I. Overview of retroviruses

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Introduction to Retroviruses
Kathryn S. Jones, Ph.D. SAIC-Frederick/NCI-Freder
ickjoneska_at_mail.nih.gov

• I. Overview of retroviruses
• A. History
• B. Shared characteristics
• C. Classification
• II. Function of different regions of the
  retroviral genome
• A. Cis acting elements
• B. Gag proteins
• C. Pol proteins
• D. Env proteins
• III. Details of life cycle
• A. Early stage
• B. Late stage

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General Introduction to Retroviruses

• Retroviruses
• - Ubiquitous found in all vertebrates
• - Large, diverse family
• - Includes HIV, FIV and FeLV
• Definition and classification of retroviruses
• - Common features- structure, composition and
  replication
• - Distinctive life cycle RNA-DNA-RNA
• - Nucleic acid is RNA in virus, and DNA in
  infected cell
• Transmission may be either
• - Horizontal- by infectious virus (exogenous
  virus) or vertical- by proviruses integrated in
  germ cells (endogenous virus)
• - Can transmit either as free viral particle or
  (for some retroviruses) through cell-cell contact

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A Little Retrovirus History (part I)

• - Francis Peyton Rous discovered the first
  retrovirus (cancer-causing chicken virus, RSV) in
  1910.
• Was derided at time.

- Won Nobel prize for the work in 1966 (at age
87).

• Prior to 1970
• Retroviruses were RNA tumor viruses
• Viruses able to cause cancer
• Had RNA genome

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A Little Retrovirus History (part II)

• Strange observations
• Infection could be stopped with DNA synthesis
  inhibitors
• Transcription inhibitors blocked replication
• Why so strange?
• At time-central dogma of molecular
  biologyDNA?RNA?Protein
• So.. RNA couldnt be template for DNA

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(No Transcript)
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A Little Retrovirus History (part III)

• 1960s Howard Temin suggested DNA provirus was
• part of replication cycleRNA?DNA?RNA?Protein
• - Originally derided
• Won Nobel prize (with Baltimore) in 1970 after
• they independently discovered RT activity in
  infected cells
• 1980 Human T-cell leukemia virus discovered,
• the first pathogenic human retrovirus.
• 1982 Human immunodeficiency virus discovered.
• 1990 First gene therapy trial involving the use
  of retroviral-based vectors in patient with a
  deficiency in adenosine deaminase (ADA).
• 2006 Xenotropic murine leukemia-related virus
  discovered.

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Retrovirus Overview
Enveloped virus with lipid bilayer and viral
spike glycoproteins.
Have outer matrix protein and inner core capsid
containing viral genome.
Genome Two copies of single stranded
positive-stranded RNA (8-10kb).
All retroviruses contain gag, pol and env
genes. Simple - only gag, pol, env Complex -
additional genes involved
in replication.
Reverse transcriptase to generate DNA
Viral genes are integrated into host genome.
Progeny virus produced using host cell
transcriptional and translational machinery.
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Retroviruses
Transmission EM
Scanning EM
3D representation of HIV virion
http//www.mcld.co.uk/hiv/?q3D20HIV
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Retrovirus Classification
Genus
Example
Genome
Avian leukemia virus
Alpharetrovirus
Simple
Betaretrovirus
Mouse mammary tumor virus
Simple
Murine leukemia virus Feline leukemia
virus Xenotropic murine leukemia-related virus
Gammaretrovirus
Simple
Human T-cell leukemia virus
Deltaretrovirus
Complex
Wall-eyed sarcoma virus
Epsilonretrovirus
Complex
HIV, SIV, FIV
Lentivirus
Complex
Human foamy virus
Spumavirus
Complex
Yeast TY-3
Metavirus
Drosophila melanogaster Gypsy
Errantvirus
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Retrovirus Genome (Diploid)

• Ranges from 7-10 kb in size (1 copy)

• Diploid 2 copies/virion

• Important in high recombination rate

From Flint et al. Principles of Virology (2000),
ASM Press
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PPT
y
( Packaging Signal)
PBS- primer binding site PPT- polypurine tract
AAAA 3
5m7GpppG
R
U5
U3
R
gag
pol
env
R - repeat sequence
PBS
U3 - promoter/enhancer
U5 - reverse transcription/ integration.
CA
SU
TM
MA
CA
NC
PRO
RT
IN
MA-Matrix CA- Capsid NC- Nucleocapsid
PRO- Protease RT- Reverse transcriptase IN-
Integrase
SU- surface envelope protein TM- transmembrane
envelope protein.
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Genome of Simple vs. Complex Retroviruses
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Retroviral Structural genes
Gene Proteins Function gag group specific
antigen (internal structural proteins) matrix
(MA), binds envelope, organization capsid
(CA), protects genome and enzymes nucleocapsid
(NC) chaperones RNA, buds pol
polymerase enzymes reverse transcriptase RNA
to DNA RNAase H (RT) degrades template
RNA protease (PR) maturation of
precursors integrase (IN) provirus
integration env envelope proteins surface
glycoprotein (SU) receptor binding
transmembrane protein (TM) virus-cell fusion
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Gag proteins
CA
MA
TM
SU
SU
Gag protein 1200-1800/virion Gag-Pol protein
100- 200/virion
Matrix (MA)- involved in binding to envelope
proteins- inner surface of membrane. Capsid
(CA)-major protein of the shell most abundant
protein in the virion, forms core
(fragile) Nucleocapsid (NC)- involved in RNA
packaging and folding also uncoating
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Pol proteins
Protease (PR)- cleaves Gag and Pol polyproteins,
required for virion maturation Reverse
transcriptase (RT)-reverse transcribes the RNA
genome, also has RNAseH activity. Has DNA
polymerase activity that can use DNA or RNA as
template. Integrase (IN)- inserts the dsDNA copy
of the viral genome into the host cell
chromosome.
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Protease

• 10 kd, dimer
• Cuts Gag polyprotein to MA,CA,NC
• Aspartyl protease
• Exquisite cleavage specificity
• Major class of anti-HIV drugs are Protease
  Inhibitors

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Reverse Transcriptase
RNA
DNA
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Env proteins
Surface glycoprotein (SU)- involved in receptor
recognition Transmembrane glycoprotein (TM)-
triggers the fusion of the viral and cellular
membranes,
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Cis-acting Elements in Retrovirus Replication

• Cis acting sequences important for
• Transcription of RNA genome and mRNAs for viral
  proteins (enhancer/promoter, cap site,
  polyadenylation sequences)
• Allowing full length (genomic) RNA to exit
  nucleus (RRE, CTE)
• 3. Reverse transcription (PBS, PPT, R U5)
• 4. Packaging genome (DMS, and packaging site ??)

Integrated proviral DNA genome
RNA genome
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Retroviral Life Cycle
Early events from viral binding and entry
until the time the DNA copy of the viral genome
is integrated into the host cells chromosome
Late events From time when integrated provirus
is expressed until virus has been released
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Retroviral Life Cycle Binding and Fusion
4

• Virus binds to cell surface
• Specific interactions occur between the Env
  proteins on the virus and specific host cell
  proteins (receptors)
• Env proteins undergo conformational change, which
  results in the fusion of the viral and cellular
  membranes
• Most use plasma membrane fusion by some use
  endocytosis and then fuse envelope with membrane
  of endosome

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Binding of Retroviruses to Target Cells
HIV
CD4
Immune system cell
Co-receptor

• Virus binds to specific receptors, via
  interaction with SU
• Different retroviruses use different receptors
• BUT small groups of viruses share receptors
• Env proteins- undergo conformational change which
  allows TM to facilitate virus-cell fusion

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Examples of Retroviral Receptors
Xenotropic/Polytropic MLV
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Integration of Provirus

• Provirus complexed with protein moves to nucleus
  pre-integration complex
• most retroviruses require cells going into
  mitosis for the breakdown of the nuclear membrane
• - productive infection only in dividing cells
• HIV and related viruses can enter intact nuclei,
  so no need for cell division
• - can productively infect nondividing cells
• Integrase is still attached cuts up the DNA of
  the cell and seals provirus in the gap
• may lead to immediate expression of viral genes
  or little or no expression (latent infection)
• when this cell divides so does the genomes and
  get daughter cells with viral genome
• irreversibleadvantage for vectors
• - can lead to insertional mutagenesis

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Latent vs. active infection
In latent infection- retroviral genome is present
but is not transcribing viral genome or mRNA for
structural proteins.
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Retroviral Life Cycle Transcription of Viral
Genome
If provirus is not latent, transcription of the
provirus occus. This produces RNA for new
retrovirus genomes and RNA that codes for the
retrovirus capsid and envelope proteins.
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Two Pathways to Retroviral Assembly

• Capsid assembly occurs at the membrane during
  budding (most retroviruses)
• 2. Capsid presassembled in cytoplasm and then
  transported to plasma membrane
  (Betaretroviruses Type B/Type D
  spumaretroviruses)

ONE single A.A. change in MA (R55W) can convert
M-PMV from type D to type C
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Retrovirus budding from a cell
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After Budding, Virus Goes from Immature to Mature
Form
Mature Form (after budding)-Core becomes more
dense-Different retroviruses have different
morphology in mature form