Viral Replication

• Viruses


  Consist of their genome that is covered in a capsid protection shell (Capsomeres)

  
• Viral genomes


  • ssDNA
  • dsDNA
  • ssRNA
  • dsRNA

  
• Viral envelopes


  • Some (not all) viruses have an additional layer derived from host cell membrane (e.g. influenza virus, HIV)

  
• Viral capsids


  • Can have different morphologies, e.g. helical & icosahedral

  
• Viral entry


  Viruses differ in their way how to enter a host cell (Endocytosis vs. Fusion), Bacteriophage only inject the genome but never enter the host cell

  
• Viruses are very specific to their host

  
• Infection of host cell consists of the very same steps, even though the specifics differ

  
• Hypotheses of how viruses evolved

  • 3 hypotheses

  
• Classification of viruses


  Depends on structure, genome organization, transcription and replication strategies

  
• All viruses must generate mRNA that can be recognized by host cell ribosomes

  
• Virus identification by electron microscopy

  The first step in identification rests on visual observation of viral morphology (not infallible)

  
• Virus identification by nucleic acid analysis

  Polymerase chain reaction (PCR) and reverse-transcriptase PCR (RT-PCR) can be used to identify viruses by genome sequence and study viral evolution patterns

  
• RT-PCR


  1. RNA is isolated

  2. Primer, RT, nucleotides are added

  3. RT makes a complementary DNA copy of the RNA

  4. Sample is heated to denature strands and inactivate RT

  5. Primers, Taq pol, and nucleotides are added

  6. Taq pol makes a second DNA strand

  7. Further PCR cycles can amplify the amount of DNA produced

  
• Viral attachment proteins and host cell receptors

  Viruses recognize host cells via interaction between viral attachments proteins and host cell receptors. Each virus has a specific range/type of cells that it can infect.

  
• Cellular receptors for selected viruses

  • CD4
  • ICAM-1
  • Bgp 1a
  • CR2
  • Sialic acid
  • OmpF

  
• Strain specificity of bacteriophage T2

  Depends on attachment protein. T2 recognizes lipopolysaccharide and the Outer Membrane Protein OmpF of E. coli K-12 strains, but not O157:H7. PP01 recognizes O157:H7, but not K-12.

  
• Host specificity (Host range) is determined by interactions between viral and cellular proteins

  
• Viral attachment proteins


  Most enveloped viruses attach by a specific viral protein (spike) embedded within the envelope, e.g. the hemagglutinin (HA) protein. Non-enveloped viruses have spikes (attachment proteins) that extend from capsid surface. For some non-enveloped viruses, the capsid directly interacts with receptor.

  
• Viral attachment protein : cellular receptor interaction is very specific. Strain specificity even exists within a single species!

  
• Homology of receptor proteins among host species dictates the likelihood of an infection
• We can very specifically design drugs to interfere with the binding
• Viral entry mechanisms


  Several different mechanisms exist for viruses to enter the fairly tight casing of an eukaryal/bacterial/archaeal cell

  
• Entry of enveloped viruses (Influenza, HIV)

  Binding of virus to receptor

  2. Endocytosis

  3. Acidification of endosomal vesicle

  4. Low pH triggers conformational change in viral protein to expose fusion peptide

  5. Fusion of viral envelope and endosomal membrane

  6. Release of genome

  
• Entry of non-enveloped viruses (reovirus)

  Virus binds to receptor on cell surface

  2. Endocytosis is triggered

  3. Low pH of endosome induces conformational changes within viral capsid proteins

  4. Exposure of pore-forming domain creates hole in plasma membrane

  
• Entry of bacteriophage genome

  Phage attachment protein interacts with host cell receptor

  2. Tail of the phage contracts

  3. Tail core penetrates through cell wall

  4. Brings DNA in contact with plasma membrane

  5. Phage pilot protein binds phage DNA and assists in allowing DNA to penetrate the plasma membrane

  
• Entry of plant viruses (tomato yellow leaf curl virus)

  Due to tough cell wall structures (plus waxy, thick cuticula), plant viruses often require damage induced by insects to infect the plant cells

  
• Viral uncoating


  Prior to replication, viral genome must become accessible. Disassembly necessary for subsequent replication. Uncoating (Capsid disassembly) occurs, e.g. through conformational changes of capsid proteins or proteolytic processing.

  
• Kinetics of a viral infection

  One-step virus multiplication curve: 1. Latent phase (Genome replication & Protein Production), 2. Rise phase (Cell lysis – virus release), 3. Plateau

  
• Classifications of viruses based on replication strategies

  • 3 DNA virus groups: dsDNA, ssDNA, dsDNA

  4 RNA virus groups: dsRNA, ssRNA (+), ssRNA (-), ssRNA using reverse transcriptase

  
• Class I: dsDNA viruses


  dsDNA serves as a template for mRNA synthesis. Genome replication in nucleus of host cell by DNA-dependent DNA polymerase. Exception: Poxvirus, replicates in cytoplasma using viral enzymes. Transcription by host cell DNA dependent RNA polymerase

  
• Class II: ssDNA viruses

  mRNA forms from dsDNA intermediate. Genome replication occurs as ssDNA à first conversion into dsDNA, which serves as template.
• DNA virus groups


  • dsDNA
  • ssDNA
  • dsDNA

  
• RNA virus groups


  • dsRNA
  • ssRNA (+)
  • ssRNA (-)
  • ssRNA using reverse transcriptase
• Before we talk about the viral replication, let's make sure we understand replication, transcription, and translation in the eukaryotic host cell

  
• Viruses can be classified based on how they replicate and generate mRNA

  
• Class I: dsDNA viruses


  • dsDNA serves as a template for mRNA synthesis
  • Genome replication in nucleus of host cell by DNA-dependent DNA polymerase
  • Exception: Poxvirus, replicates in cytoplasma using viral enzymes

  
• Class II: ssDNA viruses

  • mRNA forms from dsDNA intermediate
  • Genome replication occurs as ssDNA à first conversion into dsDNA, which serves as template for replication into ssDNA
• Class VII: ssDNA viruses (utilize reverse transcriptase)


  • dsDNA genome serves as template for mRNA generation and full-length RNA
  • Viral reverse transcriptase generates genomic DNA from ssRNA intermediate

  
• Replication of eukaryal DNA viruses

  • Viral genome replication and transcription of viral genes occur much like they do in the host cell

  
• Replication of typical eukaryal DNA viruses (e.g. Herpes virus)

  • Replication occurs in nucleus
  • Host-cell DNA polymerase is used to produce new copies of viral genome
  • Host-cell DNA-dependent RNA polymerase is used for transcription of viral DNA