans can occur and may result in a pandemic if the virus adapts for efficient replication and transmission between immunologically naïve human hosts. Fortunately, pandemics occur infrequently due to the existence of host range barriers. Poor replication of the influenza genome in the human cell nucleus is one of the main barriers that restricts avian origin influenza virus. The most common mutation which can overcome this block is the E627K mutation in the PB2 subunit of the trimeric influenza polymerase complex. Both the absence of a compatible positive factor or the presence of an inhibitory factor in human hosts have been proposed to explain why a polymerase bearing a glutamic acid at position 627 of PB2 is restricted in human cells. Avian ANP32A has been identified as a host factor which can support the activity of an avian origin viral polymerase. However, this polymerase is not compatible with the shorter human orthologues of this protein, human ANP32A and ANP32B. Since these human proteins have been implicated in supporting the activity of a human adapted influenza polymerase, it is hypothesised that the PB2 E627K mutation is an adaptation towards utilising huANP32A and -B. In this study we investigate how the ANP32A proteins are able to support polymerase activity and why species differences in this protein determine its compatibility with polymerases bearing different mutations. This was primarily examined by assessing differences in the interactions between specific ANP32A proteins and viral polymerases. We found that the interactions with trimeric polymerase complex were dependent on the species of ANP32A. These interactions were stabilised at inactive ribonucleoproteins, but dissociated as replication of the viral genome occurred. However, using multiple methods, we concluded that the ability of viral polymerase to co-opt ANP32A was determined by more than differences in interactions alone. As ANP32A can act as an adapter protein in some cases, we also investigated whether the interactome of the polymerase differed in the presence or absence of avian ANP32A. We further explored the impact of two human proteins, SRPK1 and importin alpha7, which differed in their interaction with avian origin polymerase when chicken ANP32A was co-expressed. Finally, we visualised the viral polymerase by overexpression of PA-GFP in human cells and observed differences between an avian origin polymerase and that with humanising mutations. Specifically, the avian origin polymerase appeared to form speckles in the nucleus of human cells. We explored the requirements for formation of these speckles as well as their relationship with ANP32A. This study has allowed us to gain insight into the mechanism by which ANP32A can support influenza virus polymerase. This was explored via several avenues, which led to the identification of other host factors which may affect polymerase activity. Overall, these data have enhanced our understanding of host restriction of influenza virus polymerase and the interplay between host and viral factors.
Supervisor: Barclay, Wendy ; Maertens, Goedele Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.797645  DOI:
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