Characterization of a Dual-Function Domain That Mediates Membrane Insertion and Excision of Ff Filamentous Bacteriophage

J. Mol. Biol. (2011) 411, 972–985

doi:10.1016/j.jmb.2011.07.002 Contents lists available at www.sciencedirect.com

Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

Characterization of a Dual-Function Domain That Mediates Membrane Insertion and Excision of Ff Filamentous Bacteriophage Nicholas J. Bennett, Dragana Gagic, Andrew J. Sutherland-Smith and Jasna Rakonjac⁎ Institute of Molecular BioSciences, Massey University, Palmerston North 4442, New Zealand Received 24 March 2011; received in revised form 24 June 2011; accepted 1 July 2011 Available online 13 July 2011 Edited by M. Gottesman Keywords: viral infection; phage entry; g3p; membrane proteins; pore-forming proteins

The filamentous phage Ff (f1, fd, or M13) of Escherichia coli is assembled at the cell membranes by a process that is morphologically similar to that of pilus assembly. The release of the filament virion is mediated by excision from the membrane; conversely, entry into a host cell is mediated by insertion of the virion coat proteins into the membrane. The N-terminal domains of the minor virion protein pIII have the sole role of binding to host receptors during infection. In contrast, the C domain of pIII is required for two opposite functions: insertion of the virion into the membrane during infection and excision at the termination step of assembly/secretion. We identified a 28-residue-long segment in the pIII C domain, which is required for phage entry but dispensable for release from the membrane at the end of assembly. This segment, which we named the infection-competence segment (ICS), works only in cis with the N-terminal receptor-binding domains and does not require the equivalent ICS sequences in other subunits within the virion cap. The ICS contains a predicted amphipathic α-helix and is rich in small amino acids, Gly, Ala, and Ser, which are arranged as a [small]XXX[small]XX[small]XXX[small]XXX[small] motif. Scanning Ala/ Gly mutagenesis of ICS showed that small residues are compatible with infection. Overall, organization of the C domain is reminiscent of α-helical poreforming toxins' membrane insertion domains. The unique ability of pIII to mediate both membrane insertion and excision allowed us to compare these two fundamental membrane transactions and to show that receptor-triggered insertion is a more complex process than excision from membranes. © 2011 Elsevier Ltd. All rights reserved.

*Corresponding author. E-mail address: [email protected]. Present addresses: N. J. Bennett, Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada H3A 2B4; D. Gagic, AgResearch Ltd., Grasslands Research Centre, Tennent Drive, Private Bag 11008, Palmerston North 4442, New Zealand. Abbreviations used: ICS, infection-competence segment; ssDNA, single-stranded DNA; cC, complete C domain; WTpIII, wild-type pIII; PEG, polyethylene glycol; TBS, Tris-buffered saline; TAE, Tris–acetate– ethylenediaminetetraacetic acid.

Introduction The F-pilus-specific filamentous phage of Escherichia coli (f1, fd, or M13), collectively called Ff, have been used extensively (and interchangeably) as a model system for protein translocation and insertion into the membranes of Gram-negative bacteria.1–3 They have also been the principal workhorse of phage display technology.4–6 Virion protein III (pIII hereinafter), the subject of this work, is commonly used as a platform for display of peptides and proteins. Filamentous bacteriophage of pathogenic Gram-negative bacteria have important roles in the

0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.

Membrane Insertion/Excision Domain of Ff pIII

horizontal transfer of toxin genes (Vibrio cholerae CTXφ7) and biofilm dynamics (Pseudomonas aeruginosa phage Pf48). The Ff virion, a long, somewhat flexible filament, is composed of five proteins. The major coat protein pVIII is present in about 2700 copies as a helical array of overlapping subunits, forming a tubelike structure that encloses the single-stranded DNA (ssDNA) genome.9–13 The termini of the filament contain different pairs of minor coat proteins, pVII/ pIX and pIII/pVI, each present in the virion in five copies, likely corresponding to the 5-fold rotational symmetry of Ff phage capsid.14,15 Of the five virion proteins, pVIII, pVII, pIX, and pVI are small and mostly hydrophobic, whereas pIII is relatively large and mostly hydrophilic. These five proteins are integral membrane proteins prior to their incorporation into virions. pIII is composed of three domains, N1, N2, and C (also referred to as D1, D2 and D316), separated by long, flexible glycine-rich linkers. The Nterminal domains bind to primary and secondary receptors.16–19 In Ff phage, the order of receptorbinding domains is inversely paired to the primary and secondary receptors: N2 binds the primary receptor, the tip of the F pilus, whereas N1 binds the secondary receptor, the periplasmic domain of integral inner-membrane protein TolA.16–19 pIII is the least conserved protein among different filamentous phage, particularly the two receptorbinding N-terminal domains N1 and N2, the order of which can vary in different phage.20 The primary receptors for filamentous phage are several types of pili,7,20,21 whereas the secondary receptor in at least three filamentous phage was shown to be TolA.16,22,23 The C-terminal domain is conserved among E. coli-infecting filamentous phage but not in filamentous phage infecting more distantly related species (e.g., CTXφ of V. cholerae).24 The C-terminal hydrophobic membrane anchor, however, is present in all pIII homologues. The C domain is part of the detergent-resistant virion cap complex, together with pVI. The receptor-binding steps have been well studied using genetic, biochemical, and structural approaches.16,17,23,25,26 In contrast, little is known of the post-receptor-binding events that lead to the entry process itself.18,27 Similarly, there is a wealth of information about phage assembly extrusion at the genetic and biochemical levels, and there is some structural information on the exit port in Ff.2,28–30 However, the mechanistic details of the assembly and termination process are not clear. Different sets of accessory trans-envelope protein complexes are required for these two processes: host-encoded pilus/TolQRA18,22 for infection and phage-encoded pIV/pI/pXI for assembly.28 Earlier studies found that, in Ff phage of E. coli, a small C-terminal fragment of the pIII C domain (83

973 residues) is sufficient for incorporation into the virion, whereas a slightly longer C-terminal fragment (93 residues) is required for switching from extension of the virion filament to release from the infected cell.31 Protein pVI, which co-localizes with pIII at the same end of the virion, is also required for release of the phage, and both proteins interact with the major coat protein pVIII prior to assembly into the virion.32 It was proposed that upon incorporation into the virion, a conformational change in the C domain of pIII disrupts the hydrophobic interactions of the pIII C-terminal membrane anchor (and the virion) with the membrane, catalyzing the release of the phage.31 We have now identified the Ff pIII C domain segment of 28 residues required specifically for infection and redundant for assembly. Secondaryand tertiary-structure modeling and the presence of clustered, small-residue-rich and aromatic-residuerich sequences of the pIII C-terminal domain combine features of α-helical pore-forming proteins from bacteria and membrane fusion proteins from eukaryotic enveloped viruses.

Results Experimental setup To define the portion of the C domain required for the process of phage infection, we constructed a series of nested deletion mutants containing the progressively shortened C domain of pIII from the N-terminal end and fused them to the receptorbinding N1N2 domains to produce fusions named N-tC141, N-tC132, N-tC121, N-tC111, N-tC93, and N-tC83 (Fig. 1b). The prefix N-tC stands for N1N2 domain fusion (N-) to truncated C domain (tC); the number corresponds to the size of the C-terminal fragment that each fusion contains. (For diagrams of fusions, please refer to Fig. 1b.) Using these fusions alone to test infection is complicated because the C domain is involved in both terminating assembly and stabilizing the virion.27,31 In order to separate the effect of truncated C domains on infection from their effect on assembly and virion stability, we produced composite virions that contained two different derivatives of pIII: the complete C domain (cC) of pIII and one of the N-tC fusions. The composite virions were denoted N-tC/cC (NtC141/cC, N-tC132/Cc, N-tC121/Cd, N-tC111/Cc, N-tC93/Cc, and N-tC83/Cc; Fig. 1d). The cC subunit (Fig. 1a) does not contain receptor-binding domains N1 and N2; hence, it does not contribute to the ability of virions to infect host cells.27 The N-tC fusions were expressed from plasmids, and cC was expressed from a phagemid (pYW01), for production of phagemid particles, or from a plasmid

Membrane Insertion/Excision Domain of Ff pIII

974

(a) SS

Complete C domain (cC)

Wild-type pIII (WTpIII) G

N1

(b)

N2

C

G

TM

SS

tC141

N-tC132

tC132

N-tC121

tC121

N-tC111

tC111

N-tC93

tC93

N-tC83

tC83 Virions of complemented ΔgIII helper phage VCSM13d3 or f1d3 (input phage)

WTpIII

2

WTpIII

+ WTpIII

Δ gIII helper phage

+ cC

WTpIII/cC (Positive control)

3

cC

Δ gIII helper phage

N-tC141 to N-tC83

N-tC141 to N-tC83

cC

N-tC/cC (Table 1)

4

tC141 to tC83

ΔgIII helper phage

WTpIII

+

+

cC

N-tC141

(d)

cC

TM

(c) tC mutants (tC141 to tC83)

N-tC fusions (N-tC141 to NtC83)

1

C

WTpIII

+ WTpIII

cC

+

+

tC141 to tC83

WTpIII/tC (Table 2)

ΔgIII helper phage

-

cC

cC (Negative control)

•Produced (output) virions were purified and concentrated •Titers of the infectious virions were determined •Virions were quantified •Infectivity and relative infectivity were calculated

Fig. 1. Schematic representation of experimental design. (a–c) Diagrams of the proteins used to assemble composite virions. (a) WTpIII and cC of pIII; (b) N-tC fusions (N-tC141, N-tC132, N-tC121, N-tC111, N-tC93, and N-tC83); (c) truncated C domains (tC141, tC132, tC121, tC111, tC93, and tC83). SS, signal sequence (white block arrow); N1, N1 domain (green); G, glycine-rich linkers (black line); N2, N2 domain (blue); cC, C domain (orange); TM, transmembrane anchor (orange rectangle). Dashed boxes, deleted C domain segment in the N-tC fusions and tC mutants. Shapes to the right of block diagrams symbolize the domain organization of each mutant in folded state; green, blue, and orange ovals connected by black lines represent N1, N2, and cC domains, respectively, connected by the long glycine-rich linkers. (d) Production and analysis of the composite virions. Virions in the figure are symbolized by pIII-containing caps and a small number of the neighboring major coat protein subunits (black rectangles). For simplicity, only three pIII molecules, instead of five, are shown. Top, helper phage stock (VCSM13d3 or f1d3) carrying a complete deletion of gIII in the genome but a WTpIII in the virion.33 Middle, host cells used for production of composite virions and controls: (1) WTpIII/cC, (2) N-tC/cC (infectivity data in Table 1), (3) WTpIII/tC (infectivity data in Table 2), (4) cC virions, negative control. Plasmids and products are indicated within the cells. Produced virions are shown below the cells.

(pNJB50), for production of phage. Helper phage that lack gIII entirely33 were used to produce the composite virions. A flowchart diagram of the

composite virion production is shown in Fig. 1d. The resulting progeny virions were then tested for assembly and stability, as well as for co-

Membrane Insertion/Excision Domain of Ff pIII MW (kDa) 1

2

3

4

5

6

7

8

9

(a) 85 60

50

WTpIII

N-tC fusions

40

25 20 C domain

(b)

pVI

Fig. 2. Western blotting of the virion proteins. (a) Detection of pIII using anti-pIII antibodies raised against a C-terminal decapeptide of pIII.33 (b) Detection of pVI using anti-pVI antibody.28 Virions contain pIII mutant proteins or the wild type and combinations thereof as indicated: lane 1, cC; lane 2, WTpIII/cC; lane 3, N-tC83/ cC; lane 4, N-tC93/cC; lane 5, N-tC111/cC; lane 6, NtC121/cC; lane 7, N-tC132/cC; lane 8, N-tC141/cC; lane 9, WTpIII/cC. Bands corresponding to WTpIII, N-tC fusions, and cC are indicated.

incorporation of cC and each of the N-tC fusions into the virions. Incorporation of pIII mutant proteins into the virions Poor incorporation of N-tC fusions into composite N-tC/cC virions would result in a reduction in infectivity, not because of the inability of a particular tC domain to mediate phage entry, but rather due to the absence of the receptor-binding N1N2 domains from the virions. To rule this out, we examined the composite N-tC/cC virions (N-tC141/cC, N-tC132/ cC, N-tC121/cC, N-tC111/cC, N-tC93/cC, and NtC83/cC) for the presence of N-tC subunits. Western blotting detected the N-tC subunits (N-tC141, NtC132, N-tC121, N-tC111, N-tC93, and N-tC83) in purified virions (Fig. 2a). We also assessed the presence of pVI, the protein that forms the virion cap together with pIII.32 pVI is required for proper assembly of the phage;31 its role in infection has not been determined. pVI was detected in all samples (Fig. 2b), indicating that it was incorporated efficiently into composite virions. Therefore, all composite virions contained the expected proteins. Assembly and stability of composite virions The length of Ff virions is the most significant indicator of pIII-mediated assembly and release from the membrane. If pIII-mediated release does

975 not occur, such as in N-tC83-expressing cells in the absence of cC,27 then the virion filaments continue to elongate, until they are so long that they break off the host cells by mechanical shear. Due to their large size, these broken-off virions (termed polyphage) appear as a slow-migrating smear just below the wells in native agarose electrophoresis. Wild-type f1 virions appear as a ladder of two to three bands, with the fastest-migrating band representing a virion containing a single genome (monophage) and two slower-migrating bands representing virions that contain two or three sequentially packaged genomes (oligophage). Whereas monophage account for ∼ 90% of wild-type f1 virions, a larger contribution of longer phage (oligophage) is characteristic for ΔgIII phage complemented by wildtype pIII (WTpIII) expressed from a plasmid, or for suppressed gIIIam mutants.31,34,35 In addition, the complemented ΔgIII virion samples contain lowmigrating polyphage, appearing as a smear between the wells and the ladder of oligophage. Polyphage, but not oligophage, are disassembled by the detergent Sarkosyl (Fig. 3, lane 9 versus lane 18). The polyphage presumably arise due to the limited access of pIII to some of the few hundreds of assembly complexes in each of the cells.35 Similar to the composite virions containing a combination of WTpIII and cC (WTpIII/cC; Fig. 3, lanes 1 and 10), composite N-tC/cC virions (NtC141/cC, N-tC132/cC, N-tC121/cC, N-tC111/cC, N-tC93/cC, and N-tC83/cC) migrate as a ladder of virion bands (monophage and oligophage) in the native agarose gel electrophoresis (Fig. 3a). In

(a)

(b)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 17 18 19

Polyphage

Oligophage

Monophage Free ssDNA

Fig. 3. Native agarose gel electrophoresis of the virions. Untreated (a) or Sarkosyl-treated (b) phage virions were separated by agarose gel electrophoresis. Virions contain N-tC or WTpIII in combination with cC. Lanes 1 and 10, WTpIII/cC; lanes 2 and 11, cC; lanes 3 and 12, N-tC83/cC; lanes 4 and 13, N-tC93/cC; lanes 5 and 14, N-tC111/cC, lanes 6 and 15, N-tC121/cC; lanes 7 and 16, N-tC132/cC; lanes 8 and 17, N-tC141/cC; lanes 9 and 18, WTpIII only (f1d3 helper phage stock); lane 19, purified f1d3 ssDNA.

Membrane Insertion/Excision Domain of Ff pIII

976 addition to the ladder of oligophage, samples variably contained a population of polyphage that migrated just below the wells and above the oligophage ladder. To determine whether the cap of the composite virions is structurally stable, we tested their ability to resist Sarkosyl-mediated disassembly. The ladder of oligophage bands from all tested composite virions was unaffected by Sarkosyl (Fig. 3b), showing that the composite virions have a stable virion cap. Furthermore, as we showed previously, cC not only complemented the deficiency of the N-tC fusion containing the membrane-release-incompetent tC83 fragment (NtC83) in virion release but also formed a Sarkosylresistant cap.27 In contrast to these composite virions, the oligophage ladder of simple virions containing only tC93, tC111, and tC12131 or N-tC93, N-tC111, and N-tC121 (data not shown) completely disappears upon Sarkosyl treatment. Virions in the oligophage ladder were resistant to Sarkosyl in all composite virions (N-tC141/cC, N-tC132/cC, NtC121/cC, N-tC111/cC, N-tC93/cC, and N-tC83/ cC), although some ssDNA was released by the detergent. This corresponds to clearance of the polyphage smear between the wells and the oligophage ladder, as has been seen with the WTpIIIcontaining virions (Fig. 3a versus Fig. 3b; top of the gel). In addition, assuming a random assortment of the pIII subunits during assembly, in 3% of virions, all five subunits will become N-tC fusions, which, in the case of N-tC93-, N-tC111-, and N-tC121containing virions, will result in Sarkosyl-mediated disassembly. In summary, in the majority of particles from composite virion populations, cC had a dominant stabilizing effect over the nonfunctional

N-tC83 and destabilizing N-tC93, N-tC111, and NtC121 fusions. As a result, composite virions NtC141/cC, N-tC132/cC, N-tC121/cC, N-tC111/cC, N-tC93/cC, and N-tC83/cC have overall similar physical properties, irrespective of the particular N-tC fusion they contain. A specific region of the C domain is required for Ff infection The infectivities of composite virions N-tC141/ cC, N-tC132/cC, N-tC121/cC, N-tC111/cC, NtC93/cC, and N-tC83/cC were measured to determine the ability of each progressively shorter C-terminal fragment to mediate phage entry (Table 1; Fig. 4). Virions containing only cC were used as a negative control, and composite virions containing WTpIII/cC were used as a positive control (Fig. 1a and d). Stocks of ΔgIII helper phage used to generate the N-tC141/cC, N-tC132/ cC, N-tC121/cC, N-tC111/cC, N-tC93/cC, and NtC83/cC progeny necessarily contain WTpIII in the virions to allow infection of producer cells (Fig. 1d); helper phage that remain unabsorbed contribute to the overall phage titer of the resulting lysate. This unwanted background could obscure the infectivity measurement of the newly produced phage particles. To circumvent this problem, we used a phagemid-based production system, in parallel with phage production system, since phagemid particles were not present in the helper phage stock (Table 1). The number of infectious phagemid particles in the samples was determined by transduction of the CmR marker to an indicator strain.

Table 1. Infectivity of composite N-tC/cC virions Phagemid Virion cap compositiona cC (−) WTpIII/cC (+) N-tC83/cC N-tC93/cC N-tC111/cC N-tC121/cC N-tC132/cC N-tC141/cC a

Phage

Infectious particles (pp/mL)b

Total number of particles (ge/mL)c

Infectivity (pp/ge)d

Relative infectivitye

Relative infectivityf

1.2 ± 0.6 × 106 1.7 ± 1.5 × 1010 4.9 ± 2.8 × 106 9.3 ± 6.7 × 106 1.3 ± 0.5 × 108 1.5 ± 0.8 × 1010 4.1 ± 0.1 × 1010 2.7 ± 2.0 × 1010

4.7 ± 1.2 × 1011 3.4 ± 0.7 × 1011 7.7 ± 5.2 × 1011 7.0 ± 2.8 × 1011 1.2 ± 0.7 × 1012 6.2 ± 2.2 × 1011 5.8 ± 2.4 × 1011 2.7 ± 0.9 × 1011

2.5 × 10− 6 5.1 × 10− 2 6.3 × 10− 6 1.3 × 10− 5 1.1 × 10− 4 2.4 × 10− 2 7.2 × 10− 2 1.0 × 10− 1

4.9 × 10− 5 1 1.2 × 10− 4 2.6 × 10− 4 2.1 × 10− 3 4.8 × 10− 1 1.4 2.0

7.2 × 10− 5 1 9.5 × 10− 4 2.1 × 10− 4 2.1 × 10− 3 1.9 × 10− 1 8.1 × 10− 2 8.2 × 10− 2

Virions were produced as described in Fig. 1d. The titer of infectious phagemid particles (pp), expressed as the number of CmR transductants per milliliter, obtained by spotting dilutions of the lysate on a lawn of indicator strain TG1 (CmS; see Materials and Methods). c Total number of phagemid particles (infectious and noninfectious), expressed as the number of genome equivalents (ge) per milliliter and determined as described in Materials and Methods. d Infectivity of phagemid particles, expressed as a ratio of the number of infectious particles per milliliter to the total number of particles per milliliter. e Relative infectivity of phagemid particles, expressed as a ratio of infectivity of composite N-tC/cC virions containing an internal deletion mutant pIII or negative control (cC-only virions) to the infectivity of WTpIII/cC virions (positive control). f Relative infectivity of composite phage virions, obtained using the plasmid pNJB50 (instead of the phagemid pYW01) as a source of cC and f1d3 (instead of VCSM13d3) as a helper phage. Relative infectivity was calculated by the same method used for phagemid particles. b

Membrane Insertion/Excision Domain of Ff pIII

977

100

Relative Infectivity

1 1×10 -1 1×10 -2 1×10 -3 1×10 -4 1×10 -5

Fig. 4. Relative infectivity of the composite virions. The infectivity for each N-tC/cC composite phagemid particle sample was divided by the infectivity of the WTpIII/cC positive control (Table 1) and plotted on a log scale. Each value is an average of three experiments.

The total number of phagemid particles (infectious and noninfectious) was determined by measuring the amount of encapsulated phagemid ssDNA. All samples contained similar numbers of phagemid particles as the WTpIII/cC sample, confirming that phagemid assembly and release from the inner membrane of the producer cells were equally efficient for all samples. The ability of the phagemid particles to mediate infection was expressed as infectivity, which was calculated as the ratio of the number of infectious phagemid particles to the total number of phagemid particles in each sample (Table 1). The infectivities of NtC121/cC, N-tC132/cC, N-tC141/cC, and WTpIII/ cC phagemid particles were 4 orders of magnitude higher than that of the negative control. In contrast, the infectivities of N-tC83/cC and N-tC93/cC phagemid particles, carrying the two shortest C

domains fused to N1N2 domains, were similar to that of the negative control, whereas the infectivity of N-tC111/cC was marginally increased (43-fold). The infectivities of N-tC132/cC and N-tC141/cC phagemid particles exceeded that of positive control WTpIII/cC by a factor of about 2. However, when a plasmid was used to provide cC and phage were produced instead of phagemid particles, the infectivities of N-tC132/cC and N-tC141/cC virions were reproducibly about 10-fold lower than that of WTpIII/cC or N-tC121/cC (Table 1). This unexpected result could be due to different dynamics of entry in phagemid- versus phage-containing virions or due to other unknown differences between the phage and phagemid particle production systems. To determine whether the termination-competent fusions N-tC141, N-tC132, N-tC121, N-tC111, and N-tC93 could mediate entry in the absence of in trans-expressed cC, we tested the infectivity of phage produced in the absence of cC. The infectivity of each of these virions matched that of the corresponding composite N-tC141/cC, N-tC132/ cC, N-tC121/cC, N-tC111/cC, and N-tC93/cC virions (data not shown). Therefore, although cC is required for resistance of N-tC121, N-tC111, and N-tC93 virions to Sarkosyl, it does not affect their ability to infect E. coli. Infection-competence segment mediates infection only in cis with N1N2 domains The segment between residues 121 and 93 (counting from the C terminus) is required for entry but dispensable for release; hence, we named it the infection-competence segment (ICS). The cap complex contains five subunits of pIII;36 therefore, the ICS sequence could be required only in the subunits that contain N1N2 domains and, hence, bind the receptors or in all five subunits of the virion. To test

Table 2. Infectivity of composite WTpIII/tC virions Virion cap compositiona

Infectious particles (pp/mL)b

Total number of particles (ge/mL)c

Infectivity (pp/ge)d

Relative infectivitye

WTpIII/cC WTpIII/tC83 WTpIII/tC93 WTpIII/tC111 WTpIII/tC121 WTpIII/tC132 WTpIII/tC141

7.4 ± 3.8 × 1010 5.3 ± 2.3 × 1010 3.9 ± 2.1 × 1010 3.1 ± 0.8 × 1010 8.1 ± 0.9 × 109 2.8 ± 0.2 × 1010 1.3 ± 0.1 × 1010

1.5 ± 0.03 × 1012 2.0 ± 0.2 × 1012 3.5 ± 0.9 × 1012 1.5 ± 0.03 × 1012 7.1 ± 1.8 × 1011 9.7 ± 1.4 × 1011 4.8 ± 1.3 × 1011

4.9 × 10− 2 2.6 × 10− 2 1.1 × 10− 3 2.0 × 10− 3 1.1 × 10− 3 2.9 × 10− 3 2.6 × 10− 3

1 5.4 × 10− 1 2.3 × 10− 1 4.1 × 10− 1 2.3 × 10− 1 5.9 × 10− 1 5.4 × 10− 1

a

Virions were produced as described in Fig. 1d. The titer of infectious phagemid particles (pp), expressed as the number of AmpR transductants per milliliter, obtained by spotting dilutions of the lysate on a lawn of indicator strain TG1 (AmpS; see Materials and Methods). c Total number of phagemid particles (infectious and noninfectious), expressed as the number of genome equivalents (ge) per milliliter and determined as described in Materials and Methods. d Infectivity of phagemid particles, expressed as a ratio of the number of infectious particles per milliliter to the total number of particles per milliliter. e Relative infectivity of phagemid particles, expressed as the ratio of infectivity of WTpIII/tC83–WTpIII/tC141 virions to WTpIII/cC (positive control). b

Membrane Insertion/Excision Domain of Ff pIII

978 this, we constructed composite virions that contained WTpIII in combination with truncated C domain subunits, with some containing the complete ICS (tC141, tC132, and tC121) or partial ICS (tC111) and some completely lacking the ICS (tC93 and tC83).31 None of these truncated C domain mutants encode the receptor-binding domains (Fig. 1c and d; Table 2). If the ICS were required in all subunits, the composite virions WTpIII/tC111, WTpIII/tC93, and WTpIII/tC83, containing a Cterminal fragment shorter than tC121, should have a reduced infectivity. The infectivity of composite phagemid particles WTpIII/tC111, WTpIII/tC93, and WTpIII/tC83 was, however, similar to the infectivity of particles containing an ICS in all subunits: WTpIII/cC, WTpIII/tC141, WTpIII/ tC132, and WTpIII/tC121 (Table 2). Thus, the absence of the ICS from subunits that do not interact with cellular receptors does not affect infection. Together with the data from the composite NtC141/cC, N-tC132/cC, N-tC121/cC, N-tC111/cC, N-tC93/cC, and N-tC83/cC virions (Table 1), this shows that the ICS is required in cis, covalently linked to the receptor-binding N1N2 domains, and

C132

(a)

does not function in trans. In conclusion, the ICS of the receptor-binding subunit works independently from the ICS sequences in other subunits. In the “reverse” process of virion assembly and release of composite virion N-tC83/cC, the membrane release competence sequence (between residues 83 and 93, counting from the C terminus) of the cC subunits also works independently from the corresponding membrane release competence sequences in other subunits, in combination with assembly-incompetent N-tC83 subunits (Fig. 3).27 Analysis of the ICS The ICS (121-GKLDS VATDY GAAID GFIGD VSGLA NGNG-93) was analyzed within the context of the pIII C domain. As there is no high-resolution structure of a homologue of the pIII C domain, modeling the tertiary structure of this domain using standard homology-based programs was not possible. Instead, the I-TASSER algorithm,37 which does not require coordinates of close homologues for modeling, was applied (Fig. 5a). An I-TASSER model of cC predicted an α-helical structure (Fig.

C121

ICS helix C C141 C111

N

C83

S-S bridge C154

C141

C132

110 A 103 G 114 T

C121

C111

117 S

106 G

D

113 102

D

107

D

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100

S

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111 G I 104 115

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109 116

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101

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SGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSP ICS I-TASSER Jpred C70

C1

LMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES Membrane anchor S-S bridge I-TASSER Jpred

Fig. 5. Bioinformatic analysis of the pIII C domain. (a) An I-TASSER model of the pIII C domain (a ribbon diagram). Residues limiting the C-terminal fragments (tC141–tC83) in the N-tC mutants are labeled, counting from the C-terminal end. Red, residues 154–94; blue, residues 93–1 (counting from the C terminus). (b) Helical wheel diagram of the region corresponding to predicted ICS α-helix 117-SVATDYGAAIDGFIGVS-100 (counting from the C terminus), obtained using Java Helical Wheel Applet.38 Color coding of the helical wheel residues is as follows: ocher, hydrophobic; green, polar; pink, acidic. (c) Primary sequence of the pIII C domain. Color coding of amino acids in the sequence is as follows: small residues, green (Gly, light green; Ser, green, Ala, dark green); aromatic residues Phe and Tyr, purple; acidic residues Asp and Glu, red; basic residues Lys and Arg, blue. Boxes under the sequence, ICS and membrane anchor. The S–S bridge is indicated by a line connecting two Cys residues. Secondary-structure predictions are shown below the sequence. Top line, secondary structures as predicted by the I-TASSER 3D model of the C domain. Color scheme matches the 3D model in (a). Bottom, schematic representation of the secondary-structure prediction by the Jpred algorithm.39

Membrane Insertion/Excision Domain of Ff pIII

5a and c). This model, as well as the secondarystructure prediction program Jpred,39 predicts an ICS α-helix between residues 116 and 102 (counting from the C terminus). The top-ranked template selected by the I-TASSER algorithm was the membrane insertion domain (T domain) of diphtheria toxin.40 Similar to the pIII C domain, the T domain converts from soluble to membraneintegrated form. Although the C score of the model is low (− 3.64), the conserved arrangement of amphipathic and hydrophobic α-helices and the conserved membrane insertion function of the pIII C domain and of the diphtheria toxin T domain support the model. A helical wheel plot predicts that the ICS α-helix (between residues 116 and 102, counting from the C terminus) is amphipathic (Fig. 5b). The polar face of this helix contains mainly small amino acids. Furthermore, the primary sequence contains a helix–helix interaction motif,41,42 [small]XXX[small] XX[small] (110-AAIDGFIG-103). In the I-TASSER model, this α-helix extends into a predicted coil in which the small-residue motif is extended (103GDVSGLANG-94). We constructed point mutants of six residues within the predicted ICS helix, 111GAAIDG-106, and four residues just upstream of the helix, 118-DSVA-115, replacing wild-type residues with Ala (or with Gly if the wild-type residue was Ala). All mutants were as infectious as the WT pIII (data not shown). In our mutagenesis, small residues Gly and Ala in the ICS are replaced with each other; furthermore, large residues are replaced by a small residue (Ala), without interfering in infection. Therefore, it appears that small residues in ICS are beneficial for phage infection. The lack of negative effect on infection in our ICS point mutants also showed that no single key residue, including negatively charged Asp residues, is essential for infectivity. Downstream of ICS, an S–S loop and a Cterminal membrane anchor, which are essential for all functions of pIII,43 are extremely rich in aromatic amino acids (4 out of 15 residues and 7 out of 23 residues, respectively). Aromatic amino acids are overrepresented in the viral fusion peptides that mediate host membrane insertion of enveloped eukaryotic viruses.44

Discussion Identification and properties of the ICS Previous studies have elucidated in detail the structural changes involved in the binding of Ff filamentous phage to its cellular receptors, the tip of the F pilus and the TolA domain III, via the N2 and N1 domains of pIII, respectively.16–18,45,46 However, little is known about the post-receptor-binding

979 stages of phage infection.18 During entry, the virion changes from a compact molecular complex that is highly resistant to detergents, temperature, and pH extremes, to an unstable complex that readily “dissolves” into the inner membrane of the host cells, allowing DNA entry into the cytoplasm.18 The prerequisite trigger for this transition is the “unlocking” or conformational rearrangement of the pIII C domain and exposure of its C-terminal hydrophobic membrane anchor.27 An opposite event, unique among the viruses, converts the membrane-embedded phage filament, which draws its subunits from the inner membrane and its DNA from the cytoplasm, into a tightly capped virion, devoid of the phospholipids.9 The C domain of pIII, which is required for this process, has recently been shown to be also involved in a post-receptor-binding step of infection. Furthermore, the C domain is required for infection of female (F−) cells that do not have an F plasmid.27 Therefore, the C domain acts independently of the F pilus and its retraction machinery. To begin unraveling the mechanism of phage entry that involves the C domain of pIII, we have now shown that a truncated C domain, a C-terminal fragment containing 121 residues (out of the total of 153 residues), covalently linked to the N1N2 domain (including both glycine-rich linkers), is sufficient for infection. This is a longer fragment than the one required for virion termination and release (93 Cterminal residues).31 The segment between residues 121 and 93 (counting from the C terminus) that is required for infection but dispensable for release was denoted ICS (Fig. 5c). Having different requirements for infection versus entry is consistent with the different sets of accessory proteins involved in the two processes: host-encoded F pilus/TolQRA in infection and phage-encoded pI/pXI/pIV in assembly. The ICS is modeled as an amphipathic α-helix followed by a random coil, both by the secondary-structure prediction algorithm Jpred and by the 3D modeling program I-TASSER. The whole ICS segment is rich in small amino acids Gly, Ala, and Ser, which are organized as a long, smallresidue motif, [small]XXX[small]XX[small]XXX [small]XXX[small]. These motifs often mediate interactions between transmembrane α-helices.41,47,48 Our scanning mutagenesis of the pIII ICS helix residues 111-GAAIDG-106 that replaced Ala with Gly or Gly with Ala within the AXXXG motif did not measurably affect phage infection, confirming the importance of small residues for phage entry. Potential partners of these motifs of pIII ICS are the TolQ transmembrane helices TM2 and TM3, each containing a [small]XX[small]XXX[small] motif found to be essential for TolQRA complex function and for entry of pore-forming colicins A and E1.49 Another potential interaction surface for the amphipathic ICS helix is the TolR periplasmic domain, which, in the high-resolution structure, forms a

980 hydrophobic groove along the periplasmic surface of the inner membrane that could accommodate a hydrophobic face of an amphipathic α-helix.50 The mechanism of Ff infection Using virions that contain combinations of pIII mutants containing or lacking ICS and receptorbinding domains N1N2, we showed that, for phage entry to occur, the N1N2 domains must be in the same subunit as ICS. Because filamentous phage is a virus that contains only one receptor-binding complex, investigating inter-subunit requirements for the ICS in phage entry was possible. The ICS linked to N1N2 functions “autonomously”; subunits that lack N1N2 domains neither decrease nor improve the infectivity of a virion, irrespective of whether these N1N2-negative subunits contain ICS. These findings point to a complex receptor-triggered event in which a pIII subunit that has N1N2 domains covalently linked to an ICS-containing Cterminal fragment is necessary and sufficient for unlocking of the virion and insertion of the coat proteins into the inner membrane. Several lines of evidence provide clues to the mechanism of this event. It has been reported that a soluble N1N2 fragment of Ff pIII interacts in vitro with the phage that contains only the C domain; however, the infectivity of the “reconstituted” particles in which N1N2 domains are non-covalently associated with the C domains is very low, as much as 4 orders or magnitude lower than that of the phage that contain the WTpIII.51,52 Therefore, the N1–TolA interaction is not per se sufficient for infection; rather, the N1N2 has to be covalently bound to the C domain. Furthermore, it was reported that insertion of βlactamase between the N1N2 domain and the C domain of FfpIII decreased infectivity by 2 orders of magnitude, underscoring the importance of correct N1N2–C domain distance in infection.51,52 Similarly, low but measurable infectivity was reported for chimeric constructs in which V. cholerae phage CTXφ pIII N1N2 domain was fused to the wild-type (full length) FfpIII and used to infect V. cholerae. Both Ff and CTXφ use the conserved TolQRA complexes for infection; however, the CTXφ and Ff pIII proteins themselves are not conserved (aside from general organization, including the signal sequence, three domains, and the C-terminal membrane anchor). Interestingly, the infection defect was completely corrected when the CTXφ N1N2 domain was fused directly to the Ff C domain.24 Therefore, it appears that the Ff pIII C domain works efficiently in V. cholerae when fused to the CTXφ N1N2 domain, as long as the distance between the N domain and the C domain is preserved. Low infectivity for N-pilusdependent infection was also observed in a study where the N-pilus-binding N-terminal domains of E.

Membrane Insertion/Excision Domain of Ff pIII

coli filamentous phage Ike were fused to full-length Ff pIII, resulting in the separation of the cC of fd phage from the N-pilus-binding domains of Ike.53 In contrast to the negative effect of increasing the distance between the receptor-binding domains and the ICS, we showed that shortening of the C domain, in our internal deletion mutants N141, N132, and N121, does not impair infection. Altogether, it appears that there is a limit to the N1N2–ICS distance that is tolerated in infection. It is likely that the virion unlocking mechanism, which “opens” the phage cap during infection, is directional and cannot be reversed once it is initiated. Given that pIII is not reused for the assembly of the progeny virions but is detectable as a full-length protein in the infected cells,35 it could be sequestered by the TolQRA complex, driving the conformational change in the direction of phage entry and preventing virion closure, thus avoiding entry failure. Taking into account our results, in conjunction with structural and functional data on TolQRA49,50 and the F pilus trans-envelope assembly complex, 54 we propose a model for the filamentous phage entry (Fig. 6). After binding of the pIII N2 domain and pilus retraction, the pIII N1 domain gains access to TolA (in an unknown fashion). High-affinity interaction of N1 with TolA allows in cis “activation” of ICS, which, in turn, results in interaction(s) of the C domain with the TolQRA complex and/or conformational rearrangement of the C domain that exposes the hydrophobic C-terminal helix and one or more amphipathic helices of the C domain, leading to insertion of pIII into the membrane. The loss of this pIII subunit from the cap destabilizes the remaining pIII subunits and/or exposes the hydrophobic α-helices of the major protein pVIII, which successively depolymerize into the membrane, concomitantly transferring the DNA into the cytoplasm. If the ICS is absent from the receptor-binding subunit, the infection aborts, despite successful binding of the virion to both primary and secondary receptors (Fig. 6). The pIII C domain is a membrane insertion domain In contrast to pIII in the virion, which can be considered a “soluble” form, membrane-inserted pIII is an ion-conducting pore in the liposomes, confirming that it is a pore-forming protein.55 In two large classes of membrane-inserting proteins, viral fusion proteins and pore-forming toxins, the transition from soluble to membrane-inserted form relies on receptor-induced and/or low-pH-induced conformational rearrangements to allow insertion into the phospholipid bilayer.44,56 Many of these membrane-inserting domains contain a “latched loaded spring” in their water-soluble form.57 The inducing signal typically releases the latch,

Membrane Insertion/Excision Domain of Ff pIII

981

Fig. 6. Model of Ff phage entry. (1) Binding of N2 domain (blue oval) to the tip of the F pilus (lightblue circles) and pilus retraction. (2) The retracted F pilus presumably ushers the N1 domain (green oval) of pIII into the periplasm where it protrudes through the barrel-like F pilus assembly machinery54 to gain access to the secondary receptor N-tC83 TolA III domain (brown oval). (3) N-tC93 Virion is likely liberated from the F cC N-tC111 pilus assembly machinery, while the ICS (magenta) mediates “openICS 2A ing” of the C domain (orange) and, in turn, insertion of the C-terminal WTpIII hydrophobic α-helix into the inner N-tC141 N-tC132 membrane. The inner-membrane cC N-tC121 complex TolQRA is required for this step.18 (4) Entry of phage DNA into the cytoplasm and integration of the major coat protein pVIII into the inner membrane. (2A) NonproTolAIII ductive receptor binding of N-tC83/cC, N-tC93/cC, and NtC111/cC particles. If the receptorTolAII binding subunit does not contain an ICS, infection stalls at step 2A: N1 and N2 bind to the receptors, but TolAI 1 2 4 3 DNA infection does not proceed further. TolQ TolR OM, outer membrane; IM, inner membrane. Major coat protein pVIII, black rectangles; minor coat proteins pVII and pIX, gray ovals; TolA and TolRQ, brown shapes; F pilus and the trans-envelope pilus assembly/retraction system (T4SS), light blue.54

allowing conformational rearrangements that expose multiple membrane-inserting or channelforming structural elements. In viral fusion proteins and α-helical pore-forming toxins, the unlatching typically results in formation and/or movement of at least one hydrophobic and one amphipathic α-helix.57 In the pIII C domain, a combination of the amphipathic ICS helix, followed by two downstream amphipathic helices and the hydrophobic C-terminal membrane anchor α-helix between residues 27 and 6 (counting from the C terminus), together with the fact that the diphtheria toxin α-helical membrane insertion domain was selected by the I-TASSER algorithm from the Protein Data Bank database as a template for modeling of the pIII C domain, suggests that the pIII C domain fits within the group of α-helical membrane insertion domains. Filamentous phage membrane transactions are more complex, however, than those of the poreforming toxins and viral fusion proteins. The C domain of pIII is, to our knowledge, the only protein domain that, in addition to membrane insertion, also mediates excision from the membranes. Further-

more, membrane excision is coupled to formation of a multi-protein complex—an extremely stable virion cap structure containing two other proteins, pVI and pVIII, resistant to detergents and pH extremes. The transitions and triggers that govern all these membrane transactions are very intriguing and likely much more complex in their regulation than those of eukaryotic viral membrane fusion proteins or bacterial pore-forming toxins.

Materials and Methods Bacterial and phage strains All bacterial strains used are E. coli K12 and are derivatives of strain TG1 supE Δ(hsdM-mcrB)5 (rk− mk− McrB−) thi Δ(lacproAB)[F′ traD36 lacIq Δ(lacZ)M15 proA+B+]. Phage f1d3 and VCSM13d3 carry identical complete deletion of gIII.33 To produce infectious stocks of these phage, we used the pIIIproducing strain K1976. K1976 is TG1 transformed with the complementing plasmid pJARA11233 which contains wildtype gIII under the control of the filamentous phage-induced psp promoter.

982 Construction of plasmids Plasmids pNJB30, pNJB31, pNJB32, pNJB33, pNJB34, and pNJB35 are derivatives of pJARA20035 coding for NtC fusions N-tC141, N-tC132, N-tC121, N-tC111, N-tC93, and N-tC83, respectively (Fig. 1b). The coding sequence of mutants was obtained using ligation-mediated PCR. The N1N2 terminal domain fragment (N), composed of complete N1 and N2 domains and both glycine-rich linkers (present in all N-tC fusions), was amplified using the primer pair NJB20 (GCT TTC CAT TCT GGC T) and NJB9 (CTC ATA ATC AAA ATC ACC GGA ACC A) and plasmid pJARA200 as a template. The C domain deletion series was constructed using a single reverse primer NJB19 (CCC AAG CTT TTA AGA CTC CTT ATT AC) and a series of forward primers: NJB4 (tC141; GCT AAT AAG GGG GCT ATG), NJB5 (tC132; GCC GAT GAA AAC GCG C), NJB6 (tC121; GGC AAA CTT GAT TCT GT), NJB7 (tC111; GGT GCT GCT ATC GAT GG), and NJB8 (tC93; GGT GCT ACT GGT GAT TTT GC); the template was pJARA200. The PCR products encoding an N fragment and the tC fragments were gel purified and phosphorylated (OptiKinase, USB). Each tC-fragmentencoding PCR product was then ligated to the amplified sequence encoding the N1N2 (N) at an equal molar ratio. The ligated fragments were then PCR amplified using the flanking primers NJB20 and NJB19 and the ligation product as a template. The reverse flanking primer NJB19 contains a HindIII site, while the forward flanking primer NJB20 is positioned upstream of a naturally occurring BamHI site within the N2 domain of pIII. PCR-amplified fragments were digested with BamHI and HindIII and ligated into BamHI-HindIII-digested pJARA200 to replace the C domain and a portion of the N2 domain of WTpIII with the N-tC fusions. The plasmid pNJB50 is a derivative of the cC-expressing plasmid pJARA2421 in which the tac promoter was replaced by a psp promoter. The plasmid pYW01 is a phagemid (it contains an f1 origin of replication) expressing cC under the control of the psp promoter.58 Both plasmids contain a ColD origin of replication and a CmR marker from the vector pGZ119EH.59 All constructs were sequenced to ensure accuracy. Phage infection and growth The plasmids expressing N-tC fusions or the WTpIII were transformed into strain K1981 (TG1 containing pYW01), for phagemid particle production, or strain K1994 (TG1 containing pNJB50), for phage production. The resulting double-transformed strains were propagated in 2xYT medium containing Amp (100 μg/mL) and Cm (25 μg/mL). To produce the composite virions containing a combination of an N-tC fusion or a WTpIII with Cc, we infected exponentially growing cultures [OD600 (optical density at 600 nm) = 0.2] with helper phage VCSM13d3 (for phagemid particle production) or f1d3 (for phage production), at a multiplicity of infection of 100 phage per cell, for 1 h at 37 °C. Infected cells were then separated from unabsorbed phage by centrifugation (7000g for 10 min at 37 °C), resuspended in fresh medium that contained IPTG (0.1 mM), and then incubated for a further period of 4 h to allow phage or phagemid particle production. After the incubation, the cells were removed

Membrane Insertion/Excision Domain of Ff pIII by centrifugation (7000g for 20 min at 30 °C) and the supernatant containing the released phage or phagemid particles was collected for further analysis. A series of composite phagemid particles containing WTpIII and one each of the series of truncated C domains (tC141 to tC83; Table 2) were obtained by infecting cells double transformed with phagemid pJARA14060 that expresses a full-length pIII under the control of the psp promoter and one of the previously published tC141– tC83-fragment-producing plasmids under the control of the tac promoter.31 VCSM13d3 was used as a helper phage. The negative control strain that produced only cC contained a basic vector (pBR322; AmpR) from which pJARA200 and pJARA140 were derived. Phage purification and concentration Virions in culture supernatants were concentrated by standard polyethylene glycol (PEG) precipitation (5% w/v PEG 8000 and 0.5 M NaCl). The concentrated virions were further treated with DNase I and RNase A to remove residual cellular DNA and RNA, followed by a second PEG precipitation. Precipitates were resuspended in Trisbuffered saline (TBS; 50 mM Tris and 150 mM NaCl, pH 7.5), treated with Triton X-100 (1% v/v) to remove residual inner- and outer-membrane vesicles, PEG precipitated, and resuspended in TBS. The concentrated phage suspension was centrifuged to remove any insoluble debris, and the supernatant was used for further analyses. Agarose gel electrophoresis of the phage and quantification A genome equivalent is a unit of measure of virion amount and is defined as a particle containing one encapsulated genome (or, in a multiple-length particle, a segment of the filament that corresponds to one genome). Thus, a virion particle containing 10 genomes represents 10 genome equivalents, as do 10 virion particles containing 1 genome each.35 In all experiments, the number of genome equivalents was determined from agarose gel electrophoresis of phage or phagemid ssDNA, released from SDS-disassembled virions.61 Prior to electrophoresis, virions were disassembled by incubation in SDS-containing Tris–acetate–ethylenediaminetetraacetic acid (TAE) buffer (1% SDS, 1× TAE, 5% glycerol, and 0.25% bromophenol blue, pH 8.3) at 70 °C for 20 min. After electrophoresis, phage ssDNA was stained with ethidium bromide and quantified densitometrically. Since the amount of ssDNA in a band is not linearly proportional to the intensity of the fluorescence, every gel contained a set of twofold dilutions of f1 ssDNA standard, which was used for calibration. Native virion agarose gel electrophoresis was used to separate virions of various lengths and to detect free ssDNA when the stability or size distribution of virions was analyzed.49 Samples were mixed with standard DNA loading buffer (1× TAE, 5% glycerol, and 0.25% bromophenol blue) and loaded onto agarose gels (0.6%). When the stability of the phage was analyzed, the ionic detergent Sarkosyl (0.1%) was added to the loading buffer and the sample was incubated at room temperature for 10 min prior to loading.35 The virions were separated by

Membrane Insertion/Excision Domain of Ff pIII electrophoresis at 3 V/cm for 16 h. After electrophoresis, free phage ssDNA was detected by staining the gel in ethidium bromide. To detect the virions, we disassembled them within the gel by soaking them in 0.2 M NaOH for 1 h, followed by neutralization in 0.45 M Tris, pH 7.1. The exposed virion ssDNA was then visualized by re-staining the gel with ethidium bromide.34,35 Protein electrophoresis and western blots Proteins from the phage samples were separated via SDS-PAGE, using either glycine62 or N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine63 gel system, and transferred to nitrocellulose membranes, which were then incubated with appropriate antibodies. Immunostaining52 detection was used to detect the position of antibodies. Antibody R164,33 raised against the C-terminal decapeptide of pIII (FANILRNKES), was used to detect pIII.21 Antibody 19–3832 was used for the detection of pVI. The buffer used in western blots was TBS-T (30 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 8.0). Blocking and antibody binding buffers also contained 0.5% I-Block (Applied Biosystems).

Acknowledgements We are indebted to Marjorie Russel and Peter Model for advice, for generously providing materials, and for critical reading of the manuscript. We thank Alok K. Mitra for advice on the structural biology aspects of the project, Yun Wu for technical assistance, and Julian Spagnuolo for comments on the manuscript. This work was supported by the Marsden Fund of the Royal Society of New Zealand (contract number 02-MAU-210), New Zealand Foundation for Research and Technology contract C03X0701 and Institute of Molecular BioSciences Postgraduate Fund. N.J.B. was supported by the Massey University Doctoral Scholarship.

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