CMMB 343 Virology Reading Notes

• CMMB 343 Virology Reading based on the 16th edition, sections 5.1-5.7, 11.1, 11.3-11.4, and 11.8-11.10.

Viruses

• Subcellular particles can only replicate within a living host cell.
• Viruses are obligate intracellular parasites that steal the host's energy molecules, macromolecules, (most) enzymes.
• Viruses exist in two forms/states, extracellular as an infectious virion and intracellular as a non-infectious replicating genome.
• Viruses have been found for organisms in all 3 Domains.

Virions

• Virions have a large size range.
• Poliovirus is 28 nm, about the size of a ribosome.
• Pandoravirus is 1200 nm, larger than most bacterial cocci.
• Most virions have ordered, symmetrical structures: helical, icosahedral, or complex (non-symmetrical).

Virion Structural Components

• All viruses have a protein "coat" capsid surrounding the genomic material.
• Capsids subunits are capsomeres.
• Some viruses have an envelope which is a phospholipid bilayer and often contains glycoprotein “spikes".
• Non-enveloped viruses are "naked".
• Some virions contain viral enzymes required for infection.

Viral Genomes

• Viral genomes can be DNA or RNA, single-stranded or double-stranded, linear or circular.
• Virions have a large genome size range
• Circovirus has a genome size of 1.75kb and codes for 3 genes
• Pandoravirus has a genome size of 2.5Mb and codes for 2500 genes.

Bacteriophage

• Bacteriophages are viruses of bacteria and archaea
• They are complex
• Bacteriophages attach to host cell receptors with tail fibers and inject their genome into host cytosol, leaving the capsid on the cell surface.

Culturing Phage: Plaque Assays

• A bacterial lawn is prepared on an agar plate.
• A viral solution is then spread on top.
• Plaques (clear areas) form where the bacteria have been killed.
• Counting plaque-forming units (PFU) gives estimates of titer.

Viral One-Step Growth Curve

• The viral one-step growth curve includes the eclipse, maturation, early enzymes, nucleic acid, protein coats, the latent period, the addition of the new virus and the assembly and release of the virus.

Phage Receptors

• Host cell phage receptors perform normal functions for the host cell.
• Viral evolution has made these cell components targets for attachment

The Lytic Infection

• Lytic infection is the infection cycle of virulent phage.
• The lytic cycle results in replication of the virus and host cell death.
• E.g., bacteriophage T4 (linear dsDNA) and its E. coli host.
• The first step of attachment involves tail fibers adsorbing (attaching) to LPS carbohydrates on the host's outer membrane.

T4 Penetration

• T4 tail fibers retract, bringing tail pins into contact with the lipid bilayer.
• The tail sheath contracts, pushing a tail tube through the outer membrane.
• T4 lysozyme forms a pore through the cell wall.
• T4 dsDNA moves into the cytosol.

T4 Synthesis

• During the "Early" stage of T4 protein synthesis, the host RNA polymerase transcribes a series of viral genes that are only expressed at the start of infection.
• Includes anti-sigma factor proteins that inhibit host sigma factors and phage-specific replisome.
• During the "Middle" and "late" stages of T4 protein synthesis, capsomere, tail, and tail fiber proteins are produced, with enzymes that facilitate assembly and release.

T4 Assembly and Release

• “Late” T4 genes encode a packaging motor complex that fills viral capsids with copies of the viral dsDNA
• The remaining virion components assemble
• "Late" T4 genes encode enzymes that compromise the host cell membrane and cell wall.
• The host cell lyses and progeny virions are released.

Circular Permutation

• During replication, the linear copies of T4 dsDNA are joined to generate a long concatemer.
• During assembly, the phage DNA is moved into each capsid until it is full and then cut from the rest of the molecule, a "headful" packaging mechanism.
• Because more than a genome-length of DNA fits into each capsid, the DNA is cut at different sites, and each progeny virion has a different gene order.
• The T4 genome is terminally redundant, where Genes at one end of the molecule are repeated at the other end.

Headful Packaging

• Headful packaging involves the endonuclease cutting site, resulting in circular permutation.

The Lysogenic Infection

• Lysogenic infection is the infection cycle of temperate phage that results in a latent infection where the viral genome is replicated as the host continues to grow and divide.
• E.g., bacteriophage λ (linear dsDNA) and its E. coli host.
• During lysogeny, the viral DNA becomes integrated into the host genome.
• Integrated viral DNA is called a prophage with a host integrated viral DNA called a lysogen.

λ Adsorption/Penetration

• The λ tail attaches to a host maltose transport protein.
• Penetration is like T4.
• The 5' ends of the λ DNA contain short, single-stranded, complementary, "cohesive ends."
• Following entry into the host cytosol, λ DNA circularizes forming a cos site.

To Lysogeny or not to Lysogeny

• Most infections result in lytic infection
• IF the λ repressor (cl) gene is expressed upon penetration, cl accumulates, and most λ genes are repressed, A DNA becomes a prophage.
• IF the cro repressor (cro) gene is expressed upon penetration, cro accumulates, and the cl gene is repressed entering the lytic cycle.
• A few λ genes are expressed during lysogeny to "monitor" host stress levels and induce a switch to the lytic cycle if conditions deteriorate.

λ DNA Integration/Excision

• Both & and host contain att sequences.
• λ integrase nicks the DNA at the att sites, and the viral DNA enters the host chromosome.
• If a switch to the lytic cycle is made, a λ excision enzyme cuts the prophage out of the host chromosome.

λ Synthesis

• If expressed, "early" through "late" λ protein synthesis occurs like in a T4 infection.
• The circular λ DNA is copied by rolling circle replication.
• The λ DNA is copied continuously, generating long concatemers.
• Individual λ genomes are cut from the concatemer at each cos site and packaged into assembling phage capsids.