Biology 421/521: Virology : Lecture 10, 21 October 2002

Last Updated: 10/20/02 at 3:51 PM

Outline

• Retrovirus transcription
  • Retroviral genomes
  • tRNA primers
• Reverse Transcriptase (RT)
  • Strong Stop
  • Template Exchange
  • LTRs
  • RT Structure and Activity
  • Other RTs
• Retroelements

RT Discovery

• RNA tumor viruses - Temin
• Integration into cellular DNA to form Oncogenes
• Viral Polymerases - Baltimore
• RNA Dependent DNA polymerase
• Nobel Prize 1975

Why so important?

• DNA -> DNA -> RNA -> protein
• Retrovirus
• Biotech: cDNAs
• Carcinogenesis
• AIDS

Reverse Transcription

• RTs are DNA! polymerases
• Similar structure, active site
• Need primer
• Endogenous reactions = from purified virus particles

Genome

• 2 copies (+) RNA virus
• Held together at 5' end by basepairing
• Only 1 copy is integrated = pseudodiploid
• Probably necessary for replication
• Coated with NC protein (ssb)
• Figure 7.1

tRNAs ?

• Many tRNAs packaged in virion
• Most unknown function but 1 tRNA is a primer
  • tRNA^Pro, tRNA^Lys in mammalian viruses
• Bind (basepair to primer binding site) +
• Figure 7.2

Reverse Transcriptase

• Many copies in virion (50-100)
• Can function in virion preparations
• 4 activities
  • RNA dependent DNA polymerase
  • DNA dependent DNA polymerase
  • DNA helicase
  • RNAse H (domain)
    • Exo and endo DNA-RNA hybrids

The RT - Cycle - 1

• Replication from primer to 5' end of template
  • (-) "Strong stop" DNA
  • Only ca. 100 nucleotides
• RNAse H cleaves off RNA
• Figure 7.3 (1->3)

First template exchange

• (-) ss DNA anneals to 3' end of template at "r" site (direct repeat) and extends to end of (+) template to give cDNA
• Note that cDNA has a permuted genome
• (+) template degraded by RNAse H
• Figure 7.3 (3->5)

Completion of the + DNA strand

• polypurine tract primer
• copies until pseudo-uridine of tRNA primer
• tRNA cleaved by RNAse H
• Figure 7.3 (5->8)

Second template exchange

• This free end anneals to the 3' end of the cDNA (-) strand
• The U3, R and U5 sequences are copied to and end up at both ends of the genome
  • Long terminal repeats (LTRs)
• Mostly by strand-displacement synthesis
• Figure 7.3 (9)

Overall RT

• "Destructive replication"
• Makes "provirus"
• Puts LTR in correct location for transcription and integration
• Figure 7.3

RT Enzyme Function

• Many RT genes are split up
• Figure 7.6
• Require Mg²⁺ or Mn²⁺
• Slow rates

Fidelity of RT

• Lacks 3'-5' exonuclease
• poor fidelity (like RNA-RNAPs)
  • 10^-4 to 10^-6
• Incorporates many mistakes
• Figure 7.7

RNAse H

• Needs divalent cations
• Only cleaves RNA from DNA-RNA heteroduplexes
• Leaves 3'-OH and 5' Phosphate

HIV-1 RNAse H structure

• Only 24% amino acid identity
• Almost identical structure
• Figure 7.9

HIV-1 RT structure - 1

• Primary structure
• Figure 7.8

HIV-1 RT tertiary structure

• Figure 7.10

Other RT Examples

• Hepadnaviruses, Caulimoviruses have RNA intermediates
  • retroid viruses
• In Bacteria (and Archaea???)
• May be remnant of RNA world
• Many retro-elements in genomes
• Up to 10%

Retroelements

• Endogenous proviruses
• Retrotransposons
• Retroposons (LINES)
• Retrosequences (SINES)
• Processed pseudogenes
• Figure Box 7.2

Phylogeny of Retroelements

• Figure 7.13