eTopics

eTopic 11.1 The Filamentous Phage M13: Vaccines and Nanowires

Not all viruses have icosahedral capsids. Filamentous bacteriophages consist of an extended, flexible tube of variable length. The variable-length tube is able to package varying lengths of DNA—an advantage for genetic engineering. An exciting use of the filamentous phage M13 is to engineer vaccines. The vaccine is made by recombining the M13 coat protein gene with an antigenic gene of a pathogen such as hepatitis A virus. The recombinant M13 phage then displays the hepatitis A protein in its coat. The phage acts as an immunogenic carrier for the antigens, a technique known as phage display (discussed in Chapter 12). Another impressive use of filamentous phages is to grow microscopic metal “nanowires” that conduct electricity. The wires are made using a mutant phage whose coat proteins bind metals such as gold and cobalt. Filaments form microscopic tubes of metal around the phage, and these can be used in the manufacture of tiny batteries.

Filamentous phages, like the tailed phages, must construct progeny using the materials and metabolism of a host cell. Yet the means by which they achieve this goal differ greatly from those of T4. Where T4 features remarkable complexity in its capsid, the structure of M13 is a simple helical filament (Fig. 1). Instead of the 170 genes of the T4 genome, M13 manages just as successfully with 11, including some that overlap (Fig. 2). And instead of lysing its host, M13 allows the host to continue growing, although more slowly than uninfected cells do.

Filamentous Phage Structure

The M13 capsid consists of a flexible protein cylinder that is about 150 times as long as it is wide (Fig. 1A). The cylinder is assembled around a supercoiled, single-stranded circle of DNA. The advantage of the filamentous structure is that it requires a relatively small number of different protein subunits encoded in the genome. Similar flexible filamentous structures have evolved independently in animal and plant viruses; compare, for example, Ebola filovirus and tobacco mosaic virus (see Section 6.2). Filamentous animal viruses, however, show a virulent infectious cycle completely different from the slow release of filamentous phages.

Each subunit of the M13 filament consists of a short alpha-helical peptide encoded by gene 8. The protein P8 subunits have positively charged lysine residues at the internal end to bind the negatively charged DNA. The subunits extend outward at a shallow angle, looking like bunches of bananas stacked as a long tube (Fig. 1B). The tube is tipped at one end by five copies of protein P3, a much larger and more flexible protein that binds the cell surface receptor during phage adsorption (see Fig. 2). At the opposite end of the tube are proteins P7 and P9, which enable a progeny phage particle to emerge from the cell.

The simple, flexible nature of the filamentous phage has proved useful for development of the technology of phage display. In phage display, a gene encoding a key portion of a protein domain (usually a short peptide) is cloned at the terminus of the sequence for one of the phage coat proteins, such as protein P8 or protein P3 (see Fig. 2). The recombinant phage now displays the desired protein domain on its coat, where it can be recognized by antibodies or can selectively bind to a target molecule. This technique can be used to screen a library of recombinant phages for binding partners to an antibody or to a target regulatory protein, such as a cell cycle regulator involved in tumorigenesis.

Adsorption to Host and Delivery of DNA

Phage M13 infects only F+ strains of E. coli, which possess F pili. The phage adsorbs by binding the tip protein P3 to an F pilus (Fig. 3). The pilus contracts, bringing the phage into contact with a coreceptor, or secondary receptor protein, TolA. The TolA complex is an important structural anchor of the E. coli cell envelope. Coreceptors for host attachment are a common feature of viruses, including animal viruses such as influenza virus and HIV-1.

As the M13 filament contacts the cell, it transfers its DNA across the cell envelope by an unknown mechanism. Unlike phage T4, the empty M13 filament does not remain intact outside the cell. Instead, the filament subunits disassemble and insert into the inner membrane, where they eventually join the pool of newly synthesized subunits to form progeny phages. Because most M13 assembly steps involve the membrane, we know less about the details of M13 assembly than we do about the assembly of T4, whose components are all cytoplasmic and therefore easier to study.

M13 Reproduction Does Not Kill the Host

As the infecting capsid dissolves in the inner membrane, the phage DNA (single-stranded circle) enters the cytoplasm. Phage DNA is replicated by host enzymes while host cell growth continues. Replication of the single-stranded genome starts with generation of a double-stranded replicative form (Fig. 4A). The replicative form then undergoes rolling-circle replication of the (+) strand, like phage T4. The growing concatemer (multiple repeats of the minus strand) is protected by subunits of protein P5, a single-strand binding protein. As each genome length is completed, it is nicked and circularized without overhang. The circular DNA is packaged with capsid proteins at the cell membrane.

The transition of phage M13 from single-stranded to double-stranded was important historically because of its role in DNA sequencing. When DNA-sequencing methods were first invented by Sanger, they required a template that was single-stranded. But recombinant DNA techniques required double-stranded DNA for restriction endonuclease cleavage and ligation. The most effective way to obtain large quantities of a single-stranded template was to splice a gene sequence within a double-stranded M13 replicative form. The replicative form was then used to transform cells that subsequently produced M13 phages carrying single-stranded DNA. Today, a single-stranded template is no longer required for PCR-based sequencing, but M13-based vectors remain useful for special applications, such as phage display.

Filamentous Phages Self-Assemble at the Inner Membrane

The M13 genome expresses packaging proteins, including protein P8, as well as the specialized tip proteins P3, P7, and P9 (see Fig. 2). All packaging proteins insert initially into the inner cell membrane. To mediate their assembly, a pore complex forms, composed of protein P4. P4 monomers extend through all layers of the envelope and assemble in a ring to build the pore. The positive charges on the N-terminal end of P4 help attract the negatively charged DNA into the pore. The pore guides the assembly of P8 monomers (also positively charged) and other packaging proteins around the DNA (see Fig. 4B). To avoid lysing the host cell, the pore is plugged by proteins P1 and P11 until phage assembly begins.

As the P5-coated chromosome enters the pore, the protein P5 subunits are replaced by the growing tube of P8 proteins. The final step of phage export involves capping with protein P3, which is needed for attachment and infection of the next host cell. The pore complex is then recapped with P1 and P11 to avoid lysis. The production of M13 phages slows growth of the host because of resource consumption, but it does not destroy the host cell; in fact, two of the phage’s 11 gene products (about one-sixth of its genome) are devoted to preventing host cell lysis. The evolved strategy of M13 is to maintain its current host at minimal cost, rather than to maximize its immediate number of progeny.

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Figure 1  A filamentous phage: phage M13.  A. M13 filamentous phage particles (8 nm wide, 900 nm long) with tail protein P3 gene fused to a gene encoding anti-tetanus-toxoid antibody. The protein P3-antibody was stained with tetanus toxoid-colloidal gold, observed as a black sphere at the tip of the phage filament (TEM). B. Model of filamentous phage structure consisting of a helical tube of protein P8 subunits, based on X-ray crystallography.

Source: Carolos F. Barbas et al. 1991. PNAS 79:78

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Figure 2  The M13 phage structure and genome.  Phage M13 structure with proteins color-coded to genes in the phage genome. Note: The actual phage filament is about 150 times as long as it is wide.

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Figure 3  M13 adsorption to the pilus and TolA.  Attachment to pilus of F+ cell.

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Figure 4  Assembly and export of M13 progeny phages.  A. Replication of phage M13. Most assembly is conducted by phage proteins within the cell membrane. B. Assembly for export through the pore made of P4 monomers. P4 makes a channel used to export filamentous phages without lysing the bacterial host. The single-stranded DNA of the filamentous virus is coated by P8 subunits as it passes through the membrane, and then passed through the P4 channel to emerge as mature, infectious phages.