Download Retroviruses

Introduction to Retroviruses
I. Overview of retroviruses
A. History
B. Shared characteristics
C. Classification
Kathryn S. Jones, Ph.D.
SAIC-Frederick/NCI-Frederick
[email protected]
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
biology”:DNARNAProtein
–So.. RNA couldn’t be template for DNA
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5
A Little Retrovirus History (part III)
1960s: Howard Temin: suggested DNA “provirus” was
part–of replication cycle:RNA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
matrix
Env
capsid
RNA
Scanning EM
3D representation of HIV virion:
http://www.mcld.co.uk/hiv/?q=3D%20HIV
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Retrovirus Classification
Genus
Example
Genome
Alpharetrovirus
Avian leukemia virus
Simple
Betaretrovirus
Mouse mammary tumor virus
Simple
Gammaretrovirus
Murine leukemia virus
Simple
Feline leukemia virus
Xenotropic murine leukemia-related virus
Deltaretrovirus
Human T-cell leukemia virus
Complex
Epsilonretrovirus
Wall-eyed sarcoma virus
Complex
Lentivirus
HIV, SIV, FIV
Complex
Spumavirus
Human foamy virus
Complex
Metavirus
Yeast TY-3
Errantvirus
Drosophila melanogaster Gypsy
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Retrovirus Genome (Diploid)

Retrovirus genome is +RNA

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), 10
ASM Press
y ( Packaging Signal)
5’m7GpppG R U5
gag
pol
PPT
env
U3 R AAAA 3’
PBS- primer binding site
PPT- polypurine tract
R - repeat sequence
PBS
U3 - promoter/enhancer
U5 - reverse transcription/
integration.
CA
SU
MA CA NC
TM
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),
capsid (CA),
nucleocapsid (NC)
binds envelope, organization
protects genome and enzymes
chaperones RNA, buds
pol = polymerase enzymes
reverse transcriptase +
RNAase H (RT)
protease (PR)
integrase (IN)
RNA to DNA
degrades template RNA
maturation of precursors
provirus integration
env = envelope proteins
surface glycoprotein (SU) receptor binding
transmembrane protein (TM) virus-cell fusion
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Gag proteins
CA
MA
SU
TM
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
1. Transcription of RNA genome and mRNAs for viral proteins
(enhancer/promoter, cap site, polyadenylation sequences)
2. 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
LTR
U3
R
LTR
U5
gag
pol
env
PBS (tRNA binding site)
DMS (dimer linkage site)
packaging site
U3
R U5
2nd strand primer site
RNA genome
transcription
cap
R U5
gag
pol
env
U3
R
A 19
n
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
cell’s 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
•Virus binds to4 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
Ecotropic MLV
out
out
in
in
N
CAT-1
C
(Cationic amino acid transporter)
ALV-A
Tv-A
N
XPR-1
(unknown function)
ALV-B, -D, -E
CAR1
C
HIV
CD4
CCR5
CXCR4
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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
- irreversible:advantage 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
1. 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
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from type D to type C
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
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