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• The Retroviridae are a family of enveloped (+) sense ssRNA viruses that have been intensely studied because of their association with cancers, leukemias and the AIDS syndrome • The first association of viruses with cancer was in early 1900’s with the discovery by Ellerman and Bang that leukemia could be transmitted from one chicken to another by injecting leukemia cell extracts • In 1911 Peyton Rous showed that a bacterial free filtrate from solid tumors of chickens could cause an identical cancer in chickens inoculated with the filtrate • The virus causing the leukemia was subsequently shown to be avian leukosis virus and the virus causing tumors was designated Rous sarcoma virus
• Although the discoveries by Ellerman, Bang and Rous were not well accepted at the time, 60 years later these viruses were designated retroviruses and Rous won the Nobel Prize for his work in 1963 at the age of 83 • In early 1970, Baltimore and Temin independently identified the unusual enzyme, reverse transcriptase and won 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 from cellular genes and brought us closer to understanding cancer
• Vallee and Carre (1904) - Filterable virus is cause of “Swamp Fever” of horses (EIAV) • Ellerman and Bang (1908) - chicken leukemia transferred by bacteria-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 cellular phosphokinase) • Gallo (1981) - human retrovirus (HTLV-Human T Cell Leukemia Virus) • Barre-Sinoussi (1983) - HIV isolated / shown to be the cause of AIDS
• Retroviruses (family Retroviridae) are enveloped, single stranded (+) RNA viruses that replicate through a DNA intermediate using reverse transcriptase. • This large and diverse family includes members that are oncogenic, are associated with a variety of immune system disorders, and cause degenerative and neurological syndromes.
oncos) .tumor Spuma (Latin)-foam Lenti (Latin.Retrovirus Classification Derivation of Names • • • • Retro (Latin) .slow .backwards Onco (Greek. lentus) .
Human T Cell Leukemia (HTLV) Walleye Dermal Sarcoma HIV. Harvey) Complex. Epsilonretrovirus 6. Lentivirus 7. Betaretrovirus 3.Retrovirus Classification Family: Retroviridae Genus 1. Gammaretrovirus 4. Onco Avian leucosis virus. Onco Complex Complex Bovine Leukemia. Deltaretrovirus 5. Alpharetrovirus 2. RSV Simple. Spumavirus Features Examples Simple. Visna. Onco Murine leukemia virus (Moloney. EIAV Simian Foamy Virus . Onco Mouse Mammary Tumor Virus Simple. Onco Complex.
RSV Type D (bar) Lentivirus (cone) HIV .Retrovirus Virions Thin Section EM of Some Retroviruses Type A (donut) Type B (eccentric) MMTV Type C (central) ALV.
Retrovirus structure • Retrovirus virions are 80-120 nm in diameter. have spherical morphology. which can have different shapes and has a conical shape in HIV . a phospholipid envelope with knobs • Contain around 2000 molecules of nucleocapsid (NC) protein that bind to the two copies of (+) strand RNA genome •Retroviral ribonucleoproteins are encased within a protein shell built from the capsid protein to form an internal core.
enzymes (PR.NC).CA. and exterior proteins (SU. ” .TM).Rous sarcoma virus Avian leukosis virus Retroviruses are enveloped.RT.IN). There are several different complex morphological types. and contain: two copies of RNA. internal structural proteins (MA.
HIV Structure surface transmembrane matrix protein capsid protein nucleocapsid protein RT Integrase protease .
HIV Virions Cryoelectron micrograph of mature human immunodeficiency virus type 1 (HIV-1) showing the elongated internal cores .
but does not serve as mRNA immediately after infection • Has a direct repeat (R) and unique (U) regions at both ends • All retroviruses encode Gag. Vif. Tat. Pol and Env • Lentiviral genomes encode a number of additional auxiliary proteins. Rev. suppressed by readthrough or frameshifting • Looks like mRNA. 3’ poly(A) tail • Stop codon between gag and pol. Nef. Vpr and Vpu .HIV genome organization • Single standed (+) sense RNA genome of about 10 kb • 5’ cap.
HIV integration: .
Genome of Simple vs. HIV. and WDSV are “Complex” retroviruses that contain accessory genes • Notice gag-pol-env in both simple and complex . Complex Retroviruses • ALV and MLV are “Simple” retroviruses • HTLV. HFV.
Genome of Simple vs. Complex Retroviruses .
• gag.produce transformed phenotype . pol.regulate and coordinate virus expression. and env Retroviral Proteins – 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) . function in immune escape • Oncogene products (v-Onc. in Acutely Transforming Retroviruses) .
Nucleocapsid. p24 NC. Capsid. p17 CA. Matrix.Gag Proteins MA. This varies from virus to virus . p7 – Binds to RNA – Zinc fingers • Note: retroviral proteins are sometimes designated by their apparent molecular weight.
Enzymatic Proteins: Protease • 10 kd. dimer • Cuts Gag polyprotein to MA.CA.NC • Aspartyl protease • Exquisite cleavage specificity • Major class of antiHIV drugs are Protease Inhibitors .
Enzymatic Proteins: Reverse Transcriptase • 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 RNA in RNA:DNA hybrids) – Activity encoded in different domain than polymerase .
Enzymatic Proteins: Integrase • Integrates retroviral DNA into host genome • Endonuclease activity • Drugs being developed .
Env Proteins: Transmembrane (TM) • Holds SU to retroviral envelope • Involved in membrane fusion/penetration of virus into cell .
HIV).Env Proteins: Surface (SU) • Glycoprotein (gp. also often highly variable (EIAV. followed by apparent molecular weight) • Attaches to a specific receptor on cell surface • Major neutralizing antigen on retrovirus. SU (gp120) Lipid Bilayer (derived from cell) TM (gp 41) . Hard to make vaccines.
Regulatory proteins: Tat • HIV LTR functions as a promoter in a variety of cell types in vitro • It includes an enhancer sequence that binds a number of cell type specific transcription activators .
Mechanism of Tat activation • Downstream of the site of initiation of transcription is the TAR RNA sequence which forms a stem loop that binds the 14 kd viral regulatory protein Tat • In the absence of Tat. viral transcription terminates prematurely • Tat facilitates efficient elongation • Tat protein is cytotoxic to cells in culture • Causes depolarization and degeneration of cell membranes • Transgenic expression in mice causes a disease that resembles Kaposi’s sarcoma .
the transcriptional complexes become competent to carry out transcription .Stimulation of transcription by HIV-1 Tat protein: • Before Tat is made proviral transcripts are terminated within 60 bp of the initiation site • Production of the Tat protein allows transcription complexes to synthesize full length RNA • Binding of Tat to TAR together with the cyclin T subunit of Tak leads to stimulation of phosphorylation of the largest subunit of RNA polymerase II • As a result.
Regulatory proteins: Rev • Rev Protein is an RNA binding protein that recognizes a specific sequence within the structural element in env called the Rev-responsive element (RRE) .
Vpr and Vpu are also expressed later in infection from singly spliced mRNAs and their export to the cytoplasm is Rev dependent . unspliced or singly spliced transcripts containing the RRE are exported from the nucleus • Rev facilitates synthesis of the viral structural proteins and enzymes and ensures availability of full length genomic RNA to be incorporated into new virus particles • The accessory proteins.Rev protein: • Rev activates the nuclear export of any RRE containing RNA • As the Rev concentration increases. Vif.
HIV accessory proteins Nef protein: • Translated from multiply spliced early transcripts • myristylated at its N-terminus and anchored to the inner surface of the plasma membrane • Nef deleted HIV and SIV are much less pathogenic in vivo • Nef downregulates expression of CD4 by enhancing endocytosis • Can activate CD4+ T lymphocytes by modulating signaling pathways .
initiate reverse transcription. but do not produce full-length double stranded DNA • vif inhibits antiviral action of a cytidine deaminase. which is synthesized in nonpermissive cells •This enzyme deaminates deoxycytidine to deoxyuridine and leads to endonucleolytic digestion or G to A transitions .Vif Protein: HIV accessory proteins • Viral infectivity factor • Accumulates in the cytoplasm and at the plasma membrane of infected cells • Mutant viruses lacking the vif gene were less infectious and defective in some way • vif-defective virions enter cells.
Processing of the transcripts to genome and mRNAs 8. Transit of the DNA to the nucleus 5. Synthesis of virion proteins 9. Assembly and budding of virions 10. Penetration of the virion core into the cell 3. Integration of the viral DNA into random sites in cellular DNA to form the provirus 6. Attachment of the virion to a specific cell surface receptor 2.1. Synthesis of viral RNA by cellular RNA polymerase II using the integrated provirus as a template 7. Reverse transcription within the core structure to copy the genome RNA into DNA 4. Proteolytic processing of capsid proteins Retrovirus replication cycle .
Retrovirus life cycle: .
the gp120 undergoes a conformational change that allows binding to specific subset of chemokine receptors on the cell surface.HIV binding and entry •The gp120 has a specific domain that binds to the CD4 molecule present on susceptible cells •Upon binding to CD4. the CCr5 receptor and the CXCr4 receptor .
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 .
these viruses are not cytophatic • In full-blown AIDS cases.HIV binding and entry • The use of each coreceptor corresponds to viruses with different biological properties and pathogenicity. cytophatic effects and they use the coreceptor CXCr4. • Viruses isolated at the beginning of infection use the CCr5 co-receptor. which enables it to insert into the cellular phospholipid bilayer . which is the major coreceptor for macrophagetropic strains. new viral species appear with high level of replication. which is the major receptor for T-cell tropic strains • There are also dual tropic viruses that can use both CXCr4 and CCr5 coreceptors or alternative chemokine coreceptors • The fusion between the viral membrane and the cellular membrane involves a change in conformation of gp41.
Retroviral genome •Retroviruses contain two copies of the RNA genome held together by multiple regions of base pairing • This RNA complex also includes two molecules of a specific cellular RNA (tRNAlys) that serves as a primer for the initiation of reverse transcription • The primer tRNA is partially unwound and hydrogen bonded near the 5’ end of each RNA genome in a region called the primer binding site .
Reverse transcription 1) (-) strand synthesis starts near the 5’ end of the (+) strand RNA genome with a specific host tRNA as a primer and runs out of template after ~100 nt 2) Synthesis proceeds to the 5’ end of the RNA genome through the u5 region ending after the r region. forming the (-) strand strong stop DNA (-ssDNA) .
resulting in a singlestranded DNA product 3) This facilitates hybridization with the r region at the 3’ end of the same or second RNA genome.Reverse transcription cont. resulting in the first template exchange for RT . 2) RNA portion of the RNA-DNA hybrid is digested by the RNase H activity of RT.
4) (-) strand DNA terminates at the primer binding site 5) When (-) strand elongation passes the polypurine tract (ppt) region.Reverse transcription cont. the RNA template escapes digestion by RNase H and serves as a primer for (+) strand synthesis by DNA dependent DNA polymerization (DDDP) .
Reverse transcription cont. forming the (+) strand strong stop product (+ssDNA) . 6) (+) strand synthesis then continues back to the U5 region with the (-) strand DNA as the template and terminates after copying the first 18 nt of the primer tRNA and stops.
allowing the second template exchange. 7) The tRNA is then removed by RNase H activity of RT 8) The exposed PBS anneals to the PBS sequence at the 3’ end of the (-) strand DNA. . Product of the second template exchange is a circular DNA molecule with overlapping 5’ ends.Reverse transcription cont.
9) DNA synthesis is terminated at the breaks in the template strands at the PBS and PPT ends. . producing a linear molecule with long terminal repeats (LTRs).Reverse transcription cont.
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