retroviruses 105

7/29/2019 Retroviruses 105

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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


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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


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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


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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.


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Retrovirus Classification

Derivation of Names Retro (Latin) - backwards

Onco (Greek, oncos) - tumor Spuma (Latin)-foam Lenti (Latin, lentus) - slow


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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


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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


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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


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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.


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surface

transmembrane

matrix protein

capsid proteinnucleocapsid protei

RTIntegraseprotease

HIV Structure


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Cryoelectron micrographof mature humanimmunodeficiency virustype 1 (HIV-1) showingthe elongated internalcores

HIV Virions


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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


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HIV integration:


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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


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Genome of Simple vs. Complex Retroviruses


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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


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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


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Enzymatic Proteins: Protease

10 kd, dimer Cuts Gagpolyprotein toMA,CA,NC

Aspartyl protease Exquisite cleavage

specificity Major class of anti-

HIV drugs areProtease Inhibitors


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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


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Enzymatic Proteins: Integrase

Integrates retroviralDNA into hostgenome

Endonucleaseactivity

Drugs beingdeveloped


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Env Proteins: Transmembrane (TM)

Holds SU toretroviral envelope

Involved inmembranefusion/penetration

of virus into cell

f ( )


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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)


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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


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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


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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


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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


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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


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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


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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


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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


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Retroviruslife cycle:

HIV bi di d


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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


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Coreceptors for macrophage and T-celltropic 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


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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


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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


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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


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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


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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)


R t i ti t


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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)


Re erse transcription cont


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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


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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).


retroviruses 105

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