PhiXing-it, displaying foreign peptides on bacteriophage ΦX174

AbstractAlthough bacteriophage φX174 is easy to propagate and genetically tractable, it is use as a peptide display platform has not been explored. One region within the φX174 major spike protein G tolerated 13 of 16 assayed insertions, ranging from 10 to 75 amino acids. The recombinant proteins were functional and incorporated into infectious virions. In the folded protein, the peptides would be icosahedrally displayed within loops that extend from the protein׳s β-barrel core. The well-honed genetics of φX174 allowed permissive insertions to be quickly identified by the cellular phenotypes associated with cloned gene expression. The cloned genes were easily transferred from plasmids to phage genomes via recombination rescue. Direct ELISA validated several recombinant virions for epitope display. Some insertions conferred a temperature-sensitive (ts) protein folding defect, which was suppressed by global suppressors in protein G, located too far away from the insertion to directly alter peptide display

Viral Adaptation to an Antiviral Protein Enhances the Fitness Level to Above That of the Uninhibited Wild Type

Viruses often evolve resistance to antiviral agents. While resistant strains are able to replicate in the presence of the agent, they generally exhibit lower fitness than the wild-type strain in the absence of the inhibitor. In some cases, resistant strains become dependent on the antiviral agent. However, the agent rarely, if ever, elevates dependent strain fitness above the uninhibited wild-type level. This would require an adaptive mechanism to convert the antiviral agent into a beneficial growth factor. Using an inhibitory scaffolding protein that specifically blocks X174 capsid assembly, we demonstrate that such mechanisms are possible. To obtain the quintuple-mutant resistant strain, the wild-type virus was propagated for approximately 150 viral life cycles in the presence of increasing concentrations of the inhibitory protein. The expression of the inhibitory protein elevated the strain's fitness significantly above the uninhibited wild-type level. Thus, selecting for resistance coselected for dependency, which was characterized and found to operate on the level of capsid nucleation. To the best of our knowledge, this is the first report of a virus evolving a mechanism to productively utilize an antiviral agent to stimulate its fitness above the uninhibited wild-type level. The results of this study may be predictive of the types of resistant phenotypes that could be selected by antiviral agents that specifically target capsid assembly. While viruses often acquire resistance to antiviral agents, resistance mutants generally exhibit lower fitness than the wildtype strain in the absence of the inhibitor Due to its rapid replication, bacteriophage X174 has become an attractive model system for evolutionary studies The inhibitory proteins most likely remove assembly intermediates by lowering the thermodynamic barriers that normally prevent off-pathway reactions (4, 5). Both off-pathway reactions and proper assembly involve D-D protein interactions across what will become the twofold axes of symmetry in the virion (8, 9). In the procapsid crystal structure, ␣-helix 3 of the D 2 , D 3 , and D 4 subunits mediates these interactions. Mutants resistant to the dominant lethal proteins were isolated in one-step genetic selections, and mutations mapped to either the coat or internal scaffolding proteins. These mutations may indirectly reinstate the avidity of the D protein electrostatic bonding partners required for productive morphogenesis (4, 5). However, the resistance phenotype is weak. To isolate a more robust phenotype, wild-type X174 was continually cultured through exponential phase cells expressing an inhibitory D protein. Results from this analysis indicate that the selection for resistance coselected for a level of dependence. The inhibitory protein stimulates resistant strain fitness significantly above the uninhibited wild-type level and appears to be required for efficient capsid nucleation. These results suggest that the virus evolved a mechanism to convert this potent antiviral agent into a beneficial factor and may be predictive of the types of resistant phenotypes that could be selected by antiviral agents that specifically target capsid assembly

Virology under the microscope—a call for rational discourse

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns – conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we – a broad group of working virologists – seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology

Microviridae

Members of the Microviridae comprise at least two subfamilies (Bullavirinae and Gokushovirinae), with divergent sequences from many uncultured representatives yet to be formally classified. Bullaviruses (canonical species φX174), which infect free-living bacteria, are among the fastest known replicating viruses. Gokushoviruses were originally thought to occupy a unique niche, infecting obligate intracellular bacteria; however, genomic analyses suggest that this group infects free-living hosts as well. Some gokushoviruses, unlike other members of the family, can undergo both lytic and lysogenic replication cycles. Microviridae contain small (4000–6000 bases), circular and single-stranded deoxyribonucleic acid (ssDNA) genomes of positive polarity, which are packaged inside small (∼25 nm diameter) T = 1 icosahedral capsids. The most well-known member of the Microviridae, φX174, has been fundamental in uncovering the mechanisms of DNA replication and capsid assembly and become a model system for experimental evolution. In contrast, little is known about the replication, structure and host range of gokushoviruses despite viromics indicating their ubiquity throughout the biosphere

φX174 Genome-Capsid Interactions Influence the Biophysical Properties of the Virion: Evidence for a Scaffolding-Like Function for the Genome during the Final Stages of Morphogenesis

During the final stages of φX174 morphogenesis, there is an 8.5-Å radial collapse of coat proteins around the packaged genome, which is tethered to the capsid's inner surface by the DNA-binding protein. Two approaches were taken to determine whether protein-DNA interactions affect the properties of the mature virion and thus the final stages of morphogenesis. In the first approach, genome-capsid associations were altered with mutant DNA-binding proteins. The resulting particles differed from the wild-type virion in density, native gel migration, and host cell recognition. Differences in native gel migration were especially pronounced. However, no differences in protein stoichiometries were detected. An extragenic second-site suppressor of the mutant DNA-binding protein restores all assayed properties to near wild-type values. In the second approach, φX174 was packaged with foreign, single-stranded, covalently closed, circular DNA molecules identical in length to the φX174 genome. The resulting particles exhibited native gel migration rates that significantly differed from the wild type. The results of these experiments suggest that the structure of the genome and/or its association with the capsid's inner surface may perform a scaffolding-like function during the procapsid-to- virion transition

Complete Virion Assembly with Scaffolding Proteins Altered in the Ability To Perform a Critical Conformational Switch▿

In the φX174 procapsid, 240 external scaffolding proteins form a nonquasiequivalent lattice. To achieve this arrangement, the four structurally unique subunits must undergo position-dependent conformational switches. One switch is mediated by glycine residue 61, which allows a 30° kink to form in α-helix 3 in two subunits, whereas the helix is straight in the other two subunits. No other amino acid should be able to produce a bend of this magnitude. Accordingly, all substitutions for G61 are nonviable but mutant proteins differ vis-à-vis recessive and dominant phenotypes. As previously reported, amino acid substitutions with side chains larger than valine confer dominant lethal phenotypes. Alone, these mutant proteins appear to have little or no biological activity but rather require the wild-type protein to interact with other structural proteins. Proteins with conservative substitutions for G61, serine and alanine, have now been characterized. Unlike the dominant lethal proteins, these proteins do not require wild-type subunits to interact with other viral proteins and cause assembly defects reminiscent of those conferred by the lethal dominant proteins in concert with wild-type subunits. Although atomic structures suggest that only a glycine residue can provide the proper torsion angle for assembly, mutants that can productively utilize the altered external scaffolding proteins were isolated, and the mutations were mapped to the coat and internal scaffolding proteins. Thus, the ability to isolate strains that could utilize the single mutant D protein species would not have been predicted from past structural analyses