Targeted mutagenesis of the P22 portal protein reveals the mechanism of signal transmission during DNA packaging

ABSTRACT Herpes simplex virus 1 (HSV-1) requires seven proteins to package its genome through a vertex in its capsid, one of which is the portal protein, pUL6. The portal protein is also thought to facilitate assembly of the procapsid. While the portal has been visualized in mature capsids, we aimed to elucidate its role in the assembly and maturation of procapsids using cryo-electron tomography (cryoET). We identified the portal vertex in individual procapsids, calculated a subtomogram average, and compared that with the portal vertex in empty mature capsids (A-capsids). The resulting maps show the portal on the interior surface with its narrower end facing outwards, while maintaining close contact with the capsid shell. In the procapsid, the portal is embedded in the underlying scaffold, suggesting that assembly involves a portal-scaffold complex. During maturation, the capsid shell angularizes with a corresponding outward movement of the vertices. We found that in A-capsids, the portal translocates outward further than the adjacent capsomers and strengthens its contacts with the capsid shell. Our methodology also allowed us to determine the number of portal vertices in each capsid, with most having one per capsid, but some none or two, and rarely three. The predominance of a single portal per capsid supports facilitation of the assembly of the procapsid. IMPORTANCE Herpes simplex virus 1 (HSV-1) infects a majority of humans, causing mostly mild disease but in some cases progressing toward life-threatening encephalitis. Understanding the life cycle of the virus is important to devise countermeasures. Production of the virion starts with the assembly of an icosahedral procapsid, which includes DNA packaging proteins at a vertex, one of which is the dodecameric portal protein. The procapsid then undergoes maturation and DNA packaging through the portal, driven by a terminase complex. We used cryo-electron tomography to visualize the portal in procapsids and compare them to mature empty capsids. We found the portal located inside one vertex interacting with the scaffold protein in the procapsid. On maturation, the scaffold is cleaved and dissociates, the capsid angularizes, and the portal moves outward, interacting closely with the capsid shell. These transformations may provide a basis for the development of drugs to prevent HSV-1 infections.

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Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1813204116 ◽

2019 ◽

Vol 116 (9) ◽

pp. 3556-3561 ◽

Cited By ~ 23

Author(s):

Oliver W. Bayfield ◽

Evgeny Klimuk ◽

Dennis C. Winkler ◽

Emma L. Hesketh ◽

Maria Chechik ◽

...

Keyword(s):

Conformational Changes ◽

Structural Integrity ◽

Thermus Thermophilus ◽

Dna Packaging ◽

Dna Viruses ◽

Portal Protein ◽

Single Vertex ◽

Double Stranded Dna ◽

Β Sheets

Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus. Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit β-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside.

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Alterations of the Portal Protein, gpB, of Bacteriophage λ Suppress Mutations in cosQ, the Site Required for Termination of DNA Packaging

Genetics ◽

10.1093/genetics/161.1.21 ◽

2002 ◽

Vol 161 (1) ◽

pp. 21-31 ◽

Cited By ~ 1

Author(s):

Douglas J Wieczorek ◽

Lisa Didion ◽

Michael Feiss

Keyword(s):

Dna Packaging ◽

Missense Mutations ◽

Bacteriophage Λ ◽

Portal Protein ◽

Suppression Effects

Abstract The cosQ site of bacteriophage λ is required for DNA packaging termination. Previous studies have shown that cosQ mutations can be suppressed in three ways: by a local suppressor within cosQ, an increase in the length of the λ chromosome, and missense mutations affecting the prohead’s portal protein, gpB. In the present work, revertants of a set of lethal cosQ mutants were screened for suppressors. Seven new cosQ suppressors affected gene B, which encodes the portal protein of the prohead. All seven were allelenonspecific suppressors of cosQ mutations. Experiments with several phages having two cosQ suppressors showed that the suppression effects were additive. Furthermore, these double suppressors had minimal effects on the growth of cosQ+ phages. These trans-acting suppressors affecting the portal protein are proposed to allow the mutant cosQ site to be more efficiently recognized, due to the slowing of the rate of translocation.

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The portal protein plays essential roles at different steps of the SPP1 DNA packaging process

Virology ◽

10.1016/j.virol.2004.02.012 ◽

2004 ◽

Vol 322 (2) ◽

pp. 253-263 ◽

Cited By ~ 42

Author(s):

Anabela Isidro ◽

Adriano O Henriques ◽

Paulo Tavares

Keyword(s):

Dna Packaging ◽

Portal Protein ◽

Packaging Process

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Genetics of cosQ, the DNA-Packaging Termination Site of Phage λ: Local Suppressors and Methylation Effects

Genetics ◽

10.1093/genetics/165.1.11 ◽

2003 ◽

Vol 165 (1) ◽

pp. 11-21

Author(s):

Douglas J Wieczorek ◽

Michael Feiss

Keyword(s):

Base Pair ◽

Consensus Sequence ◽

Point Mutations ◽

Dna Packaging ◽

Recognition Site ◽

Missense Mutations ◽

Bacteriophage Λ ◽

Portal Protein ◽

Virion Assembly ◽

Cis Acting

Abstract The cos site of the bacteriophage λ chromosome contains the sites required for DNA processing and packaging during virion assembly. cos is composed of three subsites, cosQ, cosN, and cosB. cosQ is required for the termination of chromosome packaging. Previous studies have shown cosQ mutations to be suppressed in three ways: by a local suppressor within cosQ; by an increase in the length of the λ chromosome; and by missense mutations affecting the prohead's portal protein, gpB. In the first study reported here, revertants of a set of cosQ mutants were screened for suppressors, and cis-acting suppressors of cosQ mutations were studied; these included second-site cosQ point mutations, base-pair insertions within cosQ, and an additional genome-lengthening suppressor. The 7-bp-long cosQ, with the sequence 5′-GGGTCCT-3′, coincides exactly with the recognition site for the EcoO109I restriction/methylation system, which has the consensus sequence 5′-PuGGNCCPy-3′. In a second study, EcoO109I methylation was found to strongly interfere with the residual cosQ function of leaky cosQ mutants. cis-acting suppressors that overcome methylation-associated defects, including a methylation-dependent suppressor, were also isolated. Models of cosQ suppression are presented.

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Purification and organization of the gene 1 portal protein required for phage P22 DNA packaging

Biochemistry ◽

10.1021/bi00406a009 ◽

1988 ◽

Vol 27 (6) ◽

pp. 1849-1856 ◽

Cited By ~ 62

Author(s):

Christopher Bazinet ◽

Julyet Benbasat ◽

Jonathan King ◽

Jose Maria Carazo ◽

Jose L. Carrascosa

Keyword(s):

Dna Packaging ◽

Portal Protein ◽

Phage P22

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

Viruses ◽

10.3390/v13081504 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1504

Author(s):

Justin C. Leavitt ◽

Eddie B. Gilcrease ◽

Brianna M. Woodbury ◽

Carolyn M. Teschke ◽

Sherwood R. Casjens

Keyword(s):

Dna Packaging ◽

Bacteriophage P22 ◽

Portal Protein ◽

Dna Molecule ◽

Double Stranded Dna ◽

Phage P22

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.

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Genetic Evidence That Recognition of cosQ the Signal for Termination of Phage λ DNA Packaging, Depends on the Extent of Head Filling

Genetics ◽

10.1093/genetics/147.1.7 ◽

1997 ◽

Vol 147 (1) ◽

pp. 7-17 ◽

Cited By ~ 3

Author(s):

David Cue ◽

Michael Feiss

Keyword(s):

Binding Sites ◽

Small Subunit ◽

Dna Packaging ◽

Missense Mutations ◽

Portal Protein ◽

Maximum Capacity ◽

Sequence Identity ◽

Different Types ◽

A Site ◽

Binding Determinants

Abstract Packaging a phage λ chromosome involves cutting the chromosome from a concatemer and translocating the DNA into a prohead. The cutting site, cos, consists of three subsites: cosN, the nicking site; cosB, a site required for packaging initiation; and cosQ a site required for termination of packaging. cosB contains three binding sites (R sequences) for gpNul, the small subunit of terminase. Because cosQ has sequence identity to the R sequences, it has been proposed that cosQ is also recognized by gpNul. Suppressors of cosB mutations were unable to suppress a cosQ point mutation. Suppressors of a cosQ mutation (cosQ1) were isolated and found to be of three sorts, the first affecting a base pair in cosQ. The second type of cosQ suppression involved increasing the length of the phage chromosome to a length near to the maximum capacity of the head shell. A third class of suppressors were missense mutations in gene B, which encodes the portal protein of the virion. It is speculated that increasing DNA length and altering the portal protein may reduce the rate of translocation, thereby increasing the efficiency of recognition of the mutant cosQ. None of the cosQ suppressors was able to suppress cosB mutations. Because cosQ and cosB mutations are suppressed by very different types of suppressors, it is concluded that cosQ and the R sequences of cosB are recognized by different DNA-binding determinants.

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Analysis of capsid portal protein and terminase functional domains: interaction sites required for DNA packaging in bacteriophage T4

Journal of Molecular Biology ◽

10.1006/jmbi.1999.2781 ◽

1999 ◽

Vol 289 (2) ◽

pp. 249-260 ◽

Cited By ~ 51

Author(s):

Hsingchi Lin ◽

Venigalla B Rao ◽

Lindsay W Black

Keyword(s):

Bacteriophage T4 ◽

Dna Packaging ◽

Functional Domains ◽

Portal Protein ◽

Interaction Sites

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Portal Protein: The Orchestrator of Capsid Assembly for the dsDNA Tailed Bacteriophages and Herpesviruses

Annual Review of Virology ◽

10.1146/annurev-virology-092818-015819 ◽

2019 ◽

Vol 6 (1) ◽

pp. 141-160 ◽

Cited By ~ 10

Author(s):

Corynne L. Dedeo ◽

Gino Cingolani ◽

Carolyn M. Teschke

Keyword(s):

Drug Target ◽

Sequence Similarity ◽

Antiviral Drug ◽

Viral Assembly ◽

Dna Packaging ◽

Capsid Assembly ◽

Portal Protein ◽

Ring Assembly ◽

Double Stranded Dna

Tailed, double-stranded DNA bacteriophages provide a well-characterized model system for the study of viral assembly, especially for herpesviruses and adenoviruses. A wealth of genetic, structural, and biochemical work has allowed for the development of assembly models and an understanding of the DNA packaging process. The portal complex is an essential player in all aspects of bacteriophage and herpesvirus assembly. Despite having low sequence similarity, portal structures across bacteriophages share the portal fold and maintain a conserved function. Due to their dynamic role, portal proteins are surprisingly plastic, and their conformations change for each stage of assembly. Because the maturation process is dependent on the portal protein, researchers have been working to validate this protein as a potential antiviral drug target. Here we review recent work on the role of portal complexes in capsid assembly, including DNA packaging, as well as portal ring assembly and incorporation and analysis of portal structures.

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