Viruses and Phages: Structure, Replication, and Life Cycles

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 known as the virion, which facilitates transmission between host cells.

• Virus: Genetic element unable to replicate independently of a host cell.

• Virus particle (virion): The extracellular form of a virus, responsible for transmission.

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

Viral Components and Activities

• Capsid: Protein shell surrounding the viral genome.

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

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

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

Viral Genomes

Viral genomes display significant diversity in structure and composition.

• May be DNA or RNA.

• 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.

• Usually smaller than cellular genomes.

Genome Type	Examples
dsDNA	Bacteriophage T4, Herpesvirus
ssDNA	Parvovirus
dsRNA	Reovirus
ssRNA (+)	Poliovirus
ssRNA (-)	Influenza virus
RNA → DNA	Retrovirus (HIV)

Classification and Morphology

• Classified by host: bacterial viruses (bacteriophages), archaeal viruses, animal viruses, plant viruses, and others.

• Viruses vary in shape and size; most are smaller than prokaryotic cells (0.02–0.3 μm).

Structure of the Virion

The virion is composed of a capsid made of protein subunits called capsomeres or protomers, arranged in a highly repetitive pattern around the nucleic acid.

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

Enveloped Viruses

• Have a lipoprotein membrane surrounding the nucleocapsid.

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

• Few enveloped plant or bacterial viruses exist due to cell wall barriers.

• Entire virion enters animal cells; enveloped viruses exit more easily.

Enzymes Inside Virions

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

• Neuraminidases: Destroy glycoproteins/glycolipids, aiding virus liberation.

• Nucleic acid polymerases: RNA replicases (RNA-dependent RNA polymerases), Reverse transcriptase (RNA-dependent DNA polymerase in retroviruses).

II. The Viral Replication Cycle

Overview of the Virus Life Cycle

Viral replication involves several distinct phases, with differences between prokaryotic and eukaryotic hosts.

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

• Penetration: Entry/injection of viral nucleic acid (prokaryotes) or entire virion (eukaryotes).

• Synthesis: Host cell machinery redirected to produce viral nucleic acids and proteins.

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

• Release: Mature virions exit host cell (lysis, budding, or excretion).

Virus Replication Dynamics

• Characterized by a one-step growth curve: rapid increase in virion count upon cell lysis.

• Eclipse period: Genome replication and protein synthesis.

• Maturation: Packaging of nucleic acids into capsids.

• Latent period: Eclipse + maturation.

• Burst size: Number of virions released per cell.

Culturing, Detecting, and Counting Viruses

• Bacterial viruses grown in liquid medium or on agar lawns; animal/plant viruses in tissue cultures.

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

• Titer: Number of infectious units per volume.

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

III. Bacteriophage T4: Replication and Regulation

Attachment and Entry

Bacteriophage T4 uses complex mechanisms to infect Escherichia coli cells.

• Attachment via tail fibers to polysaccharides on E. coli LPS layer.

• Tail fibers retract; tail pins contact cell wall.

• T4 lysozyme forms a pore in peptidoglycan.

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

Host Defense: Restriction and Modification

• Prokaryotes use restriction endonucleases to cleave foreign DNA at specific sites.

• Host DNA is protected by modification (e.g., methylation) at recognition sites.

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

• Other defenses: toxin-antitoxin systems, CRISPR-Cas immunity.

Genome Replication and Circular Permutation

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

• T4 encodes primases, helicases, and a multi-protein DNA replisome.

• Circular permutation: Genes arranged in different orders; terminal redundancy (duplicated sequences at ends).

• T4 genome first replicated as a unit, then forms concatemers (linked genomic units).

Transcription and Translation Regulation

• Virion synthesis (~30 min) ends with cell lysis and virion release.

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

  • Early proteins: DNA replication/transcription enzymes, host RNA polymerase modifiers.

  • Middle/Late proteins: Head/tail proteins, enzymes for virion assembly and release.

• T4-specific proteins modify host RNA polymerase to recognize only phage promoters; host transcription is shut down.

Lytic Cycle Only

• T4 undergoes only the lytic cycle, not lysogeny.

Packaging and Release

• Genome pumped into capsid head under ATP-driven pressure.

• Assembly in three stages: prohead formation, packaging motor assembly, DNA packaging.

• Tail and other components self-assemble; late enzymes break cell membrane and peptidoglycan, causing lysis and virion release.

Phage Lysis Mechanisms

• Some phages cause chronic infections; most cause acute lysis for virion release.

• Three key proteins:

  • Lysozyme: Degrades cell wall.

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

  • Antiholins: Regulate holin activity, timing lysis with phage assembly completion.

IV. Strategies of Phage Genome Replication

DNA Phage M13

• Circular single-stranded DNA genome.

• Replication via rolling circle mechanism:

  • Replicative form (RF) DNA synthesized from minus strand.

  • Gene II endonuclease nicks strand, initiating rolling circle replication.

  • Gene V regulates packaging of (+) strand into phage head.

Phage T7

• Encodes its own RNA polymerase, allowing specific activation of late genes.

• Genome forms concatemers (repeats); pac sites mark where monomers are cut and packaged into phage heads.

V. Phage Replication: Virulent and Temperate

Virulent vs. Temperate Phages

• Virulent phage: Only lytic cycle; immediate multiplication and host cell lysis.

• Temperate phage: Can undergo lytic or lysogenic cycles.

• Lysogeny: Phage genome integrates into host genome (chromosome/plasmid) as a prophage.

• Lysogeny maintained by phage-encoded repressor protein; inactivation triggers lytic cycle (e.g., by cell stress).

• Lysogen: Host cell harboring temperate virus.

Phage Type	Replication Pathway
Virulent	Lytic only
Temperate	Lytic or Lysogenic

VI. Lysogenic Conversion and Regulation

Lysogenic Conversion

• Temperate phage can alter host phenotype (e.g., immunity to superinfection, toxin/enzyme expression).

• Advantages: Phage remains viable, host cell survival ensured; induction triggers lytic cycle under stress.

Bacteriophage Lambda

• Linear dsDNA with cohesive ends (cos sites) that circularize upon entry.

• Replicates by theta and rolling circle mechanisms, forming concatemers.

• Cosmids: Plasmids with cos sites, used for DNA cloning and efficient bacterial transformation.

Regulation of Lytic vs. Lysogenic Pathways

• Key regulatory proteins: Cro, CI, CII.

• Cro: Activates lytic promoters, inhibits CI synthesis.

• CI: Repressor, promotes lysogeny by activating specific promoters and inhibiting lytic genes.

• CII: Activates CI transcription, stabilizes CI; its activity determines pathway choice.

• Environmental conditions (e.g., host protease activity) influence CII stability and pathway selection.

Site-Specific Recombination

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

• Integration requires integrase enzyme; recombination occurs at a short homologous core sequence.

Maintenance and Induction of Lysogeny

• CI repressor binds operator regions (OR, OL) to prevent transcription of lytic genes.

• CI synthesis is regulated to maintain lysogeny.

• Induction (e.g., DNA damage) activates RecA, which cleaves CI, derepressing lytic promoters and initiating lytic cycle.

• Cro protein prevents further CI synthesis during induction.

VII. Satellite Phages

Parasite Phages

• Phage P4 is a satellite phage, dependent on P2 for lytic development.

• P4 uses P2's head and tail proteins, but encodes Sid protein to modify P2's head size.

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

VIII. Animal Virus Infection

Classification and Entry

• Most human viral diseases are caused by RNA viruses.

• Entire virion enters animal cell; replication often occurs in the nucleus.

• Viruses bind specific host cell receptors, often tissue-specific.

• Entry via membrane fusion or endocytosis; uncoating occurs at membrane or in cytoplasm.

Infection Outcomes

• Virulent infection: Host cell lysis (most common).

• Latent infection: Viral DNA persists in host genome; virions not produced until triggered.

• Persistent infection: Virions released by budding; host cell remains alive.

• Transformation: Conversion of normal cell into tumor cell.

Retroviruses and Reverse Transcriptase

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

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

• Genome: Two identical ss(+)RNA molecules; genes include gag (structural), pol (enzymes), env (envelope proteins).

Retrovirus Replication Process

• Virion enters cell; envelope removed.

• Reverse transcription of RNA genome in nucleocapsid produces ssDNA, then dsDNA.

• dsDNA enters nucleus; integrase incorporates it into host genome as a provirus.

• Transcription of proviral DNA, assembly and packaging of genomic RNA, budding and release of new virions.