I. The Nature of Viruses

What Is a Virus?

Viruses are unique genetic elements that require a living host cell for replication. They exist in an extracellular form called a virion, which facilitates transmission between host cells.

• Virus: Genetic element that cannot replicate independently of a living (host) cell.

• Virus particle (virion): The extracellular form of a virus, responsible for transmission from one host cell to another.

• Replication/reproduction occurs only upon infection (entry into host cell).

Viral Components and Activities

• Capsid: Protein shell that surrounds the viral genome.

• Naked viruses: Viruses (e.g., most bacterial viruses) with only a capsid and no other layers.

• Enveloped viruses: Viruses (e.g., many animal viruses) with an outer layer consisting of a phospholipid bilayer (from host cell membrane) and viral proteins.

• Nucleocapsid: The combination of nucleic acid and protein in enveloped viruses.

Viral Genomes

Viral genomes are highly diverse and can be classified based on their nucleic acid type, strandedness, and structure.

• Either DNA or RNA genomes.

• Can be single-stranded (ss) or double-stranded (ds).

• Single-stranded genomes may be plus sense (same as mRNA) or minus sense (complementary to mRNA).

• Genomes may be linear or circular.

• Viral genomes are usually much smaller than those of cells.

Example: The genome of bacteriophage λ is linear dsDNA, while the genome of phage M13 is circular ssDNA.

Classification of Viruses

• Viruses can be classified based on the hosts they infect:

  • Bacterial viruses (bacteriophages)

  • Archaeal viruses

  • Animal viruses

  • Plant viruses

  • Other viruses

• Viruses vary in shape and size, but most are smaller than prokaryotic cells, typically ranging from 0.02 to 0.3 μm.

II. Structure of the Virion

Capsomeres and Self-Assembly

The virion is composed of a capsid made up of individual protein molecules called capsomeres or protomers, arranged in a precise and repetitive pattern around the nucleic acid.

• Capsids can assemble spontaneously (self-assembly) or require host cell folding assistance.

Enveloped Viruses

• Enveloped viruses have a lipoprotein membrane surrounding the nucleocapsid.

• Envelope proteins facilitate attachment and infection of animal host cells.

• Few plant or bacterial viruses are enveloped due to the presence of cell walls.

• The entire virion enters the animal cell during infection, and enveloped viruses exit more easily from host cells.

Enzymes Inside Virions

• Lysozyme: Creates holes in the cell wall for nucleic acid entry and lyses bacterial cells to release new virions.

• Neuraminidases: Destroy glycoproteins and glycolipids, aiding in the liberation of viruses from the cell.

• Nucleic acid polymerases:

  • RNA replicases: RNA-dependent RNA polymerases.

  • Reverse transcriptase: RNA-dependent DNA polymerase (in retroviruses).

III. Overview of the Virus Life Cycle

The viral replication cycle differs between prokaryotic and eukaryotic hosts, primarily in the mode of entry.

• Prokaryotes: Nucleic acid entry.

• Eukaryotes: Entire virion entry.

Phases of viral replication in a permissive host:

1. Attachment (adsorption): Virion binds to host cell surface.

2. Penetration (entry/injection): Viral nucleic acid enters the host cell.

3. Synthesis: Host cell metabolism is redirected to synthesize viral nucleic acids and proteins.

4. Assembly and packaging: Capsids are assembled and viral genomes are packaged into new virions.

5. Release: Mature virions are released from the host cell.

Virus Replication and Growth Curve

• Virus replication is characterized by a one-step growth curve—increase in virus count occurs when cells burst.

• Eclipse: Genome replication and protein translation.

• Maturation: Packaging of nucleic acids into capsids.

• Latent period: Eclipse + maturation.

• Release: Cell lysis, budding, or excretion; burst size is the number of virions released per cell.

IV. Culturing, Detecting, and Counting Viruses

• Bacterial viruses are easiest to grow (using liquid medium or agar lawns).

• Animal and plant viruses can be cultivated in tissue cultures.

• Plaque assay: Used to detect and count viruses; plaques are clear zones on lawns of host cells where infection has occurred.

• Titer: Number of infectious units per volume of fluid.

• Plating efficiency: The ratio of plaque-forming units to the total number of virions; usually less than 100% due to inactive virions or suboptimal conditions.

V. The Viral Replication Cycle: Bacteriophage T4

Attachment and Entry

• Attachment is a major factor in host specificity and requires complementary receptors on the host cell surface.

• Receptors can be proteins, carbohydrates, glycoproteins, lipids, or lipoproteins.

• For T4, tail fibers interact with polysaccharides on the E. coli LPS layer, tail pins contact the cell wall, and lysozyme forms a pore in peptidoglycan for DNA entry.

• The capsid remains outside the cell.

Restriction and Modification

• Prokaryotes have defense mechanisms against viral infection:

  • Toxin-antitoxin molecules

  • CRISPR (antiviral system)

  • Restriction endonucleases: Enzymes that cleave foreign DNA at specific sites.

  • Host DNA is protected by modification of recognition sites; restriction enzymes are specific for dsDNA.

  • Phages may evade restriction by base substitution.

Genome Replication and Circular Permutation

• Small DNA viruses use host DNA polymerase; complex viruses like T4 encode their own.

• T4 genome exhibits circular permutation (same genes in different orders) and is terminally redundant (some sequences duplicated at both ends).

• T4 DNA is first replicated as a unit, then forms a concatemer (several genomic units recombined).

Transcription and Translation of Bacteriophage T4

• Virion synthesis takes ~30 minutes, ending with cell lysis and release of new virions.

• T4 genome is divided into early, middle, and late proteins:

  • Early proteins: Enzymes for DNA replication and transcription, modification of host RNA polymerase.

  • Middle and late proteins: Structural proteins (head, tail), enzymes for virion assembly and release.

• T4-specific proteins modify host RNA polymerase to recognize only phage promoters, shutting down host transcription.

Packaging of the T4 Genome

• Genome is pumped into the capsid head under pressure using ATP.

• Packaging occurs in three stages: assembly of proheads, assembly of packaging motor, and DNA pumping.

• After DNA packaging, tail and other components are self-assembled; late enzymes break the cell wall, causing lysis and virion release.

Phage Lysis

• Some phages cause chronic infections (continuous release), but most cause acute infections (cell lysis and release of virions).

• Three key proteins:

  • Lysozyme: Degrades cell wall.

  • Holins: Form pores in the cytoplasmic membrane for lysozyme passage.

  • Antiholins: Regulatory proteins that coordinate timing of lysis.

VI. Strategies of Phage Genome Replication

DNA Phage M13

• M13 has a circular single-stranded DNA genome.

• Replication involves rolling circle replication:

  • RF (replicative form) DNA is used as a template for synthesis of new strands.

  • Gene V prevents the plus strand from being used for more RF (-) strands, aiding in packaging the (+) strand into the phage head.

Phage T7

• T7 encodes its own RNA polymerase, allowing for delayed expression of certain genes.

• T7 RNA polymerase recognizes specific promoters, turning on late genes required for assembly and release.

• Genome replication produces concatemers (repeats of genome units), which are cut at pac sites and packaged into phage heads.

VII. Summary Table: Key Features of Viruses and Phages

Additional info: This summary covers the structure, classification, replication, and detection of viruses and bacteriophages, with a focus on bacteriophage T4 and strategies of phage genome replication. These topics align with college-level microbiology curriculum, specifically chapters on viruses, viral genomics, and viral multiplication.