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The Role of DNA Sensing in Defense Against RNA Viruses.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	The Role of DNA Sensing in Defense Against RNA Viruses./
作者:	Parker, Michael Todd.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2018,
面頁冊數:	224 p.
附註:	Source: Dissertations Abstracts International, Volume: 80-02, Section: B.
Contained By:	Dissertations Abstracts International80-02B.
標題:	Microbiology. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=10927866
ISBN:	9780438194366

The Role of DNA Sensing in Defense Against RNA Viruses.
Parker, Michael Todd.

The Role of DNA Sensing in Defense Against RNA Viruses. - Ann Arbor : ProQuest Dissertations & Theses, 2018 - 224 p.

Source: Dissertations Abstracts International, Volume: 80-02, Section: B.

Thesis (Ph.D.)--Yale University, 2018.

This item must not be added to any third party search indexes.

RNA viruses are among the most exquisite examples of evolutionary prowess in nature. Their ability to rapidly adapt to nearly any cellular niche and to then continue to develop in order to thrive is unsurpassed. It is then particularly impressive that control of these viruses is quite well executed in higher organisms, speaking to a remarkable ability to keep pace in the evolutionary arms race. At the cornerstone of this ability is the delineation between self and non-self, creating a foundation of protection against agents both familiar and alien. In the past thirty years, researchers have unveiled a staggering amount about this innate (i.e. non-specific) immune system, originally theorized by Charles Janeway in 1989 to detect "certain characteristics or patterns common on infectious agents but absent from the host"1. This is known to be important for detection of nucleic acids in the cytosol, and a large body of knowledge exists in regard to detection of ribonucleic acid (RNA). Sensing of molecular patterns in the genomes of RNA viruses is the most well-documented method of restriction in this regard. However, the recently-discovered deoxyribonucleic acid (DNA) sensing pathway has been implicated in defense against RNA viruses. Discrimination between self and non-self by this pathway is more nuanced because clear methods of delineating between DNAs are not evident. This means that location (and detection of mis-localization) are key to proper induction of a response. So far, the context of and ligands responsible for DNA sensing of RNA viruses remain unclear and these questions are the foundation of this dissertation. In Chapter 2, I identify viral complementary DNA (cDNA), host cDNA, and mitochondrial DNA (mtDNA) in the cytosol of RNA virus-infected cells as potential players in this pathway. Viral cDNA is produced during infection with both positive- and negative-sense RNA viruses and in human, mouse, and hamster cells. Accumulation of viral cDNA continues throughout infection and is sensitive to treatment with reverse transcriptase (RT) inhibitors as well as degradation by endogenous DNase. cDNA to cellular mRNA is also present in cells, suggesting that host mRNA may create a concomitant self-cDNAs. Host mtDNA is present in the cytosol at steady state and during infection and is bound by the DNA sensor cyclic guanosine monophosphate [GMP]adenosine monophosphate [AMP] synthase (cGAS) as exhibited by immunoprecipitation and deep sequencing. Taken together, these results shed light on the DNA content of the cytosol and identify ligands that may coordinate DNA sensing of RNA virus infection. To determine if and how these nucleic acids are involved in restriction of RNA viruses, experiments were undertaken investigating the modulation of these DNAs and the proteins potentially involved in their sensing, the results of which are presented in Chapter 3. Experiments in THP-1 cells exhibited reliance on DNA sensing for induction of interferon stimulated genes (ISGs) in response to RNA virus infection and that endogenous DNase knockdown results in increased baseline and virus-induced ISG responses. In cells with low immunocompetence, results were mixed but generally showed modest enhancing effects on virus production during RT inhibitor treatment but not mtDNA depletion. In more immune competent settings, viral replication increases upon knockout of cGAS and the enhancement phenotype of RT inhibitors is ablated, indicating that this enhancement is dependent on cGAS and DNA sensing mechanisms. Immortalized and primary mouse fibroblasts as well as THP-1 cells showed restriction of multiple viruses in a manner dependent on the DNA binding and cyclic GMP-AMP (cGAMP) catalytic activities of cGAS. Altogether, these results indicate that cGAS coordinates DNA sensing responses to RNA viruses, likely through interactions with viral and self cDNA, in cell type- and immortalization-influenced manners. Finally, in Chapter 4, I present methods developed to detect cGAMP and utilize these to investigate the ability of cGAS variants to catalyze its production. Wild-type cGAS, but not the cGAMP-catalysis mutant E225A/D227A, was able to produce cGAMP in response to transfection of DNA. This cell-derived cGAMP was identical to synthetically derived cGAMP by mass spectrometry and was able to stimulate transcription factor phosphorylation in a DNA sensing-dependent manner. However, production of cGAMP during infection with RNA viruses was not observed by either biological or LC/MS assessment. The kinetics of genesis and loss of cGAMP were further analyzed, showing that cGAMP is generated after DNA transfection but disappears quickly in cells expressing a membrane-associated phosphodiesterase. Interestingly, investigation of the level of ISG expression at baseline revealed that wild-type cGAS stimulates smoldering, constitutive levels of expression. This, paired with the inability to detect cGAMP upon infection suggests that perhaps the role of cGAS in restriction is as a preparative measure, rather than a reactionary one. Collectively, the results presented in this dissertation impart important knowledge in regard to innate defense against RNA viruses. These data will contribute not only to further advances in the fields of innate immunity and virology, but also provide information valuable for future development of clinical and therapeutic methodologies.

ISBN: 9780438194366Subjects--Topical Terms:

536250
Microbiology.

The Role of DNA Sensing in Defense Against RNA Viruses.
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502 $a Thesis (Ph.D.)--Yale University, 2018.
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506 $a This item must not be sold to any third party vendors.
506 $a This item is not available from ProQuest Dissertations & Theses.
520 $a RNA viruses are among the most exquisite examples of evolutionary prowess in nature. Their ability to rapidly adapt to nearly any cellular niche and to then continue to develop in order to thrive is unsurpassed. It is then particularly impressive that control of these viruses is quite well executed in higher organisms, speaking to a remarkable ability to keep pace in the evolutionary arms race. At the cornerstone of this ability is the delineation between self and non-self, creating a foundation of protection against agents both familiar and alien. In the past thirty years, researchers have unveiled a staggering amount about this innate (i.e. non-specific) immune system, originally theorized by Charles Janeway in 1989 to detect "certain characteristics or patterns common on infectious agents but absent from the host"1. This is known to be important for detection of nucleic acids in the cytosol, and a large body of knowledge exists in regard to detection of ribonucleic acid (RNA). Sensing of molecular patterns in the genomes of RNA viruses is the most well-documented method of restriction in this regard. However, the recently-discovered deoxyribonucleic acid (DNA) sensing pathway has been implicated in defense against RNA viruses. Discrimination between self and non-self by this pathway is more nuanced because clear methods of delineating between DNAs are not evident. This means that location (and detection of mis-localization) are key to proper induction of a response. So far, the context of and ligands responsible for DNA sensing of RNA viruses remain unclear and these questions are the foundation of this dissertation. In Chapter 2, I identify viral complementary DNA (cDNA), host cDNA, and mitochondrial DNA (mtDNA) in the cytosol of RNA virus-infected cells as potential players in this pathway. Viral cDNA is produced during infection with both positive- and negative-sense RNA viruses and in human, mouse, and hamster cells. Accumulation of viral cDNA continues throughout infection and is sensitive to treatment with reverse transcriptase (RT) inhibitors as well as degradation by endogenous DNase. cDNA to cellular mRNA is also present in cells, suggesting that host mRNA may create a concomitant self-cDNAs. Host mtDNA is present in the cytosol at steady state and during infection and is bound by the DNA sensor cyclic guanosine monophosphate [GMP]adenosine monophosphate [AMP] synthase (cGAS) as exhibited by immunoprecipitation and deep sequencing. Taken together, these results shed light on the DNA content of the cytosol and identify ligands that may coordinate DNA sensing of RNA virus infection. To determine if and how these nucleic acids are involved in restriction of RNA viruses, experiments were undertaken investigating the modulation of these DNAs and the proteins potentially involved in their sensing, the results of which are presented in Chapter 3. Experiments in THP-1 cells exhibited reliance on DNA sensing for induction of interferon stimulated genes (ISGs) in response to RNA virus infection and that endogenous DNase knockdown results in increased baseline and virus-induced ISG responses. In cells with low immunocompetence, results were mixed but generally showed modest enhancing effects on virus production during RT inhibitor treatment but not mtDNA depletion. In more immune competent settings, viral replication increases upon knockout of cGAS and the enhancement phenotype of RT inhibitors is ablated, indicating that this enhancement is dependent on cGAS and DNA sensing mechanisms. Immortalized and primary mouse fibroblasts as well as THP-1 cells showed restriction of multiple viruses in a manner dependent on the DNA binding and cyclic GMP-AMP (cGAMP) catalytic activities of cGAS. Altogether, these results indicate that cGAS coordinates DNA sensing responses to RNA viruses, likely through interactions with viral and self cDNA, in cell type- and immortalization-influenced manners. Finally, in Chapter 4, I present methods developed to detect cGAMP and utilize these to investigate the ability of cGAS variants to catalyze its production. Wild-type cGAS, but not the cGAMP-catalysis mutant E225A/D227A, was able to produce cGAMP in response to transfection of DNA. This cell-derived cGAMP was identical to synthetically derived cGAMP by mass spectrometry and was able to stimulate transcription factor phosphorylation in a DNA sensing-dependent manner. However, production of cGAMP during infection with RNA viruses was not observed by either biological or LC/MS assessment. The kinetics of genesis and loss of cGAMP were further analyzed, showing that cGAMP is generated after DNA transfection but disappears quickly in cells expressing a membrane-associated phosphodiesterase. Interestingly, investigation of the level of ISG expression at baseline revealed that wild-type cGAS stimulates smoldering, constitutive levels of expression. This, paired with the inability to detect cGAMP upon infection suggests that perhaps the role of cGAS in restriction is as a preparative measure, rather than a reactionary one. Collectively, the results presented in this dissertation impart important knowledge in regard to innate defense against RNA viruses. These data will contribute not only to further advances in the fields of innate immunity and virology, but also provide information valuable for future development of clinical and therapeutic methodologies.
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