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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 1900s 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
Retroviridae
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
History
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
Retroviridae
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.
Retrovirus Classification
Derivation of Names
Retro (Latin) - backwards Onco (Greek, oncos) - tumor Spuma (Latin)-foam Lenti (Latin, lentus) - slow
Features
Simple, Onco Simple, Onco Simple, Onco Complex, Onco Complex, Onco Complex Complex
Examples
Avian leucosis virus, RSV Mouse Mammary Tumor Virus Murine leukemia virus (Moloney, Harvey) Bovine Leukemia, Human T Cell Leukemia (HTLV) Walleye Dermal Sarcoma HIV, Visna, EIAV Simian Foamy Virus
Type D (bar)
Retrovirus structure
Retrovirus virions are 80-120 nm in diameter, have spherical morphology, 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, which can have different shapes and has a conical shape in HIV
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 different complex morphological types.
HIV Structure
surface transmembrane
nucleocapsid protein
RT Integrase protease
HIV Virions
Cryoelectron micrograph of mature human immunodeficiency virus type 1 (HIV-1) showing the elongated internal cores
HIV integration:
ALV and MLV are Simple retroviruses HTLV, HIV, HFV, and WDSV are Complex retroviruses that contain accessory genes Notice gag-pol-env in both simple and complex
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) - regulate and coordinate virus expression; function in immune escape Oncogene products (v-Onc, in Acutely Transforming Retroviruses) - produce transformed phenotype
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
It includes an enhancer sequence that binds a number of cell type specific transcription activators
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, the transcriptional complexes become competent to carry out transcription
Rev Protein is an RNA binding protein that recognizes a specific sequence within the structural element in env called the Rev-responsive element (RRE)
Rev protein:
Rev activates the nuclear export of any RRE containing RNA
As the Rev concentration increases, 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, Vif, Vpr and Vpu are also expressed later in infection from singly spliced mRNAs and their export to the cytoplasm is Rev dependent
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
The gp120 has a specific domain that binds to the CD4 molecule present on susceptible cells Upon binding to CD4, the gp120 undergoes a conformational change that allows binding to specific subset of chemokine receptors on the cell surface, the CCr5 receptor and the CXCr4 receptor
CXCr4 is the major coreceptor for T-cell-tropic strains CCr5 is the major coreceptor for macrophage-tropic strains
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)
2) RNA portion of the RNA-DNA hybrid is digested by the RNase H activity of RT, 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, resulting in the first template exchange for RT
4) (-) strand DNA terminates at the primer binding site 5) When (-) strand elongation passes the polypurine tract (ppt) region, the RNA template escapes digestion by RNase H and serves as a primer for (+) strand synthesis by DNA dependent DNA polymerization (DDDP)
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, forming the (+) strand strong stop product (+ssDNA)
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, allowing the second template exchange. Product of the second template exchange is a circular DNA molecule with overlapping 5 ends.
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).