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1. Disenfranchised DNA: biochemical analysis of mutant øX174 DNA-binding proteins may further elucidate the evolutionary significance of the unessential packaging protein A*

   https://doi.org/10.1128/jvi.01827-23  

   Love, Samuel D; Posey, Sierra; Burch, April D; Fane, Bentley A (March 2024, Journal of Virology)

   Parent, Kristin N (Ed.)

   ABSTRACT Most icosahedral DNA viruses package and condense their genomes into pre-formed, volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded (ss) DNA micro- and parvoviruses. Before packaging, ~120 copies of the øX174 DNA-binding protein J interact with double-stranded DNA. 60 J proteins enter the procapsid with the ssDNA genome, guiding it between 60 icosahedrally ordered DNA-binding pockets formed by the capsid proteins. Although J proteins are small, 28–37 residues in length, they have two domains. The basic, positively charged N-terminus guides the genome between binding pockets, whereas the C-terminus acts as an anchor to the capsid’s inner surface. Three C-terminal aromatic residues, W30, Y31, and F37, interact most extensively with the coat protein. Their corresponding codons were mutated, and the resulting strains were biochemically and genetically characterized. Depending on the mutation, the substitutions produced unstable packaging complexes, unstable virions, infectious progeny, or particles packaged with smaller genomes, the latter being a novel phenomenon. The smaller genomes contained internal deletions. The juncture sequences suggest that the unessential A* (A star) protein mediates deletion formation.<sc>IMPORTANCE</sc>Unessential but strongly conserved gene products are understudied, especially when mutations do not confer discernable phenotypes or the protein’s contribution to fitness is too small to reliably determine in laboratory-based assays. Consequently, their functions and evolutionary impact remain obscure. The data presented herein suggest that microvirus A* proteins, discovered over 40 years ago, may hasten the termination of non-productive packaging events. Thus, performing a salvage function by liberating the reusable components of the failed packaging complexes, such as DNA templates and replication enzymes. 

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2. STRUCTURAL MECHANISMS OF POST-PACKAGING GENOME FLOW IN BACTERIOPHAGE T4

   Rao, Venigalla (July 2024, FASEB Virus Assembly)

   Cingolani, Gino (Ed.)

   Background: Genome flow is a fundamental aspect of all biological systems. In viruses, it involves movement of nucleic acid genomes into and out of a proteinaceous capsid. Viruses must recover their newly replicated genomes into a protective capsid shell (packaging) and then safely re-introduce them into a new host (ejection) to initiate infection. While the mechanisms of DNA genome packaging in large icosahedral bacteriophages (phages) and viruses have been extensively investigated, the post-packaging mechanisms involving retention, positioning, and ejection of packaged genome are poorly understood. Aims: Using the tailed phage T4 as a model, we delineated the structural and assembly intermediates involved in transitioning a DNA-full head into an infectious virion particle, and then into a genome delivering supramolecular machine. These include intermediates of neck attachment, virion assembly, and genome release into E. coli. Methods: Various intermediates produced either by mutant phage infection or recombinant protein expression have been purified and biochemically characterized. Molecular genetic approaches were used to analyze the functional significance of amino acids involved in assembly. Structures of the purified particles were determined to near atomic resolution by cryo-electron microscopy and cryo-electron tomography. Results: Following termination of headful packaging, the pressurized T4 capsid containing tightly packed genome is sealed by the assembly of neck proteins gp13 and gp14. A dramatic conformational change in the portal dodecamer is evident, which expels the packaging motor while opening sites in portal’s “clip” domain exposed outside the capsid for binding the gp13 neck protein. Unexpectedly, we discovered that a host protein Hfq, a nucleic acid binding protein, plugs the neck structure. Hfq apparently helps to further stabilize the sealed head as it awaits tail attachment. After tail attachment, a genome end, likely the last packaged DNA, descends into the tail tube and precisely positions through interaction with an N-terminal DNA-binding motif of the tape measure protein (TMP) gp29. Six coiled-coil strands of TMP form the innermost tube of phage T4 tail, connected at the top end with DNA and at the bottom end with gp48 tube and baseplate. When the tail sheath contracts and the baseplate transform from hexagon to star shape, TMP pilots the genome to the tip of the tail tube, poised for delivery. Then, when the baseplate plug is opened fully, TMP is expelled by DNA pressure and remodels into a transmembrane channel and guides the genome to flow smoothly through the E. coli membrane envelope into the cytosol. Conclusion: Our studies describe the structural transitions of a complex and large myophage T4 in unprecedented detail. The mechanisms involve symmetry matches and mismatches, morphing, conformational transitions, and molecular remodeling that lead to genome retention, genome positioning, and genome release, precisely and efficiently. 

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3. Genome organization and interaction with capsid protein in a multipartite RNA virus

   https://doi.org/10.1073/pnas.1915078117  

   Beren, Christian; Cui, Yanxiang; Chakravarty, Antara; Yang, Xue; Rao, A. L. N.; Knobler, Charles M.; Zhou, Z. Hong; Gelbart, William M. (May 2020, Proceedings of the National Academy of Sciences)

   We report the asymmetric reconstruction of the single-stranded RNA (ssRNA) content in one of the three otherwise identical virions of a multipartite RNA virus, brome mosaic virus (BMV). We exploit a sample consisting exclusively of particles with the same RNA content—specifically, RNAs 3 and 4—assembled in planta by agrobacterium-mediated transient expression. We find that the interior of the particle is nearly empty, with most of the RNA genome situated at the capsid shell. However, this density is disordered in the sense that the RNA is not associated with any particular structure but rather, with an ensemble of secondary/tertiary structures that interact with the capsid protein. Our results illustrate a fundamental difference between the ssRNA organization in the multipartite BMV viral capsid and the monopartite bacteriophages MS2 and Qβ for which a dominant RNA conformation is found inside the assembled viral capsids, with RNA density conserved even at the center of the particle. This can be understood in the context of the differing demands on their respective lifecycles: BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in isolated, membrane-bound, cytoplasmic complexes, whereas the bacteriophages exploit sequence-specific “packaging signals” throughout the viral RNA to package their monopartite genomes. 

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4. Fate of the Urinary Tract Virus BK Human Polyomavirus in Source-Separated Urine

   https://doi.org/10.1128/AEM.02374-17  

   Goetsch, Heather E.; Zhao, Linbo; Gnegy, Mariah; Imperiale, Michael J.; Love, Nancy G.; Wigginton, Krista R.; Elkins, Christopher A. (April 2018, Applied and Environmental Microbiology)

   ABSTRACT Human polyomaviruses are emerging pathogens that infect a large percentage of the human population and are excreted in urine. Consequently, urine that is collected for fertilizer production often has high concentrations of polyomavirus genes. We studied the fate of infectious double-stranded DNA (dsDNA) BK human polyomavirus (BKPyV) in hydrolyzed source-separated urine with infectivity assays and quantitative PCR (qPCR). Although BKPyV genomes persisted in the hydrolyzed urine for long periods of time ( T 90 [time required for 90% reduction in infectivity or gene copies] of >3 weeks), the viruses were rapidly inactivated ( T 90 of 1.1 to 11 h) in most of the tested urine samples. Interestingly, the infectivity of dsDNA bacteriophage surrogate T3 ( T 90 of 24 to 46 days) was much more persistent than that of BKPyV, highlighting a major shortcoming of using bacteriophages as human virus surrogates. Pasteurization and filtration experiments suggest that BKPyV virus inactivation was due to microorganism activity in the source-separated urine, and SDS-PAGE Western blots showed that BKPyV protein capsid disassembly is concurrent with inactivation. Our results imply that stored urine does not pose a substantial risk of BKPyV transmission, that qPCR and infectivity of the dsDNA surrogate do not accurately depict BKPyV fate, and that microbial inactivation is driven by structural elements of the BKPyV capsid. IMPORTANCE We demonstrate that a common urinary tract virus has a high susceptibility to the conditions in hydrolyzed urine and consequently would not be a substantial exposure route to humans using urine-derived fertilizers. The results have significant implications for understanding virus fate. First, by demonstrating that the dsDNA (double-stranded DNA) genome of the polyomavirus lasts for weeks despite infectivity lasting for hours to days, our work highlights the shortcomings of using qPCR to estimate risks from unculturable viruses. Second, commonly used dsDNA surrogate viruses survived for weeks under the same conditions that BK polyomavirus survived for only hours, highlighting issues with using virus surrogates to predict how human viruses will behave in the environment. Finally, our mechanistic inactivation analysis provides strong evidence that microbial activity drives rapid virus inactivation, likely through capsid disassembly. Overall, our work underlines how subtle structural differences between viruses can greatly impact their environmental fate. 

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5. Unprecedented Diversity of ssDNA Phages from the Family Microviridae Detected within the Gut of a Protochordate Model Organism (Ciona robusta)

   https://doi.org/10.3390/v10080404  

   Creasy, Alexandria; Rosario, Karyna; Leigh, Brittany; Dishaw, Larry; Breitbart, Mya (August 2018, Viruses)

   Phages (viruses that infect bacteria) play important roles in the gut ecosystem through infection of bacterial hosts, yet the gut virome remains poorly characterized. Mammalian gut viromes are dominated by double-stranded DNA (dsDNA) phages belonging to the order Caudovirales and single-stranded DNA (ssDNA) phages belonging to the family Microviridae. Since the relative proportion of each of these phage groups appears to correlate with age and health status in humans, it is critical to understand both ssDNA and dsDNA phages in the gut. Building upon prior research describing dsDNA viruses in the gut of Ciona robusta, a marine invertebrate model system used to study gut microbial interactions, this study investigated ssDNA phages found in the Ciona gut. We identified 258 Microviridae genomes, which were dominated by novel members of the Gokushovirinae subfamily, but also represented several proposed phylogenetic groups (Alpavirinae, Aravirinae, Group D, Parabacteroides prophages, and Pequeñovirus) and a novel group. Comparative analyses between Ciona specimens with full and cleared guts, as well as the surrounding water, indicated that Ciona retains a distinct and highly diverse community of ssDNA phages. This study significantly expands the known diversity within the Microviridae family and demonstrates the promise of Ciona as a model system for investigating their role in animal health. 

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