Elucidating the molecular mechanisms of antiviral defense systems

Chakravarti, Arpita

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https://hdl.handle.net/2142/115865 Copy

Description

Title

Elucidating the molecular mechanisms of antiviral defense systems

Author(s)

Chakravarti, Arpita

Issue Date

2022-06-13

Director of Research (if dissertation) or Advisor (if thesis)

Huang, Raven

Doctoral Committee Chair(s)

Huang, Raven

Committee Member(s)

• Cronan, John
• Nair, Satish
• Procko, Erik

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• antiviral
• defense

Abstract

Biological conflicts between organisms are a major feature across all the domains of life. These conflicts give rise to unique defensive strategies for competing and surviving in a community. Prokaryotes and eukaryotes employ a variety of such counter defenses which includes the Restriction-Modification system, CRISPR-Cas system, and stimulation of the interferon pathway in response to invading viruses and bacteria. Among these, many involve the production of nucleotide-based second messengers that trigger downstream antiviral signaling. However, the molecular mechanisms underlying many of these systems remain unknown. This study focuses on elucidating the mechanisms of three such antiviral defense systems, namely, 1) viperin mediated antiviral signaling, 2) CdnG-Cap5 antiphage system and, 3) cNMP cyclase-TIR/DUF5706 antiphage system. Viperin (Virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) belongs to the radical SAM (S-adenosylmethionine) enzyme superfamily which catalyze some of the most unique and challenging transformations in biology. Unlike other radical SAM enzymes, viperin possesses antiviral activity against a broad range of RNA and DNA viruses. However, the molecular mechanism of its antiviral activity remains elusive. Our studies indicate that recombinant fungal and archaeal viperin catalyzes the addition of 5′-deoxyadenosyl radical (5’-dA•) to the double bond of isopentenyl pyrophosphate (IPP), producing a new compound we named adenylated isopentyl pyrophosphate (AIPP). This reaction is specific for IPP, as other pyrophosphate compounds involved in the mevalonate pathway do not act as substrates. Furthermore, any chemical change in IPP reduces its ability to be an effective substrate. Mutational studies disclosed that the hydroxyl group on the side chain of Tyr-245 in fungal viperin is the likely source of hydrogen in the last step of the radical addition, providing mechanistic insight into the radical reaction catalyzed by fungal viperin. Structure-based molecular dynamics (MD) simulations of viperin interacting with IPP revealed a good fit of the isopentenyl motif of IPP to the active site cavity of viperin, unraveling the molecular basis of substrate specificity of viperin for IPP. CBASS (cyclic-oligonucleotide-based antiphage signaling systems) are a family of antiphage bacterial immune system that share ancestry with cGAS-STING innate immunity in animals. These systems are minimally composed of an oligonucleotide cyclase, which generates the signaling cyclic-oligonucleotide second messenger in response to a phage infection and the effector that is activated by the cyclic-oligonucleotide to promote cell death before phage replication is completed, in a process referred to as abortive infection. Our studies present the biochemical and structural characterization of two CBASS systems, composed of CdnG (oligonucleotide cyclase) and Cap5 (Effector), from Asticcacaulis sp. and Lactococcus lactis. We show that CdnG from Asticcacaulis sp. synthesizes 3′,2′-cGAMP in vitro, and 3’,2′-cGAMP is the biological signaling molecule that activates Cap5 for DNA degradation. Crystal structures of Cap5, together with the SAVED domain in complex with 3′,2′-cGAMP, provide insight into the architecture of Cap5 as well as molecular recognition of 3′,2′-cGAMP by the SAVED domain of Cap5. Crystal structures revealed that Cap5 exists as a homodimer, however, the structures of AsCap5 and LlCap5 homodimers exist in two different conformational states. Amino acid conservation of the SAVED domain of Cap5, together with mutational studies, led us to propose a unique mechanism of Back-to-Front stacking of two SAVED domains, mediated by 3′,2′-cGAMP, to activate HNH nuclease domain for DNA degradation. Bioinformatic analysis indicates that, like di- and oligo-cyclic nucleotides, mono-cyclic nucleotides (cyclic-NMP) may also be involved in antiphage signaling in bacteria. To date, only cyclic-UMP (cUMP) and cyclic-CMP (cCMP) have been reported to possess antiphage activity in the Pycsar (pyrimidine cyclase system for antiphage resistance) antiphage signaling system. In this study, we show that the cyclic-NMP cyclase (cNC) from Pseudomonas fluorescens (Pf) and Escherichia coli (Ec) produces cyclic-GMP (cGMP) that activates its TIR (Toll/interleukin-1 receptor)-domain or DUF5706 (domain of unknown function 5706) effectors respectively. Since, NAD+ hydrolysis by TIR-domain containing effectors have been known to be a part of antiphage system in bacteria, we predicted that the TIR-domain containing effectors could hydrolyze NAD+ upon activation by the cGMP generated by its cyclase. In vitro reconstitution assays revealed that this was indeed the case as the PfTIR1d efficiently and specifically hydrolyzed NAD+ in the presence of cGMP. On the other hand, our phage-based in vivo assays showed that the expression of E. coli proteins EccNC1d/DUF5706 resulted in very strong inhibition of plaque formation by phages by a yet to be identified mechanism. Therefore, both cyclase-effector systems employing cGMP likely possesses antiphage activity. Crystal structures of PfcNC1d revealed that it is a homodimer and shares structural similarity to Adenylyl cyclases. However, PfcNC1d shares 20% sequence identities with those Adenylyl cyclases indicating that regulation and substrate specificity of this enzyme might be different.

Graduation Semester

2022-08

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Arpita Chakravarti

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