Carolyn M. Teschke

Scientific discoveries have the greatest impact when they are understood, valued, and supported beyond the laboratory. In the final 15 minutes of this webinar, our speakers will explore the role of science advocacy in advancing research, public health, and innovation. Learn why it is important for scientists to engage with policymakers, funders, educators, and the public, and how effective advocacy helps ensure that scientific evidence informs decisions that affect communities around the world. Through their experiences, our speakers will share insights into the importance of championing science and fostering support for the research enterprise.

The dodecameric portal ring complex is a central component of double-stranded DNA viruses and tailed bacteriophages, functioning as the orchestrator of capsid assembly, the channel for DNA translocation and the docking site for tail adaptor proteins. This seminar will highlight new findings from the Teschke lab on the functional significance of a highly conserved basic motif within the portal's wing domain. Using a combination of in vivo and in vitro approaches, they will demonstrate how this motif mediates key subunit interactions required for proper dodecameric portal ring formation and subsequent incorporation into the viral capsid.

Join us for the next Journal of Virology Seminar Series, where Makayla Leroux, Ph.D., and Carolyn Teschke, Ph.D. will present their recent work on bacteriophage P22 assembly. Learning Objectives: Understand the role of the portal protein complex in double-stranded DNA viral maturation. Understand the critical protein-protein interactions necessary for portal incorporation into the viral particle during assembly. Learn how biophysical techniques can be used to characterize large macromolecular assemblies like the portal complex. Constructing the Capsid: Portal Protein Interactions in Bacteriophage P22 Talk by Felicia Goodrum, Kristin Parent, Carolyn M. Teschke, Makayla Leroux Science Advocacy in Action Talk by Felicia Goodrum, Kristin Parent, Carolyn M. Teschke, Makayla Leroux

Some double-stranded DNA viruses, such as herpesviruses and bacteriophages, utilize a powerful molecular motor to package DNA into their capsids to liquid-crystalline density. The motor is composed of large and small terminase subunits that bind to a portal complex located at a unique vertex on the icosahedral capsid. The small terminase is critical for recognition of the viral genomic DNA. The enzymatic component of the motor is the large terminase that has two functional domains necessary for ATP hydrolysis and DNA cleavage. Bacteriophage large terminase amino acid sequences are highly divergent, but their overall structural architecture and function are conserved across both tailed bacteriophages and herpesviruses. There are limited antiviral drugs for herpesvirus infections. Therefore, the viral DNA packaging motor is being investigated as an alternative drug target. In this review, we discuss the past and recent genetic, biochemical, and structural research on viral terminase motors that has illuminated their role in DNA packaging and potential as candidates for antiviral drug targeting.

The dodecameric portal ring complex plays a critical role in the assembly of most double-stranded DNA (dsDNA) viruses and bacteriophages. The fidelity of ring oligomerization and incorporation of a single 12-mer portal ring is driven by interactions with a viral scaffolding protein. Structural evidence indicates that the wing domain of the portal is a crucial site for scaffolding protein interactions. In this study, the functional significance of the highly conserved ₂₇₂ KRRR ₂₇₅ motif in the portal wing domain of bacteriophage P22 is investigated. Alteration of the entire motif to alanines results in a reduction in infectious phage production owing to a decrease in portal incorporation into procapsids in vivo . Further interrogation of the variant portal protein in vitro showed that it assembles into oligomeric assemblies larger than dodecamers, as determined by charge detection mass spectrometry. Though variant monomers have an altered secondary structure, they are still capable of assembling into rings in the presence of scaffolding protein, but it is likely that these rings adopt a modified conformation that is unfit for incorporation. These findings indicate that the ₂₇₂ KRRR ₂₇₅ motif is necessary for critical intra-subunit interactions that regulate the portal monomer’s folding into the correct conformation for oligomerization into stable 12-mer rings. IMPORTANCE: To be infectious, most double-stranded DNA (dsDNA) viruses and bacteriophages package their DNA into the capsid via the dodecameric portal protein vertex. This first requires that portal monomers assemble into a portal ring that becomes incorporated into the capsid, but this oligomerization process is not well defined. Here, we show that mutating a highly conserved basic motif, ₂₇₂ KRRR ₂₇₅ , in the bacteriophage P22 portal protein wing domain impairs proper ring assembly and incorporation, despite not being involved in inter-subunit contacts. This defect arises from altered secondary structure of the portal monomer, emphasizing the importance of specific intra-subunit interactions necessary for portal oligomerization. These results further our understanding of essential protein-protein interactions involved in viral assembly and provide insight into key interactions that can aid the development of antiviral drugs targeting portal oligomerization and incorporation.

Some double-stranded DNA viruses utilize a powerful molecular motor to package DNA 13 into their capsids to liquid-crystalline density. The motor is composed of a portal complex and 14 large and small terminase subunits. The enzymatic component of the motor is the large terminase 15 that has two functional domains necessary for ATP hydrolysis and DNA cleavage. Large terminase 16 amino acid sequences are highly divergent, but their overall structural architecture and function 17 are conserved across tailed bacteriophages and herpesviruses. Because there are limited antiviral 18 drugs for herpesvirus infections, and these exclusively target the viral DNA polymerase, cross-19 resistance is of high concern. Therefore, the DNA packaging motor is being investigated as an 20 alternative drug target. In this review, we discuss the past and recent genetic, biochemical, and 21 structural research on large terminases that has illuminated their role in DNA packaging and 22 potential as a candidate for antiviral drug targeting. 23

The 134‐residue phage L decoration protein (Dec) forms a capsid‐stabilizing homotrimer that has an asymmetric tripod‐like structure when bound to phage L capsids. The N‐termini of the trimer subunits consist of spatially separated globular OB‐fold domains that interact with the virions of phage L or the related phage P22. The C‐termini of the trimer form a spike structure that accounts for nearly all the interactions that stabilize the trimer. A Dec mutant with the spike residues 99–134 deleted (Dec_1‐98) was used to demonstrate that the globular OB‐fold domain folds independently of the C‐terminal residues. However, Dec_1‐98 was unable to bind phage P22 virions, indicating the C‐terminal spike is essential for stable capsid interaction. The full‐length Dec trimer is disassembled into monomers by acidification to pH <2. These monomers retain the folded globular OB‐fold domain structure, but the spike is unfolded. Increasing the pH of the Dec monomer solution to pH 6 allowed for slow trimer formation in vitro over the course of days. The infectious cycle of phage L is only around an hour, thereby implying Dec trimer assembly in vivo is templated by the phage capsid. The thermodynamic hypothesis holds that protein folding is determined by the amino acid sequence. Dec serves as an unusual example of an oligomeric folding step that is kinetically accelerated by a viral capsid template. The capsid templating mechanism could satisfy the flexibility needed for Dec to adapt to the unusual quasi‐symmetric binding site on the mature phage L capsid.

Icosahedral dsDNA viruses such as the tailed bacteriophages and herpesviruses have a conserved pathway to virion assembly that is initiated from a scaffolding protein driven procapsid formation. The dsDNA is actively packaged into procapsids, which undergo complex maturation reactions to form infectious virions. In bacteriophage P22, scaffolding protein (SP) directs the assembly of coat proteins into procapsids that have a T=7 icosahedral arrangement, en route to the formation of the mature P22 capsid. Other than the C-terminal helix-turn-helix involved in interaction with coat protein, the structure of the P22 303 amino acid scaffolding protein within the procapsid is not understood. Here, we present a structural model of P22 scaffolding protein encapsulated within the 23 MDa procapsid determined by magic angle spinning NMR spectroscopy. We took advantage of the 10-fold sensitivity gains afforded by the novel CPMAS CryoProbe to establish the secondary structure of P22 scaffolding protein and employed ¹⁹ F MAS NMR experiments to probe its oligomeric state in the procapsid. Our results indicate that the scaffolding protein has both α-helical and disordered segments and forms a trimer of dimers when bound to the procapsid lattice. This work provides the first structural information for P22 SP beyond the C-terminal helix-turn-helix and demonstrates the power of MAS NMR to understand higher-order viral protein assemblies involving structural components that are inaccessible to other structural biology techniques.

Tupanviruses, members of the family Mimiviridae , infect phagocytic cells. Particle uncoating begins inside the phagosome, with capsid opening via the stargate. The mechanism through which this opening takes place is unknown. Once phagocytized, metal ion flux control and ROS are induced to inactivate foreign particles, including viruses. Here, we studied the effect of iron ions, copper ions, and H ₂ O ₂ on Tupanvirus particles. Such treatments induced stargate opening in vitro , as observed by different microscopy techniques. Metal-treated viruses were found to be non-infectious, leading to the hypothesis that stargate opening likely resulted in the release of the viral seed, which is required for infection initiation. To the best of our knowledge, this is the first description of a giant virus capsid morphological change induced by transition metals and H ₂ O ₂ , which may be important to describe new virulence factors and capsid uncoating mechanisms.