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: A genetic element that cannot replicate independently of a living (host) cell.

• Virus particle (virion): The extracellular form of a virus, existing outside the host and enabling transmission.

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

Viral Components and Activities

• Capsid: The protein shell that surrounds the viral genome.

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

• Enveloped viruses: Viruses (e.g., many animal viruses) with an outer phospholipid bilayer (from the 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 by their nucleic acid type and structure.

• Either DNA or RNA genomes.

• Single-stranded (ss) or double-stranded (ds).

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

• Genomes can be linear or circular.

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

Classification and Morphology

• Viruses are 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 (range: 0.02 to 0.3 μm).

Structure of the Virion

• Capsomere/protomer: Individual protein molecules arranged in a precise, repetitive pattern around the nucleic acid, forming the capsid.

• Capsids can self-assemble or require host cell folding assistance.

Enveloped Viruses

• Have a lipoprotein membrane surrounding the nucleocapsid.

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

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

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

Enzymes Inside Virions

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

• Neuraminidases: Destroy glycoproteins and glycolipids, aiding virus liberation from the cell.

• Nucleic acid polymerases:

  • RNA replicases: RNA-dependent RNA polymerases.

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

II. The Virus Life Cycle

Overview of the Virus Life Cycle

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

• Prokaryotes: Nucleic acid entry.

• Eukaryotes: Virion entry.

Phases of viral replication in a permissive host:

1. Attachment (adsorption) of the virion

2. Penetration (entry/injection) of the viral nucleic acid

3. Synthesis of viral nucleic acid and protein by host cell metabolism redirected by the virus

4. Assembly of capsids and packaging of viral genomes into new virions

5. Release of mature virions from the host cell

Virus Replication: One-Step Growth Curve

• Characterized by a one-step growth curve—increase in virion count occurs when cells burst.

• Eclipse: Genome replication and protein translation.

• Maturation: Packaging of nucleic acids in capsids.

• Latent period: Eclipse + maturation.

• Release: Cell lysis, budding, or excretion.

• Burst size: Number of virions released per cell.

Culturing, Detecting, and Counting Viruses

• Bacterial viruses: Grown in liquid medium or as lawns on agar.

• Animal/plant viruses: Cultivated in tissue cultures.

• Plaque assay: Detects and counts viruses by clear zones (plaques) on host cell lawns.

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

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

III. The Viral Replication Cycle: Bacteriophage T4

Attachment

• Host specificity is determined by complementary receptors on the host cell surface.

• Receptors can be proteins, carbohydrates, glycoproteins, lipids, or other structures.

• Receptors often serve normal cellular functions (e.g., uptake proteins, cell-to-cell interaction, flagella, pili).

Attachment and Entry of Bacteriophage T4

• Tailed bacteriophages (e.g., T4) have complex penetration mechanisms.

• Virions attach via tail fibers to polysaccharides on the E. coli LPS layer.

• Tail fibers retract, tail pins contact the cell wall, and T4 lysozyme forms a pore in peptidoglycan.

• Tail sheath contracts, injecting viral DNA into the cytoplasm; capsid remains outside.

Restriction and Modification

• Prokaryotes use several mechanisms to resist viral infection:

  • Toxin-antitoxin molecules

  • CRISPR (antiviral defense)

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

• Host DNA is protected by modification at recognition sites.

• Restriction is specific for dsDNA; ssDNA and RNA viruses are unaffected.

• Phages may evade restriction by base substitution.

Replication of Bacteriophage T4

• Genome replication can follow various schemes.

• Small DNA viruses use host DNA polymerase; complex viruses (e.g., T4) encode their own.

• T4 encodes primases, helicases, and an 8-protein DNA replisome complex.

• Circular permutation: Same genes arranged in different orders; some sequences are terminally redundant (duplicated at both ends).

• T4 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 virion release.

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

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

  • Middle and late proteins: Head and tail proteins, 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:

  1. Assembly of proheads (head precursors)

  2. Assembly of packaging motor at the opening

  3. Pumping of double-stranded linear genome into prohead

• After head is filled, tail and other components are self-assembled.

• Late enzymes break the membrane and peptidoglycan, causing lysis and virion release.

Phage Lysis

• Some phages cause chronic infections, releasing virions without killing the host; most cause acute infections, lysing the host cell.

• Timing of lysis is crucial for maximizing phage release.

• Three key proteins involved:

  • Lysozyme: Degrades the cell wall.

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

  • Antiholins: Regulatory proteins that coordinate lysis timing.

IV. Strategies of Phage Genome Replication

DNA Phage M13: Rolling Circle Replication

• M13 is a circular single-stranded DNA phage.

• Replication involves conversion to a double-stranded replicative form (RF), followed by rolling circle replication.

• Gene II endonuclease nicks the (+) strand, initiating synthesis of new progeny strands.

• Gene V product prevents the (+) strand from being used for more RF, aiding in packaging into phage heads.

Phage T7: Phage-Encoded RNA Polymerase

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

• Late genes are transcribed by T7 RNA polymerase, ensuring proper timing of phage assembly.

T7 Phage Genome Packaging

• Genome forms concatemers (repeats of genome units linked end to end).

• Pac sites are recognized, and individual genome units are cut and packaged into phage heads.

V. Phage Replication: Virulent and Temperate

Virulent vs. Temperate Phages

• Virulent phage: Only undergoes the lytic cycle, immediately lysing the host cell.

• Temperate phage: Can undergo lytic or lysogenic cycles.

  • Lysogeny: Phage genome integrates into the host genome (as a prophage), maintained by a repressor protein.

  • Inactivation of the repressor or cell stress (e.g., DNA damage) induces the lytic cycle.

• Lysogen: Host cell harboring a temperate virus.

• Prophage: Integrated phage DNA.

Lysogenic Conversion

• Temperate phage can alter host phenotype (e.g., immunity to superinfection, expression of toxins).

• Lysogeny offers advantages: phage viability without replication, survival during low host density.

• Induction triggers the lytic cycle, releasing new phage particles.

VI. Temperate Bacteriophages and Lysogeny: Lambda Phage

Lambda Phage Genome and Replication

• Lambda is a linear dsDNA virus with cohesive ends (cos sites) that circularize upon entry into the host.

• Replication occurs via theta replication (bidirectional) and rolling circle replication (producing concatemers).

• Cos sites are recognized for packaging DNA into phage heads.

• Cosmids are plasmids with cos sites, used in molecular cloning.

Lytic Development of Lambda Phage

• Three major stages:

  1. Early gene expression (N and cro genes)

  2. Replication gene expression

  3. Late gene expression (head and tail proteins)

• Transcriptional antitermination is required for full-length mRNA synthesis.

• Early stage uses N protein; late stage uses Q protein for antitermination.

Lytic vs. Lysogenic Decision

• Cro protein: Activates lytic promoters, inhibits CI synthesis.

• CI protein: Inhibits lytic promoters, maintains lysogeny.

• CII protein: Activates CI transcription, stabilizes CI, and determines pathway choice.

• High CII activity favors lysogeny; low CII activity favors lytic growth.

Maintenance and Induction of Lysogeny

• CI repressor binds operator regions, blocking transcription of lytic genes.

• CI has two domains: one for dimer/tetramer formation, one for DNA binding.

• Induction (e.g., by DNA damage) activates RecA, which cleaves CI, allowing lytic gene expression.

• Cro protein prevents further CI synthesis, ensuring lytic development.

Site-Specific Recombination

• Lambda integrates at specific attachment sites (attP on phage, attB on host).

• Integration is mediated by integrase and requires a short homologous core sequence.

Satellite Phages

• Phage P4 is a satellite phage that depends on P2 for lytic development.

• P4 uses P2's head and tail proteins, encoding a protein (Sid) that modifies P2's head size.

• P4 can only multiply in cells lysogenic for P2 or co-infected with P2.

VII. Animal Viruses and Retroviruses

Overview of Animal Virus Infection

• Classified by genome type; most human viral diseases are caused by RNA viruses.

• Entire virion enters the animal cell, often by fusion or endocytosis.

• Replication may occur in the nucleus (for DNA viruses) or cytoplasm (for RNA viruses).

Viral Infection Outcomes in Animal Cells

• Virulent infection: Lysis of host cell.

• Latent infection: Viral genome persists without virion production; can be reactivated.

• Persistent infection: Virions released without cell lysis; cell remains alive.

• Transformation: Conversion of normal cell into a tumor cell.

Retroviruses and Reverse Transcriptase

• Retroviruses: RNA viruses that replicate via a DNA intermediate (e.g., HIV).

• Contain reverse transcriptase (RNA-dependent DNA polymerase), integrase, and protease.

• Genome: Two identical ss(+)RNA molecules.

• Key genes:

  • gag: Structural proteins

  • pol: Reverse transcriptase and integrase

  • env: Envelope proteins

Replication Cycle of Retroviruses

1. Entry and uncoating of the virion

2. Reverse transcription of one RNA genome in the nucleocapsid

3. Formation of a single DNA strand, then complementary strand to form dsDNA

4. Integration of dsDNA into host genome (provirus formation)

5. Transcription of retroviral DNA

6. Assembly and packaging of genomic RNA

7. Budding and release of new virions