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Figure 2 Ff phage genome. The location of each viral gene is indicatedby number, with the direction of transcription shown byarrow. The origin of replication lies within the intergenic region,between the genes for pIV and pII. The packaging signal PS liesbetween the — strand origin of replication and gene IV. Parts of the intergenic regionhave been shown to be dispensable reviewed in Ref.

This diagramdepicts the structural organization of M13 as a representativeof the Ff viral family. At the top of the diagram lies the distal end ofthe particle, at which viral assembly initiates. At the bottom of thefigure is the proximal or infectious end of the virus, with five copiesof the pIII anchored to the particle by five copies of the pVI. Although pX has the same amino acid sequence as the carboxyl-terminalend of pII, it has been shown to possess uniquefunctions within the viral life cycle, such as inhibition of pIIfunction 5.

The crystal structure of the protein has been solvedto 1. The crystal structure of pV has been solved to 1. The protein normally exists as a dimer and wrapsaround the single-stranded form of the viral DNA within the hostcell cytoplasm. Filamentous Bacteriophage Structure and Biology 9out of 87 residues arranged in a five-stranded antiparallelb-sheet; two antiparallel b-ladder loops protrude from thissheet. The remainder of the molecule is arranged into 3 10 -helices residues 7—11 and 65—67 , b-bends residues 21—24,50—53, and 71—74 and one five-residue loop residues 38— Themajor coat proteins of all filamentous phages are short, rangingfrom 44 to 55 amino acids, with most being encoded witha signal sequence Pseudomonas aeruginosa-infecting phagePf3, being an example of a pVIII absent a signal sequence.

The concentrationof pVIII in the inner cell membrane is very high—at least5 10 5 molecules of pVIII are exported as virions per infectedcell per doubling, making it one of the most abundant proteinsin the infected cell The distal end of thephage contains approximately five copies each of the twosmall hydrophobic proteins, pVII 33 a. In the absence of either pVII orpIX, almost no phage particles are formed 11 , and geneticevidence suggests that both are involved in initiation ofassembly These structures demonstrate that although the individualdomains are the same, they differ by a rigid body rotation ofFigure 5 Crystal structure of the two amino-terminal domains ofthe gene III protein.

Thesetwo domains can be seen as knobs at the proximal end of M13 asseen in the micrograph in Fig. Filamentous Bacteriophage Structure and Biology 11N2 with respect to N1 around a hinge located at the end of theshort antiparallel b-sheet connecting N1 and N2. V , including interactingwith both host cell factors and phage proteins requiredfor morphogenesis, and in helping in the formation of phagespecificadhesion zones.

The morphogenetic proteins pI and pIV,located in different membranes, appear to interact to form anexit structure through which the assembling viral particleextrudes from the host cell Figure1 is an electron micrograph of M13, and Fig. The coat proteins from the two classes have lengthsthat vary from 44 to 53 amino acids and sequences with similaroverall character each has an amino-terminal end rich inacidic residues, a central hydrophobic region, and a carboxyterminus rich in basic residues but little conserved sequence.

There are 12 Rodi et al. The amino terminus of pVIII is exposed to the surface,with the first four to five residues forming a flexible armthat appears to extend away from the virus. The distal or topend of the particle is assembled first, and contains approximatelythree to four copies each of pVII and pIX asdetermined by labeling studies 23 , which may form a hydrophobicplug at the top of the virus The proximal end bottom in Fig. Most of the pVIII structure appearsto be comprised of a gently curving a-helix extending fromPro6 to near the carboxyl-terminus 26— The axis of thepVIII a-helix is tilted about 20 relative to the virion axisand wraps around the virion axis in a right-handed helicalsense, as can be seen in Fig.

The amino acid sequence ofpVIII can be broken into four parts: a mobile surface segment Ala1—Asp4 or Asp5 ; an amphipathic a-helix extending fromPro6 to about Tyr24; a highly hydrophobic helix extendingfrom Ala25 to Ala35; and an amphipathic helix forming theinside wall of the coat, extending from Thr36 to Ser50 This last helical region contains four positively charged sidechains which interact with the phosphate backbone of theDNA, and the DNA bases face inward 28, The aminoterminus lies at the surface, with only the first three residuesaccessible to digestion by proteases 31 and theremainder of the phage surface is composed largely of theamphipathic helix that extends from Pro6 to about Tyr Random peptides inserted at or near the amino terminus Filamentous Bacteriophage Structure and Biology 13of pVIII have shed some light on the packing requirementsand structure of the phage particle surface.

Fiber diffractionanalysis of an insertion mutant, with a pentapeptide GQASG inserted between residues 4 and 5, indicated thatthe insert lies in an extended conformation within a shallowgroove between two adjacent a-helices on the viral surface Given the resemblance of this arrangement to the presentationof peptides by major histocompatibility antigens 33 , the authors postulate that the three-dimensional conformationof small pVIII inserts may contribute to the highimmunogenicity observed for peptides inserted into the geneVIII product of M Recent work by Fuh et al.

This display requires a minimum of 8—10 residues asa linker to render the carboxy-terminal fusion product accessibleto the surface. These data appear to be consistent withtwo possible scenarios: either the linker extends throughthe protein coat and disrupts the local packing of pVIII moleculesaround the DNA core; or alternatively, the inserts areonly tolerated near the proximal end of the virion where thecarboxyl terminus of pVIII may be closer to the virus surface.

This latter hypothesis is consistent with the fact 41 that displaywith the carboxy-terminal pVIII fusion was reducedabout fold relative to amino-terminal fusions on pIII,indicating recombinant pVIII levels of roughly 0. Filamentous phages are notoriously resistant to numerousphysicochemical assaults, including prolonged incubationat high temperatures, in nonionic detergents, in high salt,and at low pH. However, Class I viral particles have beenshown to be sensitive to inactivation by small organics suchas chloroform, with accompanying structural collapse torod-shaped particles Modeling studies, based upon fiber diffraction analysis 27,28 indicate the presence of a substantial depression orhole in a large hydrophobic surface patch at this location,with Val31 lying exposed at the bottom The V31L cloneisolated by Oh et al.

Studies on the stereochemistryof this site suggest that a chloroform molecule canindeed fit into the Val31 hole These data are consistentwith that of Weiss et al. Thisend contains the packaging sequence PS and is the part ofthe phage assembled first. Gaoet al. Single-chain Fv scFv libraries have been displayed as fusions with the amino terminusof pIX Antibodies directed against pVIdo not interact with intact phage, suggesting that pVI issomewhat buried within the viral particle A partialmodel for this end of the virus was postulated by assumingthat the amino-terminal half of pVI is structurally homologousto two pVIII molecules 24 as shown in Fig.

This potentially unstable situation couldbe ameliorated by a fold of pVI, much of which forms anamphipathic surface layer, up and around these last twopVIII rings, holding the virion together and helping to maintainviral stability. The remaining parts of pVI may have aportion of pIII folded around them, sequestering them awayfrom solvent and antibody access.

Contrast-enhanced electronmicrographs of negatively stained microphage particles E. Bullitt and L. Makowski, unpublished results exhibit aslightly enlarged diameter near the proximal end, consistentwith this model. The C-terminus of pVI has been successfullyused as a vehicle for display 50 , suggesting that it is accessibleon the virion surface.

The gene III protein is the largest and most structurallycomplex component protein of filamentous phage. It consistsof three distinct domains, separated by two glycine-rich linkers residues 68—87 and — , which appear to make portionsof pIII somewhat flexible. In electron micrographs ofnegatively stained phage, the two amino-terminal domains[N1 or D1 and N2 or D2 ] can be seen as knobby structuresat the end of the virion see Fig.

The carboxyterminal residues of pIII, domain D3 or CT, are proposedto interact with pVIII to form the proximal end of the viral particle,as they remain associated after disruption of the viruswith detergents 38, The crystal structures of both N1and N2 from M13 and fd have been determined 13,53,54 see Fig. The amino terminus of the mature pIII was the firstlocation used for the display of foreign proteins and peptideson M13 55, It is still the most commonly used position.

Contrary to common belief, however, polypeptides fused tothe carboxy-terminus of the M13 gene-3 minor coat proteinmay be functionally displayed on the phage surface Ina phagemid display system, carboxy-terminal fusion throughoptimized linker sequences resulted in display levels comparableto those achieved with conventional amino-terminalfusions.

The details of the structure of pIII, as visualized inthe crystallographic analysis of the two N-terminal domains,suggest an innocuous structural environment remote fromthe host cell binding site and generally favorable to insertionsthat would have little impact on the function of pIII see Sec. VII discussion.

Solid-state NMR studies of the filamentousphage Pf1 57 demonstrated a uniform conformationfor the phosphate backbone, whereas in the Ff phage, Filamentous Bacteriophage Structure and Biology 17the phosphates take on a larger number of different orientations.

Fiber diffraction studies of the intact virion 27,32 suggesta lack of sufficient space within the capsid shell for aB-DNA duplex, suggesting that the DNA in the virion hasa somewhat extended conformation. Mutations resulting ina change in the net charge of the C-terminal, basic region ofpVIII 1 behaved in a manner indicating a direct but nonspecificelectrostatic interaction between the DNA and the coatprotein.

This was dramatically demonstrated by a series ofmutations in which Lys48 was converted to an unchargedamino acid. The PS for filamentousphage was originally identified within the intergenic regionof f1 shown in Fig. Hence, the DNA within filamentousphage particles is oriented within the virion such that thePS is always at the distal end A hairpin structurehas been shown to exist within the virion, as it can be crosslinkedwith psoralen The PS in its single-stranded formcan be drawn as an imperfect hairpin of 32 bp with a smallbulge.

Not only can the hairpin below the bulge be deletedwith no loss of function, but so also can a portion of theupper hairpin as long as the lower is present. In addition,loop sequences at the tip of the hairpin can be altered, andshort insertions added, with no loss of function. Studies publishedin 12 on PS — mutants showed that althoughvery few phage particles were produced in the absence ofa PS, suppressor mutations allowing higher packaging 18 Rodi et al.

The PS of phages Ike and f1, which exhibitlittle sequence similarity, are functionally interchangeable A construct with a perfect self-complementary segmentof DNA functioned poorly for packaging. Taken together,these studies point towards the conclusion that, while aduplex is essential for the function of the PS in filamentousphage, additional features are also involved. Multipletranscripts are produced, with six mRNAs encoding pVIIIand four encoding pV, allowing for the production of largeamounts of these proteins to coat the emerging virusand intracellular ssDNA genomes, respectively for a more Filamentous Bacteriophage Structure and Biology 19detailed discussion of transcription, see Ref.

The pV dimer binds to DNA in a highly cooperative mannerwithout marked sequence specificity at physiological pH 63— Protein V exhibits two distinct modes of DNA binding,dictated by salt concentration, and leading to either acooperatively saturated complex through nonspecific bindingor an unsaturated complex through specific binding. The radius of gyration of the DNA in thecomplex is much lower than that of the protein, indicatingthat the DNA is closer to the axis of the structure.

Each turnof the helix contains roughly eight pV dimers, with two antiparallelssDNA strands occupying the interior of the superhelix A model for the pV—ssDNA complex, consistent withthese observations and with the 1. This model places the antiparallel DNA single strands withinthe grooves formed by the b-strands that loop out from thebody of the pV dimer, as illustrated in Fig.

Synthesis of Viral ProteinsThe replication proteins pII, pV, and pX are synthesized byhost cell machinery and reside within the cytoplasm. Themechanisms of their membrane insertion are unknown, butall three appear to be inserted into the inner membrane withtheir carboxyl termini on the cytoplasmic side and theiramino termini on the periplasmic side.

The gene III protein is synthesized with an 18 amino acidsignal peptide, which is removed after membrane insertion. Membrane translocation is Sec-dependent see Ref. Most of its mass including domains N1, N2, and most ofCT resides in the periplasm. The membrane-anchoringdomain consists of the carboxy-terminal part of D3 and is alsoinvolved in attaching pIII to the viral particle see Fig. Procoat pVIII prior to cleavage by signal peptidase appears to form dimers within the membrane by packingalong the hydrophobic face of its amphipathic helix andextending through the membrane-spanning region During morphogenesis, this L-shaped conformation transformsinto the elongated, largely a-helical structure it takeson in the intact phage particle see Fig.

This figure is a schematic diagram of theincorporation of coat proteins into a growing Pf1 virion. Reprintedfrom Ref. Onceassociated with the inner membrane, procoat appears to havea U-shaped configuration, with two transmembrane helices,extending from positions —15 to —2 and from 21 to 39 76 ,bracketing an amphipathic helix that extends along the periplasmicsurface of the inner membrane.

Tyr21 and Tyr24appear to act as anchors to position the second transmembranehelix within the membrane Signal peptidase cleavageleaves the mature pVIII in an L-shaped configuration 78 with five flexible amino-terminal residues, residues8—16 forming an amphipathic helix extending along the membranesurface, and residues 25—45 making up a transmembranea-helix that extends into the cytoplasm and include aportion of the basic carboxy-terminal region of pVIII involvedin protein—DNA interactions 79 see Fig.

The extent ofthe carboxy-terminal transmembrane helix is probably not 22 Rodi et al. The different extents of this helix reportedby the two studies quoted above may reflect different conditionsunder which the measurements were made. Proteins I, IV, and XI are all required for phage assembly,but are not present in the intact virus particle. ProteinsI and XI are synthesized without signal peptides and areinserted into the inner membrane in a SecA-dependent manner They each span the membrane once and are orientedwith their carboxy-terminal 75 residues lying within the periplasm Protein I has a amino acid amino-terminalcytoplasmic domain, all but 10 amino acids of which are missingin pXI see Fig.

Plasmid-driven production of excess pIresults in a loss of membrane potential and cessation of hostcell growth, suggesting that it can form some type of channelsthrough the cytoplasmic membrane 82— Excess pXI doesnot appear to have such an effect. The amino acid cytoplasmic domainof pI contains an ATP binding site and may be activelyinvolved in assembly and extrusion of the phage particle.

The gene IV protein is synthesized with a 21 amino acidsignal peptide and is translocated into the periplasm via theSec system 16, Protein I has been shown to associate with pIV andthis interaction has been implicated in the formation of Filamentous Bacteriophage Structure and Biology 23Figure 7 Viral morphogenesis through the pIV multimer. Thisfigure depicts the roles of various viral- and host cell-encoded proteinsduring viral assembly.

Modified from Ref. These complexesare believed to be located at specific adhesion zones, wherethe inner and outer bacterial membranes are in closer contact thanin nearby areas. Recent 24 Rodi et al. Viral MorphogenesisV.

Preassembly complex and the initiationof assemblyAssembly of phage particles begins when the PS interactswith the assembly complex formed by pI, pIV, and pXI andhost cell thioredoxin at localized adhesion zones 11,18 seeFig. This complex spans the inner and outer membranesand the periplasm and provides both a platform for phage particleassembly and a pathway for phage particle extrusionwithout lysis of the host cell.

Protein IV forms the outer membraneportion of the complex, pI and pXI form the inner membraneportion, and the amino-terminal region of pI and hostcell thioredoxin form the cytoplasmic portion of the complex. Overexpression of pIV has no effect on host cell viability,indicating that the channel formed by pIV is closed in theabsence of other phage proteins In the presence of pIand pXI and amber mutants of pVII or pIX, assemblysites are formed, followed closely by cessation of bacterialgrowth, suggesting the loss of transmembrane potential Although pIV from f1 and Ike are not functionally interchangeable,when both pIV and pI are exchanged between thetwo phage types, some heterologous phage particles areformed, suggesting that pI and pIV interact to promoteassembly This interaction is confirmed by the existenceof paired compensatory mutations in the respective periplasmicdomains of pI and pIV Efforts to model the phage ends provide some clues to theprocess involved in the initiation of assembly.

Following formation of the assembly complex,elongation of the phage particle can begin. Although thioredoxin in its reduced state is a potent reductantof protein disulfide bonds, it is not the redox activity ofthe protein which is required for assembly as mutations ofits two active site cysteine residues do not prevent its participationin phage assembly One clue to its role in phageassembly is that during infection by the lytic phage T7,thioredoxin complexes with T7 DNA polymerase, conferringprocessivity on the polymerase, allowing it to polymerizethousands of nucleotides without dropping off the DNA template It is conceivable that complex formation of thioredoxinwith pI enhances processivity in the filamentousphage assembly process also by stabilizing pI binding toDNA The 10 residues of pI and pXI which face thecytoplasmic surface of the inner membrane possess an amphiphiliccharacter similar to the 10 carboxy-terminal residues ofpVIII that interact with the DNA within the intact virion,suggesting that these residues may interact with ssDNA thathas just been stripped of pV, facilitating conformational shiftingtowards interaction with pVIII Because the helicalsymmetry of the DNA in the pV complex is different from thatin the viral complex, the two structures must rotate relativeto one another during the elongation process.

Studies of pVIII structure both in the intact virion andwithin detergent micelles by X-ray diffraction, solid-stateNMR, neutron diffraction, and Raman spectroscopy haveshown that a significant conformational shift must occurwithin pVIII for the protein to be incorporated into the growingphage particle 27,78,79,94— In addition, each pVIIImolecule must now interact with the ssDNA genome and withmany other copies of pVIII, and thus, must exchange hydrophobicinteractions that anchor it within the membrane forhydrophobic interactions that stabilize it within the phageparticle see Fig.

The mobility of the residues in the hinge region of themembrane-bound form is likely to enable a smooth transformationfrom the membrane-bound form to the phage conformation 79, Three separate research groups have performed mutationalstudies which suggest that certain small residues suchas Ala7, Ala10, Ala18, Leu14, Gly34, and Gly38 are not easilymutated in pVIII or can only be substituted with other smallresidues 45,79, A model for elongation of phage particlesbased upon both NMR and X-ray diffraction studies of both Filamentous Bacteriophage Structure and Biology 27position and mobility has been proposed which takes intoaccount the roles of these highly conserved residues Both pIII and pVI are anchored in the cytoplasmicmembrane and cannot be coimmunoprecipitated from nonionicdetergent extracted membranes, whereas once they arewithin intact phage particles they coimmunoprecipitatefollowing the same treatment 38,, , inferring a stronginteraction once within the virus.

These observations implythat pVI is needed to both stabilize the proximal end of thevirus, and attach pIII to the proximal end of the virus particle. This is confirmed by the fact that anti-pVI antibodies donot interact with intact phage Protein III molecules are anchored within the cytoplasmicmembrane via residues — at their carboxyl termini,with most of their residues located within theperiplasm 1— The amino-terminal domain residues1— mediates infection see next section , whereas thecarboxy-terminal domain D3 or CT; residues — isinvolved in releasing the phage from the membrane and incapping the virion.

Stable but noninfectious phage can beformed with a truncated pIII residues — , which comprisesa portion of the N2 domain, the second glycine-richspacer and the whole CT domain , Recent studies 28 Rodi et al. Prior to termination, pIII is anchored within thecytoplasmic membrane by a carboxy-terminal membraneanchor, placing the vast majority of the protein within theperiplasm Protein VIII is similarly oriented with its carboxylterminus in the cytoplasm and amino terminus withinthe periplasm , Both prior to and after incorporationinto the virion, the two proteins interact, but after incorporationthe amino terminus of pIII points away from pVIII.

This flipping motion on thepart of the C2 subdomain could then disrupt phage—membrane hydrophobic interactions, by allowing C2 to coverup a hydrophobic portion of the pIII carboxyl terminus, andthus resulting in release of the assembled phage from the host Filamentous Bacteriophage Structure and Biology 29Figure 8 Model for termination of viral assembly. This diagramdepicts a model proposed by Rakonjac et al. A conformational shift within pIII is theorizedas the primary driving force for phage release, based upon thephenotype of a series of pIII deletion mutants.

Modified fromRef. This model is corroborated by the fact that a very shortcarboxy-terminal fragment of pIII which lacks the C2 subdomainjust amino-terminal to the membrane anchor can beincorporated into the membrane-associated viral particle,but cannot detach the virus from host cells Additionally,carboxy-terminal fusions of greater than 7 amino acid residueshave been found to prevent termination of phage assembly,suggesting that although the carboxyl terminus may notbe tightly packed, it may be within an enclosed environment J.

Rakonjac and P. Model, unpublished results. The Infection ProcessFilamentous phage infection is a two-step process: i recognition—during which the virus binds to its primary bacterialcell surface receptor, the distal tip of the F-pilus; followedby ii translocation—which involves pilus retraction,capsid protein integration into the bacterial cell membrane, 30 Rodi et al.

Only one or a few F-pili are present on the surface of abacterial cell. Both pilus structural proteins and the proteinsrequired for pili assembly are plasmid encoded, with the geneslying within the tra operon, or transfer region, of the conjugativeplasmid. Different filamentous phages have specificitiesfor different pilus types. The F-pilusconsists of a helical array of pilin subunits of 8 nm diameterwith a 2 nm lumen Assemblyof the intact F-pilus requires the activity of 11 tra gene products This assembly process is temperature-sensitive;thus, F-specific phage fails to infect or form plaques onbacteria grown at temperatures below around 32 C.

Infection of a male E. This stepis followed by pilus retraction into the host cell, with pilinsubunits believed to depolymerize into the host cell innermembrane , It is not known whether phage bindinginitiates pilus retraction, or if phages are brought to the membraneas a consequence of normal cycles of pilus retractionand repolymerization A single L70S substitution atthe last amino acid of F-pilin knocks out both pilus assemblyand Ff infection The tolQ and tolR genes were Filamentous Bacteriophage Structure and Biology 31subsequently identified and shown to be not only essential tosusceptibility to certain classes of colicins, but for filamentousphage infection as well — The Tol system appears tobe involved in other types of macromolecular import, given itsstructural and sequence homologies to the TonB—ExbB—ExbDcomplex , Bacterial cells possessing functional pIIIeither from a filamentous phage infection or encodedby a plasmid 10 show increased tolerance to the effect ofE-type colicins, suggesting that pIII and E colicins have identicalor closely adjacent binding sites on the Tol complex.

Inaddition, overexpression of pIII, its amino-terminal fragment 10, or proteins possessing the TolA D3 domain , induces outer membrane leakiness in E. This figureshows a schematic view of phage infection demonstrating howpilus-mediated separation of N1 from N2 at the amino terminusof pIII frees up N1 for interaction with the D3 domain of thecoreceptor TolA, thus mediating viral entry into the host cell.

Reprinted from Ref. The cocrystal structure of the D3 domain ofthe coreceptor TolA with the amino-terminal domain of pIII hasbeen solved to 1. Note that the amino-terminal residue of pIII arrow lies far awayfrom the interaction surface between the two proteins. The mechanism by which capsid proteins are integratedinto the host cell cytoplasmic membrane and viral DNA isuncoated and translocated into the bacterial cytoplasm is largelyunknown.

Gene VIII proteins which have become associatedwith the inner membrane can later be reutilized inthe assembly of progeny phage particles, as their insertioninto the membrane occurs in a manner which gives themthe same topology as newly synthesized pVIII molecules — It has been suggestedthat the CT domain of pIII may be involved in the formationof an entrance pore for DNA translocation ,perhaps as a mirror image of the mechanism by which pIIImediates termination of assembly see Fig.

This would be analogousto the mechanism of entry of eukaryotic viruses into hostcells, via the unmasking of hydrophobic fusogenic peptides In support of this hypothesis, it has been shown thatthe insertion of a b-lactamase domain between the aminoand carboxyl termini of pIII thus disrupting their properdistance decreases infectivity by two orders of magnitude Efficiency as a Biological Strategyfor SurvivalConsidering the effect of insertion mutations on M13 requiresan examination of the relationship between M13 and its E.

M13 is not a lytic phage. Rather, it parasitizes the host,being carried from generation to generation and producinganywhere from to progeny phage per cell per doublingtime 2,3. As discussed above, this phage production representsa serious metabolic load for the infected E. Given this negative effect on host growth, any host mutationthat resulted in resistance to phage would seem to be highlyfavorable, and resistant host should quickly outgrow infectedhost.

These mutations are not observed to occur, suggestingthat the phage has evolved strategies for preventing its hostfrom developing resistance. What form do these strategiestake? Mutations that knock out expression of any of the viralgenes with the exception of pII result in killing of the host cell 2, This effect appears to be due to the accumulation ofphage-encoded proteins III and I in the cytoplasmic membranethat results in the degradation of host cell membranes.

Theseobservations suggest that any mutation in either the host cellgenome or phage-encoded proteins that results in the haltingor even the slowing of phage morphogenesis may lead to thebuildup of pI and pIII in the host cell, and subsequently tothe death of the host. Similarly, any mutation in a viral proteinthat blocks or slows phage assembly in such a way as to allowthe accumulation of pI or pIII may also lead to host cell death.

If this is the case, inserts that slow phage production may berapidly censored from a phage-displayed library if they resultin the buildup of pI and pIII in the host cell membrane. M13appears to have evolved to prevent the development of resistancein its natural host: any mutation that significantly slowsits production represents a fatal mutation.

A detailed examinationof the diversity and censorship patterns of phage displaylibraries , reinforces this hypothesis. Phage Population DiversityA number of groups have investigated the biochemical diversityof phage display constructs using various methods includingrestriction digestion pattern analysis of small numbers ofgroup members and colony hybridization with primers — Statistical methods have been developed and usedto quantitate and annotate the sequence diversity of combinatorialpeptide libraries on the basis of small numbers or of sequences.

Application of these methods in the analysisof commercially available M13 pIII-based phage displaylibraries demonstrated that these libraries behave statisticallyas though they correspond to populations containingroughly 4. Analysis of amino acidoccurrence patterns in these libraries shows no demonstrableinfluence on sequence censorship by E. This is in contrast to a clear effect of metaboliclimitations to the diversity of pVIII libraries These sequence limitations can primarilybe attributed to two steps during viral assembly: signal peptidasecleavage and incorporation of the recombinant proteininto the growing virion from the bacterial inner membrane.

Protein SynthesisInserts into the minor structural proteins of M13 are not likelyto significantly disrupt or slow their synthesis, and this has 36 Rodi et al. However, the sheer numbers of pVIII molecules thatmust be synthesized for viral production put the major structuralprotein of M13 in a separate category. The protein synthesisapparatus of the infected host cell produces from 5 10 5 to5 10 6 copies of pVIII per doubling time.

It is well establishedthat rare codons are used by the E. Rodi and Makowski analyzed the relative occurrenceof codons in a pVIII pentapeptide library constructed with a 32codon code. Seven amino acids have multiple codons in alibrary of this form. For four of them, a statistically significantcorrelation between frequency of codon use in the library andabundance of their respective tRNA was observed. These resultsindicate that the presence of rare codons in an insert into pVIIIsignificantly decrease the likelihood that the insert will be successfullyexpressed on the surface of the phage.

Proteins VIIIand V are the only phage proteins synthesized in very large numbersas required for their functions. All other phage proteins areexpressed at relatively low levels. These facts are reflected in thelack of rare codons in these proteins. The presence of theserare codons undoubtedly contributes to the low expression levelsof these proteins in host cells.

Consequently, the presence of rarecodons in an inserted sequence is likely to have a significant effecton the levels at which a protein is expressed. Protein Insertion in the Inner MembraneIt has been demonstrated multiple times that excess positivecharges at the amino-terminal end of a membrane protein Filamentous Bacteriophage Structure and Biology 37reduces its transport across the membrane , , due tothe electrical component of the proton motive force pmf In addition, it has been demonstrated that anarginine-specific restriction exists that may be due to theinteraction of the translocating protein with the SecY protein.

Rodi et al. Peters et al. No polylysinemutants were constructed in this study. Furthermore, theirattempts to rescue export by the addition of negativelycharged residues i. Export rescue of the arginine-rich mutants wasachieved, however, by infection of prlA mutants. The prlAphenotype has been shown to be the result of mutationswithin the SecY protein, the largest subunit of the SecYEGtranslocase complex. Furthermore, prlA4 relieved translocation blockagecaused by certain folded structures.

It has been demonstratedthat in order to be translocated, secretory proteins need to beat least partially unfolded , These observations suggestthat mutants that rescue export of the arginine-richmutants also provide for export of bulkier, folded structuresnot translocated by wild-type strains. Protein ProcessingAfter insertion into the inner host cell membrane, the signalpeptides of pIII and pVIII must be cleaved by signal peptidasein order for the proteins to be available for incorporation intothe assembling virus particle.

There is evidence for sequence 38 Rodi et al. Furthermore, except for the first position, there is a significantoverabundance of proline in combinatorial peptidesdisplayed at the amino terminus of the mature pIII There is also a dramatic overabundance of proline over mostof the length of the peptides in the pIII-displayed libraries This correlates with both the weak preference for peptideswith a high propensity for b-turns in these populationsand a preference of signal peptidase for b-turn conformations.

Whether the observed overabundance of proline in theselibraries is due to the preferred three-dimensional motif forsignal peptidase substrates or to a later step in morphogenesiscannot be determined from this data alone. Maliket al. Allthese data point to the conformational nature of the incorporatedpeptide being an influence on the efficiency of signalcleavage and consequent inclusion within the display librarypopulation.

Display in the PeriplasmE. Consequently, inserts atthe amino terminus of any of the structural proteins may becrosslinked prior to assembly if they contain a cysteine residue. Work done by Haigh and Webster 73 has shown that,prior to incorporation into the growing virion, the close Filamentous Bacteriophage Structure and Biology 39proximity of pVIII molecules within the inner membraneresults in a high degree of crosslinking between singlecysteines in different pVIII molecules, thus precluding theirparticipation in viral morphogenesis.

A similar effect hasbeen observed in pIII libraries. The almost complete absenceof odd numbers of cysteine residues in an amino-terminal pIIIdisplay library resulted in significant censorship of thatlibrary. This crosslinking may be intramolecular, involvingone of the other cysteines in the pIII and potentially resultingin the misfolding of the pIII , or it may be intermolecular,resulting in dimerization which would preclude incorporationof the pIII into the growing virus particle.

Viral MorphogenesisInserts that interfere with the protein—protein interactionsthat occur during viral morphogenesis have the potential todecrease or eliminate viral production. In spite of a great dealof evidence suggesting that inserts can disrupt viral assembly,the mechanism of this disruption is not well characterizedfor any specific case.

This may involve the interaction of viralproteins as they move actively or passively through thepI—pXI—thioredoxin complex associated with the inner membrane,or through the pIV pore in the outer membrane. Fromthat position, it would seem relatively unlikely for them todisrupt protein—protein interactions involving pI or pXI. Theycould, however, pose a problem for phage during extrusionthrough the outer membrane pIV pore. Display on pVIII, however, could beseverely limited by the pore.

Iannolo et al. This seems consistentwith the demonstration that a peptide inserted near theamino terminus of mature pVIII occupies a shallow grooveon the surface of the phage particle and that this groove iscapable of accommodating about eight residues The presumptiveconclusion is that peptides that cannot be accommodatedby the shallow groove will not allow passage of theparticle through the outer membrane pore.

Other data, however, suggest that this may not be thecorrect interpretation. If theextrusion of the virus through the outer membrane is sorestrictive as to prevent the display of relatively short peptideson every pVIII, how can the extrusion of phage displayinga limited number of large proteins on pVIII be possible?

Given how little we know about the process, we cannotexclude the possibility that the mechanics of viral extrusionwill allow for export of a few large displayed proteins butexclude export of virions displaying many short peptides,somewhat akin to moving large irregular pieces of furniturethrough a small doorway.

Equally compatible with these data is the theory that theouter membrane pore is not so tight as to exclude virions displayingpeptides about 10 or more amino acids in length, butthat the exclusion of longer peptides may be due to limitationsimposed by other phases of the phage life cycle. It is possiblethat the metabolic demand upon E. It has also been pointed out that pVIII is involvedin special interactions at the two ends of the virus particle 24, —those needed to attach pVI, pVII, and pIX to theparticle—and that these interactions may have specialrequirements on either length or nature of the insert that cannotbe readily met.

An insertthat disrupts this interaction may represent a fatal mutationif every pVIII harbors it, but not in a hybrid phage with nativepVIII present to perform that function. Infection ProcessFor those viral particles that escape host cell one i. The cocrystallization of thesetwo purified domains, as shown in Fig. Given theposition of the amino terminus of pIII, only a fully extendeddodecapeptide would be likely to impart significant interferenceof the infection process.

The position of both the TolAbinding site and the intermolecular interface with pIII—N2lie opposite the amino terminus, explaining why fusions ofpeptides and proteins to the amino terminus of pIII do not precludephage infection Since the utility of a library is proportionalto the diversity of the library, it is important to have quantitativemeasures of diversity to assess library quality.

Scott and Smith 56 calculated the probability of peptidesbeing present in a library of 2. Cwirla et al. They further observed that roughly mostamino acids occurred at most positions in their hexapeptidelibrary, leading them to conclude that viral morphogenesisdid not impose severe constraints on the diversity of theirlibrary. The effect of viral morphogenesis is, in fact, observableand significant but not severe DeGraaf et al.

They analyzedthe sequence of 52 clones selected at random from apopulation of 2 10 6 individual clones and demonstrated thatthe frequency of amino acid occurrence in this library had arough correlation with that expected from the number of distinctcodons corresponding to each amino acid. They furtheridentified the presence of of the dipeptides theoreticallypossible in the 52 decapeptides selected and analyzed.

Since only dipeptides are included in this limited populationof 52 decapeptides, this observation is not significantlydifferent from that expected from random sampling. Althougheach of these analyses was motivated by the need to measurethe diversity, or complexity, of a peptide library, each fallsshort of a true quantitative measure of library diversity. In the latter scenario,if the copy numbers of the members present in the populationare dramatically different, the diversity is intrinsically lower.

Real phage-displayed peptide populations invariably containunequal numbers of different peptides and any useful measureof sequence diversity must take this into account. Experimentsthat utilize sequence information from limited numbers of populationmembers to estimate peptide population diversity cannotprovide accurate estimates of completeness, since very raremembers of the population will inevitably go unsampled.

Filamentous Bacteriophage Structure and Biology 43Limited sequence information, however, is capable ofestimating the functional diversity of a peptide library. Their measure of library diversity is directlylinked to the probability of selecting the same library membertwice during random selection from the population.

Thesum in Eq. For combinatorial peptide libraries, this diversity can bereadily estimated from the frequency of occurrence of eachamino acid at each position in the library Furthermore,by combining a quantitative estimation of the diversity ofpeptide libraries with peptide sequence pattern analysis, it 44 Rodi et al. As some of the amino acids arecoded by two or three codons, this gives an enhanced abundanceof certain residues such as leucine six codons andglycine four codons.

Let us use a genetically random dodecapeptideinsert at the amino terminus of pIII as a test case fordiversity quantitation and censorship source assignment. Anin silico computationally constructed dodecapeptide librarybased upon a 32 codon code behaves statistically as thoughonly The second largest quantitative effect on diversity ofcombinatorial peptide libraries on M13 is the almost completeabsence of odd numbers of cysteine residues , McConnell et al.

Within randomly selected peptide sequencesfrom a dodecamer library, although one would expect to see Work done by Haigh andWebster 73 has shown that pVIII molecules are close enoughtogether within the inner membrane prior to incorporationinto the growing virion that single cysteine residues have atendency to crosslink between different pVIII molecules, precludingtheir participation in viral morphogenesis. It is also possiblethat single cysteine residues may occasionally be displayedin a structural context that allows them to avoid thecrosslinking activity of the E.

Finally, an antiarginine bias within the first half of therandom peptide sequence contributes a relatively minor additionalbias see discussion above. If we approximate that biasto maximal levels i. The obligate steps of membrane insertion and signalpeptidase cleavage result in patterns of censorship that arereflected in the statistical properties of the libraries.

Quantitatively,however, it is viral morphogenesis that is the predominantbiological source of sequence bias within the randomdodecapeptide library analyzed above. Comparisonof the amino acid composition of in silico constructedhuman proteome-derived and codon-dictated libraries alongsideactual in vivo-assembled random peptide librariesdemonstrates that the sequence biases seen in real-life phagelibraries do not bias them either towards or away from thecomposition or statistical properties of real proteins as comparedto computationally constructed random libraries These biases simply increase the chance of some motifsand decrease the chance of other motifs being observed.

Thepredominant reasons for this are the methods of construction i. A more diverse population could be obtained by usingspecifically constructed trinucleotide cassettes for the synthesisof random inserts. Propagation within a prlA host with its loosened translocasecomplex would also contribute to a more complex populationof displayed peptides on M13, although a quantitationof this effect is difficult to estimate.

Consideration of the phage—host biology provides significantguidance for the design of improved phage-displayedlibraries. Given the impact and widespread application ofthe existing libraries, incorporation of improvements based Filamentous Bacteriophage Structure and Biology 47on an understanding of the biology of the phage—host systemshould substantially improve the success rates for experimentsinvolving the use of these libraries as outlined in theremainder of this volume.

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