Reverse Transcriptase & Retroviruses

History of Retroviruses: First Phase

• 1908: Discovery of chicken leukemia virus (Bang and Ellerman)

• 1911: Discovery of Rous sarcoma virus (Rous, Nobel Prize 55 years later)

• Called tumor viruses

• Found to have RNA genomes

History of Retroviruses: Second Phase Discovery (RT)

• Howard Temin: “tumor viruses” caused permanent changes at the cellular level (transformation)

  • Provirus hypothesis: Viral DNA integrates into the host genome

• David Baltimore:

  • (+) RNA Virus: No RdRp in particle

  • (-) RNA Virus: RdRp in particle

  • An enzyme that copies RNA to DNA must be in the virus particle (Reverse Transcriptase) 

Retroviridae

Orthoretrovirinae: Alpha (like Avian sarcoma leukosis virus), Beta (like mouse mammary tumor virus), Gamme (like Marine leukemia virus), Delta (like Human T-lymphotropic virus), Epsilon (Walleye epidermal hyperplasia virus), and Lentivirus (HIV)

Spumaretrovirinae: like Simian foamy virus

Simple: Alpha, Beta, and Gamma

Complex: Delta, Epsilon, Lentivirus, and Spum

Simple Retrovirus

Contains (+) ssRNA, Integrase, Reverse Trascriptase, and Protease

Capsid → Nueclocapsid → Matrix → Envelope → Surface Protein

Simple Retrovirus Expression from Provirus

LTR → gag (core) → pol (enzymes) → env (envelope) → LTR

gag

core (matrix, P10, capsid, nucleocapsid, protease)

• protease cuts out all of the other proteins and itself as the genome is produced as a multiprotein

pol

enzymes (RT and IN)

env

evelope proteins (transmembrane proteins)

spliced in cytoplasm envelope and precursor

Simple Retrovirus Replication Cycle

1. Attachment and Aborption

2. Procapsid forms and allows nt influx

3. dsDNA formed in procapsid in the cytoplasm

4. Procapsid genome enters through nuclear pore

5. Provirus integrated into host genome

6. Transcription and translation through host machinery

7. Translation of the multiprotein

8. Protein complex formed

9. Viral genome replication***

10. mRNA sent out of nucleus for envelope proteins

11. Translation of envelope proteins

12. Passes through ER and Golgi

13. Envelope buds out

14. Assembly through budding

15. Maturation of virus through protein cleavage

Unspliced Retrovirus mRNAs transportation to the cytoplasm

Constitutive transport elements (CTE) sequence which binds the host Nfx1 splicing protein and tricks the cellular proteins into exporting un-spliced viral mRNA to the cytoplasm

Maturation Process of Simple and Complex Retrovirus Particles

The viral proteases cleaves the Gag polyproteins, which triggers a major structural rearrangement of the particle. This process is essential for converting the immature particle into a mature, infectious form. Maturation process is a major target of aitretroviral drugs

Steps in Retrovirus Life Cycle*****

1. Receptor binding virus is taken in by fusion at the surface (other retrovirus are taken in by endocytosis) fusion between envelope and cell membrane

2. Core particle the genome never un-coat and is copied to dsDNA by reverse transcription within that sub-viral particle in the cytoplasm 

3. Retrovirus dsDNA enters the nucleus and is integrated and is refered as the proviral DNA

4. Sitting in the chromosomal DNA the proviral DNA is transcribed and produce non splice genomic RNA

5. Splice mRNAs are translated as precursor proteins that are encapsidated (immature virion)

6. Retrovirus particles mature via major protein structural rearrangements (mature infective virion)

Complex Retrovirus: HIV-1

• Structural genes: gag, pol, env

• Regulatory gene: tat (facilitates transcription)

• Mediate mRNA transport: rev (nuclear export of un-spliced or spliced transcripts)

• Accessory gene: vif (block antiviral innate responses)

• Accessory gene: vpr (arrest cell cycle and enhance viral gene expression)

• Accessory gene: vpu (allows effective release of viral particles)

• Accessory gene: nef (downregulated CD4 and MHC1 expression)

Complex Retrovirus: Human T-Cell Leukemia Virus Type 1 (HTLV-1)

tax and HBZ: regulatory proteins related to oncogenesis of Adult T-cell Leukemia (ATL)

p21, p12, p12, p30: accessory proteins modulate immune responses

Rex: mediate transport of un-spliced mRNAs to the cytoplasm

HIV-1 Reverse Transcriptase Structure

• RT is composed of 2 subunits: p66 and p51 (structure resembles a hand?)

• First Activity: use RNA as a template to produce DNA (requires nt)

• Synthesize of DNA from RNA (RdRp)

• Cofactors needed for reverse transcriptase activity are divalent metal cations, typically magnesium ions 

Lacks processivity: slows polymerase activity

Different to DNA polymerases RTs do not remain attached to the template-primer duplex “poor processivity”

Lacks prrof reading activigity: high error prone

lacks 3’ to 5’ exonuclease of DNA pol I that excise miss-paired nt. Incorporate mistakes as frequently as 1 per 70 copies at some template positions and as infrequently as 1 per 10⁶ copies at others

RNAse H

Cleaves RNA only when it’s in a double-stranded configuration

RNA can be in RNA:RNA or RNA:DNA; double-stranded. No DNA:DNA

Generate by endonucleolytic cleavages