Biology 421/521: Virology : Lecture 11, 23 October 2002

Last Updated: 10/25/02 at 12:45 PM

Outline

• Retrovirus integration
• Retroid Viruses

Retrovirus structure

• U5 and U3 (unique)
• gag, pol and env genes
• LTRs
• Appendix Figure 9

Retrovirus replication cycle

• Virus replication in cytoplasm
• Virus intasome imported into nucleus
• Transcribed
• Appendix Figure 10

Viral Integrase (IN)

• Unique to retroviruses for ANIMAL viruses
• Present in many bacteriophage and archaeal viruses
• Packaged in virion
• Many copies
• Present at 3' end of pol gene
• Figure 7.6 (not 7.7)

Viral integration

• Loss of 2 basepairs from viral genome
• 4-6 basepair direct repeat in target sequence
• Provirus 5'-TG......CA-3'
• Often in inverted repeat

Integration model

• Nicked ends
  • Give 2 bp 5' overhang
  • 3' = CA (required
• Random insertion
  • Gives gapped intermediate
• In a multimeric complex
  • DNA wrapped around structure
  • "nucleoprotein preintegration complexes"
• Figure 7.15

Specificity of Integration

• IN appears to be non-specific for host site
• in vivo, however more specificity is seen
• Maybe to bent DNA (chromatin
• Figure 7.16

Disintegration

• Reversal of integration reaction
• Excision (in bacteriophage)
• Just needs IN

Domain structure of IN

• 3 domains
• N-term Zn2+ (not finger)
  • Some DNA binding (non-specific)
• Central catalytic core domain
  • All activities
  • RNAse H fold
• C-term (beta-sheet)
  • DNA binding
• Figure 7.18

"Intasome"

• Some 3D structures (gamma-delta resolvase) have been solved
• Viral DNA strands are bent and symmetric
• Probably a tetramer
• Figure 7.20

Host proteins

• Required for many bacteriophage
• Needed to impede self-integration
• Probably bend DNA

Hepadnaviruses

• Hepatitis B virus
• Appendix Fig 11

Hepadnavirus genome

• 2 ss DNA strands
• slightly overlapping
• 2 direct repeats DR1 and DR2
• 5' end (-) has polymerase attached
• (+) strand has a RNA cap at 5' end

Hepadnavirus Replication

• (-) strand resistent to actinomycin D
• (+) strand inhibited
• genome becomes ds in nucleus
• pregenome RNA exported and reverse transcribed
• some partial viruses reimported into nucleus
• genome not replicated by host!
• Figure 7.22

Genome structure

• Pregenome RNA
  • longer than template
    • contains repeat
    • DR1 and epsilon
      • primer for (-) strand synthesis
      • encapsidation signal
• Figure 7.23

P-protein

• Similar to retroviral RT
• Serves as protein primer
• binds to epsilon structure at 5' end
• Figure 7.26 (1-2)

Replication 2

• P-protein with (-) strand translocates to 3' end (DR1)
• DR1 copied and RNAse H degrades (+) strand except 5' end with cap
• 5' end cap is used for (+) strand primer
• Figure 7.26 (3-5)

Replication 3

• (+) strand primer (RNA) translocates to 3' end
• Makes short DNA copy
• Circularizes
• Primer is maintained and replication is not completed
• Figure 7.26 (6)

Comparisons of retroid viruses

• Hepadnaviruses have incomplete circular genomes
• Retroviruses have linear genomes
• Figure 7.29

Review

• Retroviruses
  • RT structure and function
  • Genome structure
  • tRNA primer
  • Retrovirus transcription
  • RT fidelity
  • RNAse H
• Retroelements
  • Phylogeny and origin
• Retroviral Integration
  • How does it work
  • Specificity
  • Intasome
• Hepadnaviruses
  • Genome
  • Replication