Cleavage leads to expansion of bacteriophage P4 procapsids in vitro

Wang, Sifang; Chandramouli, Preethi; Butcher, Sarah J.; Dokland, Terje

doi:10.1016/s0042-6822(03)00375-1

Cited by 9 publications

(5 citation statements)

References 29 publications

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“…Presumably, the majority of the procapsids isolated from wild-type phage lysates represents a mature form of the procapsids, ready for DNA packaging and capsid expansion. We previously found that P4 procapsids could be artificially expanded by cleavage of gpN by trypsin (Wang et al, 2003). In contrast, the present results suggest that protein cleavage and capsid maturation and expansion are disconnected processes in vivo.…”

Section: Biochemical Characterization Of P2 and P4 Capsidscontrasting

confidence: 91%

Incorporation of scaffolding protein gpO in bacteriophages P2 and P4

et al. 2008

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Scaffolding proteins act as chaperones for the assembly of numerous viruses, including most double-stranded DNA bacteriophages. In bacteriophage P2, an internal scaffolding protein, gpO, is required for the assembly of correctly formed viral capsids. Bacteriophage P4 is a satellite phage that has acquired the ability to take control of the P2 genome and use the P2 capsid protein gpN to assemble a capsid that is smaller than the normal P2 capsid. This size determination is dependent on the P4 external scaffolding protein Sid. Although Sid is sufficient to form morphologically correct P4-size capsids, the P2 internal scaffolding protein gpO is required for the formation of viable capsids of both P2 and P4. In most bacteriophages, the scaffolding protein is either proteolytically degraded or exits intact from the capsid after assembly. In the P2/P4 system, however, gpO is cleaved to an N-terminal fragment, O(*), that remains inside the mature capsid after DNA packaging. We previously showed that gpO exhibits autoproteolytic activity, which is abolished by removal of the first 25 amino acids. Co-expression of gpN with this N-terminally truncated version of gpO leads to the production of immature P2 procapsid shells. Here, we use protein analysis and mass spectroscopy to show that P2 and P4 virions as well as procapsids isolated from viral infections contain O(*) and that cleavage occurs between residues 141 and 142 of gpO. By co-expression of gpN with truncated gpO proteins, we show that O(*) binds to gpN and retains the proteolytic activity of gpO and that the C-terminal 90 residues of gpO (residues 195-284) are sufficient to promote the formation of P2-size procapsids. Using mass spectrometry, we have also identified the head completion protein gpL in the virions.

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Section: Biochemical Characterization Of P2 and P4 Capsidscontrasting

confidence: 91%

Incorporation of scaffolding protein gpO in bacteriophages P2 and P4

et al. 2008

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“…In the presence of both gpO and Sid, Sid takes precedence, leading to the formation of T =4 capsids (Wang et al, 2006). Consistent with this observation, gpO interacts only transiently with gpN, while Sid is stably associated with the procapsids until expansion occurs (Wang et al, 2003; Chang et al, 2008). …”

Section: Discussionsupporting

confidence: 66%

“…Alternatively, cleavage might destabilize intersubunit interactions in preparation for expansion. Indeed, experimental removal of the N-terminal 29 residues by trypsin treatment was previously shown to cause spontaneous capsid expansion of P4 procapsid shells (Wang et al, 2003). The presence of gpO might play a role in delaying the expansion until DNA packaging occurs [the P4 procapsids that were expanded in vitro (Wang et al, 2003) did not contain gpO].…”

Section: Discussionmentioning

confidence: 99%

Structure and size determination of bacteriophage P2 and P4 procapsids: Function of size responsiveness mutations

Dearborn

Laurinmäki

Chandramouli

et al. 2012

Journal of Structural Biology

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“…Maturation was first documented by EM in 1976 using T4 polyheads (Laemmli et al, 1976), and since then it has been described at various resolutions in a number of other dsDNA bacteriophage (Conway et al, 2001; Dokland and Murialdo, 1993; Gan et al, 2006; Jiang et al, 2003; Wang et al, 2003). Regardless of the phage analyzed, there are common structural differences between pro phage capsids and mature capsids.…”

Section: Resultsmentioning

confidence: 99%

Bacteriophage Lambda Stabilization by Auxiliary Protein gpD: Timing, Location, and Mechanism of Attachment Determined by Cryo-EM

et al. 2008

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Summary We report the cryoEM structure of bacteriophage lambda and the mechanism for stabilizing the 20Å thick capsid containing the dsDNA genome. The crystal structure of the HK97 bacteriophage capsid fits most of the T=7 lambda particle density with only minor adjustment. A prominent surface feature at the 3-fold axes corresponds to the cementing protein gpD, necessary for stabilization of the capsid shell. Its position coincides with the location of the covalent cross-link formed in the docked HK97 crystal structure, suggesting an evolutionary replacement of this gene product in lambda by autocatalytic chemistry in HK97. The crystal structure of the trimeric gpD, in which the fourteen N-terminal residues required for capsid binding are disordered, fits precisely into the corresponding EM density. The N-terminal residues of gpD are well ordered in the cryoEM density adding a strand to a beta-sheet formed by the capsid proteins and explaining the mechanism of particle stabilization.

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Cleavage leads to expansion of bacteriophage P4 procapsids in vitro

Cited by 9 publications

References 29 publications

Incorporation of scaffolding protein gpO in bacteriophages P2 and P4

Incorporation of scaffolding protein gpO in bacteriophages P2 and P4

Structure and size determination of bacteriophage P2 and P4 procapsids: Function of size responsiveness mutations

Bacteriophage Lambda Stabilization by Auxiliary Protein gpD: Timing, Location, and Mechanism of Attachment Determined by Cryo-EM

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