retroviruses 105
TRANSCRIPT
-
7/29/2019 Retroviruses 105
1/41
The Retroviridae are a family of enveloped (+) sense ssRNA
viruses that have been intensely studied because of theirassociation with cancers, leukemias and the AIDS syndrome
The first association of viruses with cancer was in early1900s with the discovery by Ellerman and Bang that
leukemia could be transmitted from one chicken to anotherby injecting leukemia cell extracts
In 1911 Peyton Rous showed that a bacterial free filtratefrom solid tumors of chickens could cause an identical
cancer in chickens inoculated with the filtrate The virus causing the leukemia was subsequently shown tobe avian leukosis virus and the virus causing tumors wasdesignated Rous sarcoma virus
Retroviridae
-
7/29/2019 Retroviruses 105
2/41
Although the discoveries by Ellerman, Bang and Rouswere not well accepted at the time, 60 years later theseviruses were designated retroviruses and Rous won theNobel Prize for his work in 1963 at the age of 83
In early 1970, Baltimore and Temin independentlyidentified the unusual enzyme, reverse transcriptase andwon the Nobel Prize in 1975 for their work
Their discovery shattered the central dogma of molecular biology which stated the flow of genetic information was
from DNA to RNA In 1989, Bishop and Varmus won the Nobel Prize for elucidating that retroviral oncogenes are derived fromcellular genes and brought us closer to understanding
cancer
Retroviridae
-
7/29/2019 Retroviruses 105
3/41
History Vallee and Carre (1904) - Filterable virus is cause of Swamp
Fever of horses (EIAV)
Ellerman and Bang (1908) - chicken leukemia transferred bybacteria-free filtrate (virus) Rouse (1911) - Rous sarcoma in chicken (RSV) Work in chicken and mice 1930s - 1970s: RNA tumor viruses Temin (1962) - Hypothesized Provirus state Huebner and Todaro (1969) - the Viral Oncogene Hypothesis Temin and Baltimore (1970) - discovery of Reverse
Transcriptase Bishop and Varmus ( 1978) - oncogenes are derived from
cellular genes involved in growth ( src product is cellularphosphokinase)
Gallo (1981) - human retrovirus (HTLV-Human T Cell LeukemiaVirus)
Barre-Sinoussi (1983) - HIV isolated / shown to be the cause ofAIDS
-
7/29/2019 Retroviruses 105
4/41
Retroviridae
Retroviruses (family Retroviridae ) areenveloped, single stranded (+) RNA virusesthat replicate through a DNA intermediateusing reverse transcriptase.
This large and diverse family includesmembers that are oncogenic, are associatedwith a variety of immune system disorders,and cause degenerative and neurologicalsyndromes.
-
7/29/2019 Retroviruses 105
5/41
Retrovirus Classification
Derivation of Names Retro (Latin) - backwards
Onco (Greek, oncos) - tumor Spuma (Latin)-foam Lenti (Latin, lentus) - slow
-
7/29/2019 Retroviruses 105
6/41
Retrovirus Classification Family : Retroviridae
Genus Features Examples1. Alpharetrovirus Simple,
OncoAvian leucosis virus, RSV
2. Betaretrovirus Simple,
Onco
Mouse Mammary Tumor
Virus3. Gammaretrovirus Simple,
OncoMurine leukemia virus(Moloney, Harvey)
4. Deltaretrovirus Complex,Onco
Bovine Leukemia, HumanT Cell Leukemia (HTLV)
5. Epsilonretrovirus Complex,Onco
Walleye Dermal Sarcoma
6. Lentivirus Complex HIV, Visna, EIAV
7. Spumavirus Complex Simian Foamy Virus
-
7/29/2019 Retroviruses 105
7/41
Retrovirus VirionsThin Section EM of Some Retroviruses
Type A (donut)
Type B ( eccentric )MMTV
Type C (central
)ALV,RSV
Type D (bar)
Lentivirus ( cone )HIV
-
7/29/2019 Retroviruses 105
8/41
Retrovirus structure
Retrovirus virions are 80-120 nm in diameter, havespherical morphology, a phospholipid envelope with knobs
Contain around 2000 molecules of nucleocapsid (NC)protein that bind to the two copies of (+) strand RNA genomeRetroviral ribonucleoproteins are encased within a proteinshell built from the capsid protein to form an internal core,which can have different shapes and has a conical shape inHIV
-
7/29/2019 Retroviruses 105
9/41
Rous sarcoma virus Avian leukosis virus
Retroviruses are enveloped, and contain: two copies of RNA;internal structural proteins (MA,CA,NC); enzymes (PR,RT,IN);and exterior proteins (SU,TM). There are several differentcomplex morphological types.
-
7/29/2019 Retroviruses 105
10/41
surface
transmembrane
matrix protein
capsid proteinnucleocapsid protei
RTIntegraseprotease
HIV Structure
-
7/29/2019 Retroviruses 105
11/41
Cryoelectron micrographof mature humanimmunodeficiency virustype 1 (HIV-1) showingthe elongated internalcores
HIV Virions
-
7/29/2019 Retroviruses 105
12/41
Single standed (+) sense RNA genome of about 10 kb
5 cap, 3 poly(A) tail
Stop codon between gag and pol, suppressed byreadthrough or frameshifting
Looks like mRNA, but does not serve as mRNA immediatelyafter infection
Has a direct repeat (R) and unique (U) regions at both ends
All retroviruses encode Gag, Pol and Env
Lentiviral genomes encode a number of additional auxiliaryproteins, Tat, Rev, Nef, Vif, Vpr and Vpu
HIV genome organization
-
7/29/2019 Retroviruses 105
13/41
HIV integration:
-
7/29/2019 Retroviruses 105
14/41
Genome of Simple vs. Complex Retroviruses
ALV and MLV areSimple retroviruses
HTLV, HIV, HFV, andWDSV are Complex
retroviruses thatcontain accessorygenes
Notice gag-pol-env inboth simple andcomplex
-
7/29/2019 Retroviruses 105
15/41
Genome of Simple vs. Complex Retroviruses
-
7/29/2019 Retroviruses 105
16/41
Retroviral Proteins gag , pol , and env
Gag protein proteolytically processed into MA (matrix) CA (capsid) NC (nucleocapsid)
Pol protein encodes enzymes PR (protease) RT (Reverse Transcriptase which has both DNA polymerase and
RNase H activities) IN (Integrase)
Env protein encodes SU surface glycoprotein
TM transmembrane protein Accessory genes (in Complex Retroviruses) - regulateand coordinate virus expression; function in immuneescape
Oncogene products (v-Onc, in Acutely TransformingRetroviruses) - produce transformed phenotype
-
7/29/2019 Retroviruses 105
17/41
Gag Proteins MA, Matrix, p17
CA, Capsid, p24
NC, Nucleocapsid, p7
Binds to RNA Zinc fingers Note: retroviral proteins are
sometimes designated by their apparent molecular weight.This varies from virus to virus
-
7/29/2019 Retroviruses 105
18/41
Enzymatic Proteins: Protease
10 kd, dimer Cuts Gagpolyprotein toMA,CA,NC
Aspartyl protease Exquisite cleavage
specificity Major class of anti-
HIV drugs areProtease Inhibitors
-
7/29/2019 Retroviruses 105
19/41
Enzymatic Proteins: ReverseTranscriptase
DNA Polymerase Activity Requires primer with 3 OH termination Template either RNA or DNA
Requires Mg++
(or Mn++
) Lacks proof-reading function; high error rate(10 -4 errors per base)
RNase H Activity (Nuclease specific for RNAin RNA:DNA hybrids) Activity encoded in different domain than
polymerase
-
7/29/2019 Retroviruses 105
20/41
Enzymatic Proteins: Integrase
Integrates retroviralDNA into hostgenome
Endonucleaseactivity
Drugs beingdeveloped
-
7/29/2019 Retroviruses 105
21/41
Env Proteins: Transmembrane (TM)
Holds SU toretroviral envelope
Involved inmembranefusion/penetration
of virus into cell
f ( )
-
7/29/2019 Retroviruses 105
22/41
Env Proteins: Surface (SU) Glycoprotein (gp, followed by apparent molecular
weight)
Attaches to a specific receptor on cell surface Major neutralizing antigen on retrovirus, also
often highly variable (EIAV, HIV). Hard to makevaccines.
SU (gp120)
TM (gp 41)
Lipid Bilayer(derived from cell)
-
7/29/2019 Retroviruses 105
23/41
HIV LTR functions as a promoter in a variety of celltypes in vitro
It includes an enhancer sequence that binds a number of cell type specific transcription activators
Regulatory proteins: Tat
-
7/29/2019 Retroviruses 105
24/41
Downstream of the site of initiation of transcription is theTAR RNA sequence which forms a stem loop that bindsthe 14 kd viral regulatory protein Tat
In the absence of Tat, viral transcription terminatesprematurely
Tat facilitates efficient elongation
Tat protein is cytotoxic to cells in culture
Causes depolarization and degeneration of cellmembranes
Transgenic expression in mice causes a disease thatresembles Kaposis sarcoma
Mechanism of Tat activation
-
7/29/2019 Retroviruses 105
25/41
Stimulation of transcription by HIV-1 Tat protein:
Before Tat is made proviral
transcripts are terminated within60 bp of the initiation site
Production of the Tat proteinallows transcription complexes tosynthesize full length RNA
Binding of Tat to TAR together with the cyclin T subunit of Takleads to stimulation ofphosphorylation of the largestsubunit of RNA polymerase II
As a result, the transcriptionalcomplexes become competent tocarry out transcription
R g l t t i R
-
7/29/2019 Retroviruses 105
26/41
Rev Protein is an RNA binding protein that recognizes aspecific sequence within the structural element in envcalled the Rev-responsive element (RRE)
Regulatory proteins: Rev
-
7/29/2019 Retroviruses 105
27/41
Rev activates the nuclear export of any RREcontaining RNA
As the Rev concentration increases, unspliced or singly spliced transcripts containing the RRE areexported from the nucleus
Rev facilitates synthesis of the viral structuralproteins and enzymes and ensures availability of fulllength genomic RNA to be incorporated into new virusparticles
The accessory proteins, Vif, Vpr and Vpu are alsoexpressed later in infection from singly splicedmRNAs and their export to the cytoplasm is Revdependent
Rev protein:
HIV i
-
7/29/2019 Retroviruses 105
28/41
Nef protein:
Translated from multiply
spliced early transcripts myristylated at its N -terminusand anchored to the innersurface of the plasma membrane
Nef deleted HIV and SIV aremuch less pathogenic in vivo
Nef downregulates expression
of CD4 by enhancingendocytosis
Can activate CD4+ Tlymphocytes by modulating
signaling pathways
HIV accessory proteins
HIV i
-
7/29/2019 Retroviruses 105
29/41
Vif Protein:
Viral infectivity factor
Accumulates in the cytoplasm and at the plasmamembrane of infected cells
Mutant viruses lacking the vif gene were lessinfectious and defective in some way
vif -defective virions enter cells, initiate reversetranscription, but do not produce full-length doublestranded DNA
vif inhibits antiviral action of a cytidine deaminase,which is synthesized in nonpermissive cells
This enzyme deaminates deoxycytidine todeoxyuridine and leads to endonucleolytic digestion
or G to A transitions
HIV accessory proteins
Retrovirus replication cycle
-
7/29/2019 Retroviruses 105
30/41
1. Attachment of the virion to a specific cell surface receptor
2. Penetration of the virion core into the cell
3. Reverse transcription within the core structure to copy thegenome RNA into DNA
4. Transit of the DNA to the nucleus
5. Integration of the viral DNA into random sites in cellularDNA to form the provirus
6. Synthesis of viral RNA by cellular RNA polymerase IIusing the integrated provirus as a template
7. Processing of the transcripts to genome and mRNAs
8. Synthesis of virion proteins
9. Assembly and budding of virions
10. Proteolytic processing of capsid proteins
Retrovirus replication cycle
R i
-
7/29/2019 Retroviruses 105
31/41
Retroviruslife cycle:
HIV bi di d
-
7/29/2019 Retroviruses 105
32/41
The gp120 has a specific domain that binds to the
CD4 molecule present on susceptible cellsUpon binding to CD4, the gp120 undergoes aconformational change that allows binding tospecific subset of chemokine receptors on the cellsurface, the CCr5 receptor and the CXCr4 receptor
HIV binding and entry
C t f h d T ll
-
7/29/2019 Retroviruses 105
33/41
Coreceptors for macrophage and T-cell- tropic Strains of HIV
CXCr4 is the major coreceptor for T-cell-tropic strains
CCr5 is the major coreceptor for macrophage-tropic strains
HIV bi di d
-
7/29/2019 Retroviruses 105
34/41
The use of each coreceptor corresponds to viruses withdifferent biological properties and pathogenicity.
Viruses isolated at the beginning of infection use the CCr5co-receptor, which is the major coreceptor for macrophage-tropic strains, these viruses are not cytophatic
In full -blown AIDS cases, new viral species appear withhigh level of replication, cytophatic effects and they use thecoreceptor CXCr4, which is the major receptor for T-celltropic strains
There are also dual tropic viruses that can use both CXCr4and CCr5 coreceptors or alternative chemokine coreceptors
The fusion between the viral membrane and the cellular membrane involves a change in conformation of gp41, which
enables it to insert into the cellular phospholipid bilayer
HIV binding and entry
R i l
-
7/29/2019 Retroviruses 105
35/41
Retroviruses contain twocopies of the RNA genome held
together by multiple regions ofbase pairing
This RNA complex alsoincludes two molecules of aspecific cellular RNA (tRNA lys )that serves as a primer for theinitiation of reverse transcription
The primer tRNA is partiallyunwound and hydrogen bondednear the 5 end of each RNAgenome in a region called theprimer binding site
Retroviral genome
R t i ti
-
7/29/2019 Retroviruses 105
36/41
1) (- ) strand synthesis starts near the 5 end of the (+)strand RNA genome with a specific host tRNA as a primerand runs out of template after ~100 nt
2) Synthesis proceeds to the 5 end of the RNA genomethrough the u5 region ending after the r region, forming the(-) strand strong stop DNA (-ssDNA)
Reverse transcription
R t i ti t
-
7/29/2019 Retroviruses 105
37/41
2) RNA portion of the RNA-DNA hybrid is digested bythe RNase H activity of RT, resulting in a single-stranded DNA product
3) This facilitates hybridization with the r region at the3 end of the same or second RNA genome, resultingin the first template exchange for RT
Reverse transcription cont.
R t i ti t
-
7/29/2019 Retroviruses 105
38/41
4) (-) strand DNA terminates at the primer binding site
5) When (-) strand elongation passes the polypurinetract (ppt) region, the RNA template escapes digestionby RNase H and serves as a primer for (+) strandsynthesis by DNA dependent DNA polymerization(DDDP)
Reverse transcription cont.
R t i ti t
-
7/29/2019 Retroviruses 105
39/41
6) (+) strand synthesis then continues back to the U5region with the (-) strand DNA as the template andterminates after copying the first 18 nt of the primer tRNA
and stops, forming the (+) strand strong stop product(+ssDNA)
Reverse transcription cont.
Re erse transcription cont
-
7/29/2019 Retroviruses 105
40/41
7) The tRNA is then removed by RNase H activity of RT
8) The exposed PBS anneals to the PBS sequence at the 3end of the (-) strand DNA, allowing the second templateexchange.
Product of the second template exchange is a circular DNA
molecule with overlapping 5 ends.
Reverse transcription cont.
Reverse transcription cont
-
7/29/2019 Retroviruses 105
41/41
9) DNA synthesis is terminated at the breaks in thetemplate strands at the PBS and PPT ends,producing a linear molecule with long terminalrepeats (LTRs).
Reverse transcription cont.