Bacteriophage T5 attacking an E. coli. Inset shows bacteriophage T4 injecting its DNA into E. coli.

LECTURE 10: BACTERIAL VIRUSES - SOME DISCOVERIES/APPLICATIONS

A. Phage PhiX174

1. "A dwarf among viruses" - PhiX174 is extremely small

• a. Genome: ~5000 nucleotides
• b. Coat: contains only one type of protein with 60 subunits present per virion
• c. Shape: icosahedral

2. PhiX174 genome: "an intriguing puzzle"

• a. Upon preliminary examination, the genome appeared "too short" - there did not seem to be enough DNA to encode all the proteins that PhiX174 produces. It was found that the genome contains overlapping genes (Figure 6.17) that are translated in different reading frames or using alternate start codons.
• b. The genome is composed of DNA but the number of purines does not equal the number of pyrimidines, as expected in Watson-Crick base-pairing. This observation was explained when it was found that the genome exists as single-stranded DNA.
• c. Both the DNA and the protein coat of PhiX174 enter the host cell.

3. Overview of infection

• a. PhiX174 virions carry (+)strand ssDNA. Host enzymes replicate injected the ssDNA, forming dsDNA called the replicative form (RF). Polycistronic (+)strand mRNA is transcribed from this dsDNA. Viral proteins are translated from this mRNA.
• b. Genome replication occurs by rolling circle replication (Figure 6.18). First, the gene A protein nicks a site called the origin on the (+)strand of the RF dsDNA and remains bound to the 5' end. The 3' hydroxyl group at the nick is used to initiate DNA replication around the RF circle, displacing (+)strand DNA along the way. Gene A protein cuts off unit lengths of genome and ligates the ends together to produce progeny (+)strand ssDNA circles. This process continues many times resulting in the swift replication of a large number of complete genomes.
• c. Coat proteins and (+)strand ssDNA self-assemble in nascent viral particles. The gene E protein is required for cell lysis prior to release.

B. Phage M13 (Zinder 1961)

This phage was isolated from the sewers of New York based on its ability to recognize and bind the sex pili of F+ E. coli.

• a. Genome: ssDNA, circular; goes through a dsDNA RF intermediate form during replication
• b. Shape: filamentous, 6 nm x 860 nm
• c. The surprise: continuous production of viral particles in the absence of host lysis. As viral particles exit the cells, they are covered with coat proteins embedded in the cell membrane of the host (Figure 6.19). The orientation of particle exit is always the same, with the gene A protein leading the way out.

4. Applications:

• a. Fred Sanger (1970s) used M13 as a tool for DNA sequencing; his methods are still widely used. The DNA to be sequenced is cloned into the double-stranded RF DNA. Recombinant M13 genomes are introduced into cells and progeny phage containing single-stranded DNA is produced and harvested. The single-stranded DNA is purified from the phage and used as a template in Sanger's dideoxy method of DNA sequencing. The phage are simply supplying the DNA to be sequenced in single-stranded form, as required in Sanger's sequencing procedure.
• b. Greg Winter (1990s) has used M13 to produce "phage-displayed antibodies." The goal was to develop an efficient method to produce a pure stock of an antibody capable of recognizing a specific antigen of interest. An "antibody library" of random sequences is first produced by PCR and the constructs are fused to the M13 gene A in the RF DNA. The fused, artificial antibodies will be "displayed" on the surface of M13 particles because the gene A protein is naturally located on the surface of the phage. Specific phages of the library capable of recognizing the antigen of interest can be isolated by passing the phage library over a column packed with the antigen attached to a solid support. Phages displaying antibodies not recognizing the antigen are simply washed away. Infection of E. coli with virions that have bound to the column will amplify phage of the type that recognize antigen. Successive passages through the antigen column will "select" for the antibodies that bind most strongly. Note - The phage-display approach can be used to enhance other types of macromolecular interactions.

C. Phage MS2 (Zinder)

Another phage from the sewers of New York that recognizes the sex pili of F+ E. coli.

• 1. Shape: icosahedral, small
• 2. Genome: (+)strand ssRNA; contains only ~3.6 x 10³ nucleotides

MS2 was the first virus identified that carries its genome as RNA.

Upon infection, the (+)strand ssRNA is replicated, producing (-)strand ssRNA using the phage-encoded enzyme replicase, an RNA-dependent RNA polymerase. The (-)strand ssRNA is then used as a template to generate many copies of (+)strand ssRNA for use as mRNA. Inhibition of host RNA polymerase demonstrated that a viral enzyme was responsible for producing (-)strand ssRNA from (+)strand ssRNA.

3. Clear regulation of translation - a result of RNA adopting alternate/strategic structures:

• a. Only one copy of the maturation (A) protein is produced each time a (+)strand ssRNA is produced. The ribosome binding site (RBS) for the maturation protein open reading frame (ORF) is only available for binding as soon as it is produced - a ribosome hops on and translates the sequence while the rest of the (+)strand ssRNA is still being synthesized. After more RNA is produced, the RBS is masked by base-paring with another sequence. As a result, the maturation protein can no longer be translated and only one copy of the protein will have been produced.
• b. The concentration of coat proteins produced is very high. The RBS and start codon of the coat protein ORF are prominently displayed and easily accessible to the translation machinery. As a result, the coat proteins are produced in massive quantities. This makes sense since many copies of the coat protein are needed for the assembly of progeny phage.
• c. As the coat protein accumulates, translation of the replicase ORF decreases. The coat protein acts as a translation repressor by binding to a region near the replicase RBS. As a result, levels of coat protein exceed levels of replicase. This makes sense; not many copies of the replicase protein are needed since it can act catalytically.
• d. To make the lysis protein, the system depends on a "mistake" - a frameshift error. The open reading frames for the coat protein and the lysis protein overlap (Figure 6.16). The RBS and start codon of the lysis protein are obscured by ribosomes translating the coat protein ORF. Occasionally, translation of the coat protein prematurely terminates as a result of the ribosome "mistakenly" failing to maintain the proper reading frame. This opens up a short window of opportunity in which the lysis protein ORF can be translated. As with the replicase protein, it makes sense that the lysis protein should be produced in small amounts since it can act catalytically.