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

Where and when does reverse transcription occur?

• Each retroviral particle contains 50-100 molecules of RT

• In infected cells the onset of viral dsDNA synthesis occurs upon viral entry once the capsid is sufficiently degraded and is permeable to triphosphate nucleotides

• Viral DNA synthesis occurs in subviral particles

Diploid Retroviral Genome

• (+) sense RNA dimer

• 5’ Cap and 3’ Poly A tail

• Two cellular tRNA molecules at the 5’ end

• Genome coated with nucleocapsid protein

What does HIV contain an RNA dimer?

• Although the genome is an RNA dimer the substrate of RT is always only one RNA strand to produce dsDNA copy 

• The RT randombly flickers back and forth between the two RNA copies 

• Dimer genome allowed to patch together one complete DNA by recombination (increases the chance of viable RNA and DNA)

• Two copies of all genes: copy-choice

• The RT randomly flickering increases the probabilities to obtain a functional genome to be built given the virus genetic robustness

tRNA binding to RNA genome

RT requires the binding of a tRNA to the primer binding site (pbs) → pbs

Hair pin structure around the pbs + tRNA-clover leaf structure gives 18 nt of the tRNA anneal to the pbs

RT Mechanism from RNA to dsDNA

Retroviral DNA synthesis begins by synthesis of the (-) DNA strand from the tRNA primers

• Initiation of (-) strand DNA synthesis

• The 5’ end of the viral RNA genome is degraded by the RNase H activity of RT as the (-) strand DNA is synthesized (template exchange)

• Complementary regions (R sequences) of (-) strand DNA and RNA allow template exchange

• Annealing between the R sequences of the 5’ and 3’ ends allows 1st template exchange, DNA (-) synthesis

• The RNA genome degrades as the DNA (-) is synthesized

• RNA Polyurine track ppt (A/G rich region)

• DNA (-) and ppt duplex is used as primer for DNA (+) synthesis

• The PBS sequence is copied twice (from the RNA genome and from tRNA primer)

• Complementary PBS sequences provides a circular DNA template

• Synthesis of the (+) DNA strand can now continue using (-) strand DNA as a temmplate

Steps of Reverse Transcription***

1. (-) DNA strand synthesis from tRNA primer

2. (+) DNA synthesis starts from poly-urine tract (ppt) and continues from (-) DNA strand once PBS sequence is copied twice

3. Two strong stops of the RT enzyme facilitated by r/R and PBS/PBS complementarity results in template exchanges

4. Major product of reverse transcriptase found in infected cells is a linear duplex DNA copy of the viral genome with additional sequences at each end LTRs (Long terminal repeats) is the substrate for integration and is known as proviral DNA

Integration of Retroviral DNA

The last 2 bases in the 3’ end of each strand are cleaved by the integrase

The removal of these bases frees the OH-group in the adenine, which is required for the ligation with the host DNA

Integrase mediates the host DNA cut and ligation

Cellular enzymes are required to complete the integration process by repairing the integration intermediate

Integration is Not Random

HIV DNA preference to integrate into sequences that are wrapped around a nucleosome (in areas with increased transcription)

Provirus Generation

There is no viral DNA replication and no viral RNA replication in retroviruses

• One DNA produced from 2 RNAs by reverse transcription

• Promoters (LTR) are generated during the RT

• Proviral DNA is transcribed using the host transcription machinery to synthesize many copies of viral mRNA

• Viral mRNA is translated into viral proteins or incorporated into virus particles

Provirus is a permanent part of host genome

• There is not a specific mechanism for precise excision of provirus

• Cell genomes are literred with ancient and modern retroelements

• Endogenization process by which retrovirus elements are maintained and transferred in the germ line

Retro-Elements

• Proviral DNA integrated into the germline transmitted vertically give rise to endogenous retroviruses

• Often replication defective due to accumulation of mutations (all are defective in humans)

• Although replicaiton defective these are mobile elements that still moving because the RT

• Around 42% of the human genome comprises mobile genetic elements, including endogenous provirus and other retroelements

• Less than 0.5% are currently active in the human genome

HIV-1

Lentivirus

First isolated in 1983

A blood test was developed in 1984

Confirmation of lentivirus characteristics was done through electron microscopy and sequence analysis

HIV-1 and HIV-2

Complex retroviruses with similar genome organization that differ in the expression of accessory genes. HIV-2 has an  addition gene vpx which alleviates transcription repression

HIV-2 infected individuals have a longer and slower progression to AIDS

HIV Coreceptors

• To enter a CD4 cell, virus first attach to CD4 receptor, then attach to co-receptor CCR5 or CxCR4

• CxCR4 - major co-receptor for T cell tropic HIV

• CCR5 - major co-receptor for M tropic HIV

• HIV is usually R5-tropic (uses CCR5) during the early stages of infection

• Later the virus switch to using onle CxCR4 (X4-tropic)

HIV Control

• There is not a cure yet: virus cannot be clear for infected individuals

• No vaccine

• Infected people must take antivirals forever

• Antiviral drugs facilitate drug-resistant viruses

• Drugs are expensive

Is a preventative vaccine against HIV feasible?

• HIV-1 can escape neutralizing antibodies, evading the immune response

• High diversity in the envelope protein is the main factor explaining difficulties in achieving an effective HIV-1 vaccine

Hepadnavirus: Hep B Virus

• Found in many body fluids (blood, sweat, saliva, semen, vaginal secretions, breast milk)

• Transmission mother to infant at time of birth, sexually, parenteral transmission through shared needles, and blood transfusions

• Significant fraction of persistently infected individuals develop

  • chronic hepatitis, cirrhosis, and hepatocellular carcinome (HCC)

A DNA Virus with Reverse Transcriptase

• Gapped, circular, dsDNA

• 3.4 kbp

• (-) strand: full length but nicked with 5’ end bound P protein Reverse transcriptase

• (+) strand: partial length with 5’ CAP end RNA strand

• DR1 and DR2: direct repeats are terminal duplications that are produced by copying a portion of the covalently circular DNA twice

Hepadnavirus Replication Cycle

• Gapped dsDNA genome enter nucleus and is repaired by DNA repair machienry to a covalent closed circular (CCC) DNA

• CCC acquires cellular histones and persists in the nucleus as a non-replicating micro-chromosome from which RNA PoL II transcribes a 3.5 kbp pre-genome (+) cap RNA

• Viral capsid and P protein (reverse transcriptase RT) produced 

• The (+) pre-genome RNA and P protein assemble in a subviral core particle 

• RT commences (No integration) 

• Virus can be released into the ER and obtain viral envelope glycoproteins and eventually are secreted as progeny virus particles or redirected to the nucleus (recycle)

Complex Retrovirus different to Simple Retrovirus has genes that code for _____ and _____ viral proteins

regulatory and accessory viral proteins

Why does the retrovirus RT molecule promote recombination?

Because template switching during DNA strand synthesis (copy choice)

The endogenization of a retrovirus requries infection of _______ so it can be transmitted to the progeny

germinal cells