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Phage Assembly Pathways - to the Memory of Lindsay Black

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A special issue of Viruses (ISSN 1999-4915). This special issue belongs to the section "Bacterial Viruses".

Deadline for manuscript submissions: closed (31 December 2022) | Viewed by 39308

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Prof. Dr. Andreas Kuhn

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Institute of Microbiology and Molecular Biology, University of Hohenheim, Stuttgart, Germany
Interests: filamentous phages; protein
Special Issues, Collections and Topics in MDPI journals

Dr. Julie Thomas

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Thomas H. Gosnell School of Life Sciences, College of Science, Rochester Institute of Technology, Rochester, NY, USA
Interests: giant phages; phage structure/assembly; genomics; genetics; proteomics; host–phage interactions
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Most fascinating in protein biochemistry are the complex assembly systems that we know from phage and virus particles. Phage assembly occurs within the prokaryotic host cell and involves replicated viral proteins and genetic material being transformed into infectious progeny. The assembly of each bacteriophage particle results from a self-triggered process that is exquisitely controlled by a series of conformational cascades. In general, the assembly process is initiated by an oligomeric protein that recruits defined partner proteins in consecutive steps creating an assembly product of increasing complexity. The driving mechanism for this process is hidden in the intrinsic conformational flexibility of each protein which drives the assembly reaction forward. Therefore, numbers of phage assembly steps have been able to be reconstituted in vitro without any energetic input from the host.

Much of what we currently know regarding phage assembly is derived from the study of the classic phage genetic systems, such as that of T4. Yet, even for such model systems there remain major unresolved questions. For example, for the many of the more recently discovered phages, there is a myriad of questions regarding how their virions assemble. To address these questions an impressive array of experimental approaches available. These include the recent advent of novel technologies such as high-resolution tomography and cryo-electron microscopy which when combined with biochemical methods allow us to follow these processes in molecular detail and possibly even at an atomic resolution.

This special issue is dedicated to Lindsay W. Black. Lindsay was a great leader in the field of phage assembly. Sadly, Lindsay passed away earlier this year. For over 40 years, Lindsay dissected the molecular processes by which the T4 head assembles in his laboratory at the University of Maryland. Using the T4 system, Lindsay became a central figure in the field of DNA packaging and he continued to make contributions to this field for most of his career. Lindsay was also fascinated by other aspects of phage assembly and replication and made significant contributions to various other areas, including virus structure, and prohead assembly and maturation. Lindsay was a brilliant scientist and a true friend to many. To honor Lindsay’s legacy, we welcome submissions to this special issue that focuses on Lindsay’s research passion, phage assembly.

Prof. Dr. Andreas Kuhn
Dr. Julie Thomas
Guest Editors

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Keywords

capsid assembly
DNA packaging
portal assembly
host receptor
tail contraction
ejectosome assembly
DNA translocation
lysis control

Published Papers (12 papers)

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Research

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36 pages, 6110 KiB

Open AccessEditor’s ChoiceArticle

Apprehending the NAD⁺–ADPr-Dependent Systems in the Virus World

by Lakshminarayan M. Iyer, A. Maxwell Burroughs, Vivek Anantharaman and L. Aravind

Viruses 2022, 14(9), 1977; https://doi.org/10.3390/v14091977 - 07 Sep 2022

Cited by 6 | Viewed by 3095

Abstract

NAD⁺ and ADP-ribose (ADPr)-containing molecules are at the interface of virus–host conflicts across life encompassing RNA processing, restriction, lysogeny/dormancy and functional hijacking. We objectively defined the central components of the NAD⁺–ADPr networks involved in these conflicts and systematically surveyed 21,191 completely sequenced viral proteomes representative of all publicly available branches of the viral world to reconstruct a comprehensive picture of the viral NAD⁺–ADPr systems. These systems have been widely and repeatedly exploited by positive-strand RNA and DNA viruses, especially those with larger genomes and more intricate life-history strategies. We present evidence that ADP-ribosyltransferases (ARTs), ADPr-targeting Macro, NADAR and Nudix proteins are frequently packaged into virions, particularly in phages with contractile tails (Myoviruses), and deployed during infection to modify host macromolecules and counter NAD⁺-derived signals involved in viral restriction. Genes encoding NAD⁺–ADPr-utilizing domains were repeatedly exchanged between distantly related viruses, hosts and endo-parasites/symbionts, suggesting selection for them across the virus world. Contextual analysis indicates that the bacteriophage versions of ADPr-targeting domains are more likely to counter soluble ADPr derivatives, while the eukaryotic RNA viral versions might prefer macromolecular ADPr adducts. Finally, we also use comparative genomics to predict host systems involved in countering viral ADP ribosylation of host molecules. Full article

(This article belongs to the Special Issue Phage Assembly Pathways - to the Memory of Lindsay Black)

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20 pages, 2010 KiB

Open AccessArticle

Tryptophan Residues Are Critical for Portal Protein Assembly and Incorporation in Bacteriophage P22

by Brianna M. Woodbury, Tina Motwani, Makayla N. Leroux, Lauren F. Barnes, Nicholas A. Lyktey, Sanchari Banerjee, Corynne L. Dedeo, Martin F. Jarrold and Carolyn M. Teschke

Viruses 2022, 14(7), 1400; https://doi.org/10.3390/v14071400 - 27 Jun 2022

Cited by 2 | Viewed by 1867

Abstract

The oligomerization and incorporation of the bacteriophage P22 portal protein complex into procapsids (PCs) depends upon an interaction with scaffolding protein, but the region of the portal protein that interacts with scaffolding protein has not been defined. In herpes simplex virus 1 (HSV-1), conserved tryptophan residues located in the wing domain are required for portal-scaffolding protein interactions. In this study, tryptophan residues (W) present at positions 41, 44, 207 and 211 within the wing domain of the bacteriophage P22 portal protein were mutated to both conserved and non-conserved amino acids. Substitutions at each of these positions were shown to impair portal function in vivo, resulting in a lethal phenotype by complementation. The alanine substitutions caused the most severe defects and were thus further characterized. An analysis of infected cell lysates for the W to A mutants revealed that all the portal protein variants except W211A, which has a temperature-sensitive incorporation defect, were successfully recruited into procapsids. By charge detection mass spectrometry, all W to A mutant portal proteins were shown to form stable dodecameric rings except the variant W41A, which dissociated readily to monomers. Together, these results suggest that for P22 conserved tryptophan, residues in the wing domain of the portal protein play key roles in portal protein oligomerization and incorporation into procapsids, ultimately affecting the functionality of the portal protein at specific stages of virus assembly. Full article

(This article belongs to the Special Issue Phage Assembly Pathways - to the Memory of Lindsay Black)

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11 pages, 3221 KiB

Open AccessArticle

The M13 Phage Assembly Machine Has a Membrane-Spanning Oligomeric Ring Structure

by Maximilian Haase, Lutz Tessmer, Lilian Köhnlechner and Andreas Kuhn

Viruses 2022, 14(6), 1163; https://doi.org/10.3390/v14061163 - 27 May 2022

Cited by 2 | Viewed by 3054

Abstract

Bacteriophage M13 assembles its progeny particles in the inner membrane of the host. The major component of the assembly machine is G1p and together with G11p it generates an oligomeric structure with a pore-like inner cavity and an ATP hydrolysing domain. This allows the formation of the phage filament, which assembles multiple copies of the membrane-inserted major coat protein G8p around the extruding single-stranded circular DNA. The phage filament then passes through the G4p secretin that is localized in the outer membrane. Presumably, the inner membrane G1p/G11p and the outer G4p form a common complex. To unravel the structural details of the M13 assembly machine, we purified G1p from infected E. coli cells. The protein was overproduced together with G11p and solubilized from the membrane as a multimeric complex with a size of about 320 kDa. The complex revealed a pore-like structure with an outer diameter of about 12 nm, matching the dimensions of the outer membrane G4p secretin. The function of the M13 assembly machine for phage generation and secretion is discussed. Full article

(This article belongs to the Special Issue Phage Assembly Pathways - to the Memory of Lindsay Black)

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36 pages, 28617 KiB

Open AccessArticle

Evolution of Phage Tail Sheath Protein

by Peter Evseev, Mikhail Shneider and Konstantin Miroshnikov

Viruses 2022, 14(6), 1148; https://doi.org/10.3390/v14061148 - 26 May 2022

Cited by 8 | Viewed by 2973

Abstract

Sheath proteins comprise a part of the contractile molecular machinery present in bacteriophages with myoviral morphology, contractile injection systems, and the type VI secretion system (T6SS) found in many Gram-negative bacteria. Previous research on sheath proteins has demonstrated that they share common structural features, even though they vary in their size and primary sequence. In this study, 112 contractile phage tail sheath proteins (TShP) representing different groups of bacteriophages and archaeal viruses with myoviral morphology have been modelled with the novel machine learning software, AlphaFold 2. The obtained structures have been analysed and conserved and variable protein parts and domains have been identified. The common core domain of all studied sheath proteins, including viral and T6SS proteins, comprised both N-terminal and C-terminal parts, whereas the other parts consisted of one or several moderately conserved domains, presumably added during phage evolution. The conserved core appears to be responsible for interaction with the tail tube protein and assembly of the phage tail. Additional domains may have evolved to maintain the stability of the virion or for adsorption to the host cell. Evolutionary relations between TShPs representing distinct viral groups have been proposed using a phylogenetic analysis based on overall structural similarity and other analyses. Full article

(This article belongs to the Special Issue Phage Assembly Pathways - to the Memory of Lindsay Black)

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19 pages, 2447 KiB

Open AccessArticle

New Insights into the Structure and Assembly of Bacteriophage P1

by Miguel F. Gonzales, Denish K. Piya, Brian Koehler, Kailun Zhang, Zihao Yu, Lanying Zeng and Jason J. Gill

Viruses 2022, 14(4), 678; https://doi.org/10.3390/v14040678 - 25 Mar 2022

Cited by 6 | Viewed by 3554

Abstract

Bacteriophage P1 is the premier transducing phage of E. coli. Despite its prominence in advancing E. coli genetics, modern molecular techniques have not been applied to thoroughly understand P1 structure. Here, we report the proteome of the P1 virion as determined by liquid chromatography tandem mass-spectrometry. Additionally, a library of single-gene knockouts identified the following five previously unknown essential genes: pmgA, pmgB, pmgC, pmgG, and pmgR. In addition, proteolytic processing of the major capsid protein is a known feature of P1 morphogenesis, and we identified the processing site by N-terminal sequencing to be between E120 and S121, producing a 448-residue, 49.3 kDa mature peptide. Furthermore, the P1 defense against restriction (Dar) system consists of six known proteins that are incorporated into the virion during morphogenesis. The largest of these, DarB, is a 250 kDa protein that is believed to translocate into the cell during infection. DarB deletions indicated the presence of an N-terminal packaging signal, and the N-terminal 30 residues of DarB are shown to be sufficient for directing a heterologous reporter protein to the capsid. Taken together, the data expand on essential structural P1 proteins as well as introduces P1 as a nanomachine for cellular delivery. Full article

(This article belongs to the Special Issue Phage Assembly Pathways - to the Memory of Lindsay Black)

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17 pages, 4365 KiB

Open AccessArticle

Structural Studies of the Phage G Tail Demonstrate an Atypical Tail Contraction

by Brenda González, Daoyi Li, Kunpeng Li, Elena T. Wright, Stephen C. Hardies, Julie A. Thomas, Philip Serwer and Wen Jiang

Viruses 2021, 13(10), 2094; https://doi.org/10.3390/v13102094 - 18 Oct 2021

Cited by 4 | Viewed by 2578

Abstract

Phage G is recognized as having a remarkably large genome and capsid size among isolated, propagated phages. Negative stain electron microscopy of the host–phage G interaction reveals tail sheaths that are contracted towards the distal tip and decoupled from the head–neck region. This is different from the typical myophage tail contraction, where the sheath contracts upward, while being linked to the head–neck region. Our cryo-EM structures of the non-contracted and contracted tail sheath show that: (1) The protein fold of the sheath protein is very similar to its counterpart in smaller, contractile phages such as T4 and phi812; (2) Phage G’s sheath structure in the non-contracted and contracted states are similar to phage T4’s sheath structure. Similarity to other myophages is confirmed by a comparison-based study of the tail sheath’s helical symmetry, the sheath protein’s evolutionary timetree, and the organization of genes involved in tail morphogenesis. Atypical phase G tail contraction could be due to a missing anchor point at the upper end of the tail sheath that allows the decoupling of the sheath from the head–neck region. Explaining the atypical tail contraction requires further investigation of the phage G sheath anchor points. Full article

(This article belongs to the Special Issue Phage Assembly Pathways - to the Memory of Lindsay Black)

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14 pages, 997 KiB

Open AccessArticle

Intravirion DNA Can Access the Space Occupied by the Bacteriophage P22 Ejection Proteins

by Justin C. Leavitt, Eddie B. Gilcrease, Brianna M. Woodbury, Carolyn M. Teschke and Sherwood R. Casjens

Viruses 2021, 13(8), 1504; https://doi.org/10.3390/v13081504 - 30 Jul 2021

Cited by 4 | Viewed by 2685

Abstract

Tailed double-stranded DNA bacteriophages inject some proteins with their dsDNA during infection. Phage P22 injects about 12, 12, and 30 molecules of the proteins encoded by genes 7, 16 and 20, respectively. After their ejection from the virion, they assemble into a trans-periplasmic conduit through which the DNA passes to enter the cytoplasm. The location of these proteins in the virion before injection is not well understood, although we recently showed they reside near the portal protein barrel in DNA-filled heads. In this report we show that when these proteins are missing from the virion, a longer than normal DNA molecule is encapsidated by the P22 headful DNA packaging machinery. Thus, the ejection proteins occupy positions within the virion that can be occupied by packaged DNA when they are absent. Full article

(This article belongs to the Special Issue Phage Assembly Pathways - to the Memory of Lindsay Black)

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14 pages, 13902 KiB

Open AccessArticle

Membrane Insertion of the M13 Minor Coat Protein G3p Is Dependent on YidC and the SecAYEG Translocase

by Farina Kleinbeck and Andreas Kuhn

Viruses 2021, 13(7), 1414; https://doi.org/10.3390/v13071414 - 20 Jul 2021

Cited by 4 | Viewed by 2218

Abstract

The minor coat protein G3p of bacteriophage M13 is the key component for the host interaction of this virus and binds to Escherichia coli at the tip of the F pili. As we show here, during the biosynthesis of G3p as a preprotein, the signal sequence interacts primarily with SecY, whereas the hydrophobic anchor sequence at the C-terminus interacts with YidC. Using arrested nascent chains and thiol crosslinking, we show here that the ribosome-exposed signal sequence is first contacted by SecY but not by YidC, suggesting that only SecYEG is involved at this early stage. The protein has a large periplasmic domain, a hydrophobic anchor sequence of 21 residues and a short C-terminal tail that remains in the cytoplasm. During the later synthesis of the entire G3p, the residues 387, 389 and 392 in anchor domain contact YidC in its hydrophobic slide to hold translocation of the C-terminal tail. Finally, the protein is processed by leader peptidase and assembled into new progeny phage particles that are extruded out of the cell. Full article

(This article belongs to the Special Issue Phage Assembly Pathways - to the Memory of Lindsay Black)

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Review

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30 pages, 10878 KiB

Open AccessReview

Bacteriophage T4 Head: Structure, Assembly, and Genome Packaging

by Venigalla B. Rao, Andrei Fokine, Qianglin Fang and Qianqian Shao

Viruses 2023, 15(2), 527; https://doi.org/10.3390/v15020527 - 14 Feb 2023

Cited by 8 | Viewed by 5331

Abstract

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. Full article

(This article belongs to the Special Issue Phage Assembly Pathways - to the Memory of Lindsay Black)

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39 pages, 3405 KiB

Open AccessReview

Bacteriophage P22 Capsid as a Pluripotent Nanotechnology Tool

by Victor Alejandro Essus, Getúlio Silva e Souza Júnior, Gabriel Henrique Pereira Nunes, Juliana dos Santos Oliveira, Bruna Mafra de Faria, Luciana Ferreira Romão and Juliana Reis Cortines

Viruses 2023, 15(2), 516; https://doi.org/10.3390/v15020516 - 13 Feb 2023

Cited by 6 | Viewed by 2566

Abstract

The Salmonella enterica bacteriophage P22 is one of the most promising models for the development of virus-like particle (VLP) nanocages. It possesses an icosahedral T = 7 capsid, assembled by the combination of two structural proteins: the coat protein (gp5) and the scaffold protein (gp8). The P22 capsid has the remarkable capability of undergoing structural transition into three morphologies with differing diameters and wall-pore sizes. These varied morphologies can be explored for the design of nanoplatforms, such as for the development of cargo internalization strategies. The capsid proteic nature allows for the extensive modification of its structure, enabling the addition of non-native structures to alter the VLP properties or confer them to diverse ends. Various molecules were added to the P22 VLP through genetic, chemical, and other means to both the capsid and the scaffold protein, permitting the encapsulation or the presentation of cargo. This allows the particle to be exploited for numerous purposes—for example, as a nanocarrier, nanoreactor, and vaccine model, among other applications. Therefore, the present review intends to give an overview of the literature on this amazing particle. Full article

(This article belongs to the Special Issue Phage Assembly Pathways - to the Memory of Lindsay Black)

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17 pages, 3596 KiB

Open AccessReview

The Beauty of Bacteriophage T4 Research: Lindsay W. Black and the T4 Head Assembly

by Andreas Kuhn and Julie A. Thomas

Viruses 2022, 14(4), 700; https://doi.org/10.3390/v14040700 - 28 Mar 2022

Cited by 6 | Viewed by 4633

Abstract

Viruses are biochemically complex structures and mainly consist of folded proteins that contain nucleic acids. Bacteriophage T4 is one of most prominent examples, having a tail structure that contracts during the infection process. Intracellular phage multiplication leads to separate self-directed assembly reactions of proheads, tails and tail fibers. The proheads are packaged with concatemeric DNA produced by tandem replication reactions of the parental DNA molecule. Once DNA packaging is completed, the head is joined with the tail and six long fibers are attached. The mature particles are then released from the cell via lysis, another tightly regulated process. These processes have been studied in molecular detail leading to a fascinating view of the protein-folding dynamics that direct the structural interplay of assembled complexes. Lindsay W. Black dedicated his career to identifying and defining the molecular events required to form the T4 virion. He leaves us with rich insights into the astonishingly precise molecular clockwork that co-ordinates all of the players in T4 assembly, both viral and cellular. Here, we summarize Lindsay’s key research contributions that are certain to stimulate our future science for many years to come. Full article

(This article belongs to the Special Issue Phage Assembly Pathways - to the Memory of Lindsay Black)

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Other

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11 pages, 1443 KiB

Open AccessHypothesis

Enteric Chromosomal Islands: DNA Packaging Specificity and Role of λ-like Helper Phage Terminase

by Helios Murialdo and Michael Feiss

Viruses 2022, 14(4), 818; https://doi.org/10.3390/v14040818 - 15 Apr 2022

Cited by 2 | Viewed by 1749

Abstract

The phage-inducible chromosomal islands (PICIs) of Gram-negative bacteria are analogous to defective prophages that have lost the ability to propagate without the aid of a helper phage. PICIs have acquired genes that alter the genetic repertoire of the bacterial host, including supplying virulence factors. Recent work by the Penadés laboratory elucidates how a helper phage infection or prophage induction induces the island to excise from the bacterial chromosome, replicate, and become packaged into functional virions. PICIs lack a complete set of morphogenetic genes needed to construct mature virus particles. Rather, PICIs hijack virion assembly functions from an induced prophage acting as a helper phage. The hijacking strategy includes preventing the helper phage from packaging its own DNA while enabling PICI DNA packaging. In the case of recently described Gram-negative PICIs, the PICI changes the specificity of DNA packaging. This is achieved by an island-encoded protein (Rpp) that binds to the phage protein (TerS), which normally selects phage DNA for packaging from a DNA pool that includes the helper phage and host DNAs. The Rpp–TerS interaction prevents phage DNA packaging while sponsoring PICI DNA packaging. Our communication reviews published data about the hijacking mechanism and its implications for phage DNA packaging. We propose that the Rpp–TerS complex binds to a site in the island DNA that is positioned analogous to that of the phage DNA but has a completely different sequence. The critical role of TerS in the Rpp–TerS complex is to escort TerL to the PICI cosN, ensuring appropriate DNA cutting and packaging. Full article

(This article belongs to the Special Issue Phage Assembly Pathways - to the Memory of Lindsay Black)

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