LECTURE 9: BACTERIAL VIRUSES, PART I

I. Overview of Viruses

• 1890s definition/description: contagium vivum fluidum.
• 1915 (modern) definitions: virus - a subcellular entity capable of entering specific cells and of reproducing only in such cells.
• virion - an extracellular viral particle.
• phage (bacteriophage) - a virus that infects a type of bacterial cell.
  
  Viruses are obligate genetic parasites - they take over cellular machinery in order to replicate more viruses. Their hosts include a wide variety of animal, plant and bacterial cells.

A. Composition of viruses

1. Size: 0.02 - 0.3 um
2. Genome: ~10³ - 10⁵ bp. The nucleic acid can be DNA or RNA, either of which may be single- or double-stranded. The genome may be up to 20 - 50% of the weight of the virus.
3. Protein: few proteins are carried inside the virus; inside their host cells, viruses produce additional proteins needed to express their genes, replicate their nucleic acid, and lyse the host.
4. Lipids: some viruses have a lipid bilayer, usually derived from the host cell membrane; viruses often insert their own proteins into this bilayer "envelope".

As a consequence of their streamlined, compact organization, viruses assume regular symmetrical shapes.
e.g. TMV (Tobacco Mosaic Virus) - helical (Figure 6.2).
T4 - complex, with an icosahedral head and tail structure (Figure 6.5c). T4 was used in the "blender experiment" of Hershey and Chase. Enveloped viruses - many contain an icosahedral head within a lipid bilayer (Figure 6.3)

B. Replication of viruses

Virions are metabolically inert - they cannot replicate without first entering their host cell.

1. Attachment

• a. Specific - the virion attaches to a specific receptor protein on the host cell. The receptors have "normal" functions for the host - e.g. pili, flagella, transport proteins. Hosts may modify the target proteins to protect themselves from viral attack, but the viruses usually can compensate for this and recognize the modified protein.
• b. Non-specific - the virion enters the cell by a non-specific insertion process such as phagocytosis.

2. Penetration

• a. Phagocytosis - especially true of animal viruses
• b. Phages must penetrate bacterial cell walls in addition to the lipid bilayers of the host. T4 attaches to its host (E. coli) using tail fibers. A small hole is made in the cell wall using the enzyme lysozyme. It then contracts its sheath and its DNA is injected into the cell (Figure 6.11).

C. Kinetics of viral growth

The one-step growth curve (Figure 6.10) indicates a typical course of events during a viral infection (Figure 6.9) of a cell. Initially, during the eclipse stage, no new viral particles are produced. During this time, viral genes are being expressed. If infected cells are harvested for virions at this time, no infectious particles are recovered. The maturation stage begins as new viral particles are assembled. Burst size refers to the number of particles produced from the infection of a single cell.

D. Co-opting the host machinery

1. Transcription - generally, viral mRNA is produced rapidly after the nucleic acid penetrates the cell.By convention, the (+)strand corresponds to the mRNA sequence. Depending upon the nature of the nucleic acid carried by the virus, there are different mechanisms to generate (+)strand mRNA.

• a. dsDNA - if the virus carries dsDNA, it can simply be transcribed directly to make mRNA. However, transcription often involves the use of viral RNA polymerase which is usually more active than host polymerase, ensuring that viral RNA is produced in large quantities.
• b. ssDNA - if the virus carries ssDNA, it is first replicated using the host DNA replication machinery resulting in the formation of dsDNA. Subsequently, the dsDNA is transcribed, usually via viral RNA polymerase as described above.
• c. (-)strand ssRNA - viruses that carry (-)strand ssRNA use the viral enzyme replicase (an RNA-dependent RNA polymerase) to produce large quantities of the (+)strand RNA which can then be used as mRNA.
• d. (+)strand ssRNA - if the virus carries (+)strand ssRNA, this can be directly translated by host machinery. One of the proteins thus produced is a viral RNA-dependent RNA polymerase which produces (-)strand RNA using the injected (+)strand RNA as a template. These (-)strand RNAs can be used to generate more copies of the (+)strand RNA. This mechanism effectively amplifies the amount of (+)strand messages within the cell.
• e. retroviruses also carry (+)strand ssRNA, but their method to amplify the message is quite different from that described above. They use the viral enzyme reverse transcriptase, an RNA-dependent DNA polymerase, to synthesize ssDNA which is then converted into dsDNA by the same enzyme or by the host DNA polymerase. The resultant dsDNA may be inserted into the host DNA. Transcription of this dsDNA results in the production of vast quantities of (+)strand mRNA.

2. Translation

Viral particles are capable of self-assembly. This indicates that all the instructions necessary for putting together new viral particles is contained within the sequence and structure of the component proteins and nucleic acid.

E. Quantitation of viruses

1. Plaque assay method (d'Herelle, ~1920) (Figure 6.6)

Mix ~10⁷ bacteria with dilutions of the phage in soft agar; incubate.

2. Results - a lawn of bacteria will grow in the agar.

• a. Plaques - clear areas in the lawn where bacteria have been lysed by viral infection
• b. The concentration of infectious phage in the dilutions is proportional to the number of plaques produced.

3. Interpretations/Implications:

• a. One virus leads to one plaque.
• b. This allows the purification of a pure stock of phage by isolation of the viruses from a single plaque.

4. Variations for eukaryotic viruses

Some eukaryotic cells can be grown in cultures as monolayers.

• a. Viral infection of monolayers leads to plaques if the virus lyses the host cells.
• b. Alternatively, viral infection of monolayers may cause cells to grow uncontrollably forming piles of cells called "foci of infection." This happens when the virus transforms the host cell rather than lyses it.

F. Purification of viruses

Purification of anything requires that an assay be available to monitor the progress of the purification.

1. The assay used for detection during the purification of viruses is the plaque assay.

2. Methods used are similar to those employed in the purification of large proteins:

• a. Precipitation from various salt solutions - separation is based on differential solubility in salt solutions
• b. Gel filtration - separation is based on size
• c. Density gradient centrifugation - separation is based on density