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The effect of pattern recognition receptor RIG-I variant expression during mammalian- or avian-adapted influenza A infection and adaptation in the mouse.
Influenza A virus infections are common all over the world and cause substantial damage on health and economy by seasonal outbreaks. The infectious disease flu becomes even more life threatening when followed by a secondary bacterial infection, for which especially children and immune-suppressed peo...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2023
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| Zusammenfassung: | Influenza A virus infections are common all over the world and cause substantial damage on health and economy by seasonal outbreaks. The infectious disease flu becomes even more life threatening when followed by a secondary bacterial infection, for which especially children and immune-suppressed people are susceptible. Today, the annually adapted influenza vaccination is an important tool to prevent outbreaks of flu. Medical treatments of acute infections are limited with the exceptions of therapeutically targeting the viral proteins neuraminidase and M2. A deeper understanding of the molecular mechanisms of the IAV pathogenicity and antiviral defense mechanisms, as well as their interaction, can help to refine vaccination strategies and direct therapeutic options to generate more target specific and effective antiviral drugs. The pattern recognition receptor RIG-I is one of the most important sensors of the innate immune system to detect foreign RNAs. Upon the binding of RNA ligands like the panhandle structures of influenza A virus, RIG-I amongst others mediates the expression of interferon type 1 and interleukin-1β in an ATPase dependent manner. Additionally, the binding of RIG-I to the viral panhandle structures is confirmed in vitro as another antiviral function by blocking the access for the viral polymerase, as firstly described by Friedemann Weber in the year 2015.
The main aim of this thesis was to investigate the contribution of the different antiviral effects of RIG-I against the influenza A virus in a mouse infection model. Furthermore, additional insights about the RIG-I blocking function should be gained. Therefore, a mouse line deficient in RIG-I signaling and another one lacking RIG-I expression were established with the help of genetic engineering. Additionally, two recombinant influenza strains harboring an adaptation in the viral polymerase gene either to mammalian hosts or to avian hosts (polymerase subunit 2 codon 627K and 627E) were generated. Both virus strains were validated for different quality features. The recombinant virus strains were used to perform an infection study using RIG-I wild type, signaling-deficient and knockout mice. Investigating the effect of RIG-I variant expression on parameters like weight reduction, lung virus titer, loss of lung barrier integrity, interferon and cytokine concentration in response to the influenza A infection, new insights in the antiviral functions of RIG-I were gained.
The established mouse lines expressing signaling-deficient or no RIG-I did not develop any detectable burden by their genotype. The RIG-I-mediated interferon-α induction was found to be abolished in bone marrow derived macrophages of mice with signaling deficiency in RIG-I as well as RIG-I knockout mice while it was intact in RIG-I wild type mouse derived cells, as expected. Hence, an unburdened mouse line with RIG-I signaling deficiency and one with a RIG-I knockout were generated. The RIG-I PM line is the first of its kind. While the generation of a mammalian-adapted recombinant influenza A strain was successful from the beginning, the generation of the avian-adapted strain was not successful in mammalian cells. A sufficient replication of both strains was achieved in the DF-1 chicken cell line. The received stock preparations showed similar abilities and the stability of their respective genotype was confirmed over several passages in different cell lines. While the infection of mice with the generated recombinant influenza A strains led to a significant change in observed infection parameters and cytokine signaling, only a weak effect of RIG-I variant expression on the infection parameters was detectable. Additionally, the results deliver hints for a RIG-I dependent induction of IFN-γ by RIG-I, which was not described in detail yet. The data also suggest that the antiviral functions of RIG-I may be potently inhibited by the viral nonstructural protein 1 and the mammalian-adapted polymerase variant. The validation of the genetic stability of the virus strain with the avian-adapted polymerase variant in vivo indicates a significant back mutation to the original mammalian-adapted genotype over the course of the infection. This was significantly affected by the time post infection and the type of RIG I variant expression. A lower rate of back mutation than in RIG-I wild type mice was detected in mice with RIG-I signaling deficiency and the lowest in mice with RIG-I knockout. These findings suggests that both the RIG-I signaling functions and the RIG-I blocking function mediate a selective pressure on the influenza A polymerase subunit 2 codon 627. This conclusion is supported by the results of an in vitro competitive infection assay with both virus variants together, showing a replication advantage of the mammalian-adapted polymerase variant over the avian-adapted variant that is affected by the type of RIG-I expression.
Taken together, the results of this study deliver deep insights into the interaction between the innate pattern recognition receptor RIG-I and the influenza A virus. The findings suggest that the presence of RIG-I forces viral polymerase variants common in avian to adapt to a mammalian host. Further, the study delivers additional data confirming a RIG-I-mediated antiviral effect by blocking the access of the viral polymerase to the panhandle structures of viral RNAs. Additionally, the data suggests a high potency of the nonstructural protein 1 and the mammalian-adapted polymerase subunit 2 codon 627K to prevent the effects of RIG-I. The finding that RIG-I may contribute to interferon-γ release could be interesting and should be investigated in future studies, since this interaction is poorly described in the literature, but connects two important features of the innate immune system. |
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| DOI: | 10.17192/z2023.0544 |