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1. Structures of a large prolate virus capsid in unexpanded and expanded states generate insights into the icosahedral virus assembly

   https://doi.org/10.1073/pnas.2203272119  

   Fang, Qianglin; Tang, Wei-Chun; Fokine, Andrei; Mahalingam, Marthandan; Shao, Qianqian; Rossmann, Michael G.; Rao, Venigalla B. (October 2022, Proceedings of the National Academy of Sciences)

   Many icosahedral viruses assemble proteinaceous precursors called proheads or procapsids. Proheads are metastable structures that undergo a profound structural transition known as expansion that transforms an immature unexpanded head into a mature genome-packaging head. Bacteriophage T4 is a model virus, well studied genetically and biochemically, but its structure determination has been challenging because of its large size and unusually prolate-shaped, ∼1,200-Å-long and ∼860-Å-wide capsid. Here, we report the cryogenic electron microscopy (cryo-EM) structures of T4 capsid in both of its major conformational states: unexpanded at a resolution of 5.1 Å and expanded at a resolution of 3.4 Å. These are among the largest structures deposited in Protein Data Bank to date and provide insights into virus assembly, head length determination, and shell expansion. First, the structures illustrate major domain movements and ∼70% additional gain in inner capsid volume, an essential transformation to contain the entire viral genome. Second, intricate intracapsomer interactions involving a unique insertion domain dramatically change, allowing the capsid subunits to rotate and twist while the capsomers remain fastened at quasi-threefold axes. Third, high-affinity binding sites emerge for a capsid decoration protein that clamps adjacent capsomers, imparting extraordinary structural stability. Fourth, subtle conformational changes at capsomers’ periphery modulate intercapsomer angles between capsomer planes that control capsid length. Finally, conformational changes were observed at the symmetry-mismatched portal vertex, which might be involved in triggering head expansion. These analyses illustrate how small changes in local capsid subunit interactions lead to profound shifts in viral capsid morphology, stability, and volume. 

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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. Bacteriophage T4 Head: Structure, Assembly, and Genome Packaging

   https://doi.org/10.3390/v15020527  

   Rao, Venigalla B.; Fokine, Andrei; Fang, Qianglin; Shao, Qianqian (February 2023, Viruses)

   Bacteriophage (phage) T4 has served as an extraordinary model to elucidate biological structures and mechanisms. Recent discoveries on the T4 head (capsid) structure, portal vertex, and genome packaging add a significant body of new literature to phage biology. Head structures in unexpanded and expanded conformations show dramatic domain movements, structural remodeling, and a ~70% increase in inner volume while creating high-affinity binding sites for the outer decoration proteins Soc and Hoc. Small changes in intercapsomer interactions modulate angles between capsomer planes, leading to profound alterations in head length. The in situ cryo-EM structure of the symmetry-mismatched portal vertex shows the remarkable structural morphing of local regions of the portal protein, allowing similar interactions with the capsid protein in different structural environments. Conformational changes in these interactions trigger the structural remodeling of capsid protein subunits surrounding the portal vertex, which propagate as a wave of expansion throughout the capsid. A second symmetry mismatch is created when a pentameric packaging motor assembles at the outer “clip” domains of the dodecameric portal vertex. The single-molecule dynamics of the packaging machine suggests a continuous burst mechanism in which the motor subunits adjusted to the shape of the DNA fire ATP hydrolysis, generating speeds as high as 2000 bp/s. 

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4. A thermophilic phage uses a small terminase protein with a fixed helix–turn–helix geometry

   https://doi.org/10.1074/jbc.RA119.012224  

   Hayes, Janelle A.; Hilbert, Brendan J.; Gaubitz, Christl; Stone, Nicholas P.; Kelch, Brian A. (March 2020, Journal of Biological Chemistry)

   Tailed bacteriophages use a DNA-packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component of this DNA-packaging machinery acts as a molecular matchmaker that recognizes both the viral genome and the main motor component, the large terminase (TerL). However, how TerS binds DNA and the TerL protein remains unclear. Here we identified gp83 of the thermophilic bacteriophage P74-26 as the TerS protein. We found that TerS P76-26 oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. A cryo-EM structure of TerS P76-26 revealed that it forms a ring with a wide central pore and radially arrayed helix–turn–helix domains. The structure further showed that these helix–turn–helix domains, which are thought to bind DNA by wrapping the double helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA-binding domain imposed strong constraints on how TerS P76-26 can bind DNA. Finally, the TerS P76-26 structure lacked the conserved C-terminal β-barrel domain used by other TerS proteins for binding TerL. This suggests that a well-ordered C-terminal β-barrel domain is not required for TerS P76-26 to carry out its matchmaking function. Our work highlights a thermophilic system for studying the role of small terminase proteins in viral maturation and presents the structure of TerS P76-26 , revealing key differences between this thermophilic phage and its mesophilic counterparts. 

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5. The Effects of Packaged, but Misguided, Single-Stranded DNA Genomes Are Transmitted to the Outer Surface of the ϕX174 Capsid

   https://doi.org/10.1128/JVI.00883-21  

   Ogunbunmi, Elizabeth T.; Roznowski, Aaron P.; Fane, Bentley A. (August 2021, Journal of Virology)

   Sandri-Goldin, Rozanne M. (Ed.)

   ABSTRACT Most icosahedral viruses condense their genomes into volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded DNA (ssDNA) viruses. ssDNA genome packaging combines elements found in both double-stranded DNA (dsDNA) and ssRNA systems. Similar to dsDNA viruses, the genome is packaged into a preformed capsid. Like ssRNA viruses, there are numerous capsid-genome associations. In ssDNA microviruses, the DNA-binding protein J guides the genome between 60 icosahedrally ordered DNA binding pockets. It also partially neutralizes the DNA’s negative phosphate backbone. ϕX174-related microviruses, such as G4 and α3, have J proteins that differ in length and charge organization. This suggests that interchanging J proteins could alter the path used to guide DNA in the capsid. Previously, a ϕXG4J chimera, in which the ϕX174 J gene was replaced with the G4 gene, was characterized. It displayed lethal packaging defects, which resulted in procapsids being removed from productive assembly. Here, we report the characterization of another inviable chimera, ϕXα3J. Unlike ϕXG4J, ϕXα3J efficiently packaged DNA but produced noninfectious particles. These particles displayed a reduced ability to attach to host cells, suggesting that internal DNA organization could distort the capsid’s outer surface. Mutations that restored viability altered J-coat protein contact sites. These results provide evidence that the organization of ssDNA can affect both packaging and postpackaging phenomena. IMPORTANCE ssDNA viruses utilize icosahedrally ordered protein-nucleic acids interactions to guide and organize their genomes into preformed shells. As previously demonstrated, chaotic genome-capsid associations can inhibit ϕX174 packaging by destabilizing packaging complexes. However, the consequences of poorly organized genomes may extend beyond the packaging reaction. As demonstrated herein, it can lead to uninfectious packaged particles. Thus, ssDNA genomes should be considered an integral and structural virion component, affecting the properties of the entire particle, which includes the capsid’s outer surface. 

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