Retroviridae • 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

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

Retrovirus Classification Family: Retroviridae Genus Features Examples 1. Alpharetrovirus Simple, Onco Avian leucosis virus, RSV 2. Betaretrovirus Mouse Mammary Tumor Virus 3. Gammaretrovirus Murine leukemia virus (Moloney, Harvey) 4. Deltaretrovirus Complex, Onco Bovine Leukemia, Human T Cell Leukemia (HTLV) 5. Epsilonretrovirus Walleye Dermal Sarcoma 6. Lentivirus Complex HIV, Visna, EIAV 7. Spumavirus Simian Foamy Virus

Retrovirus Virions Thin Section EM of Some Retroviruses Type A (donut) Type B (eccentric) MMTV Type C (central) ALV,RSV Type D (bar) Lentivirus (cone) HIV

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

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 different complex morphological types. ”

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

HIV genome organization • Single standed (+) sense RNA genome of about 10 kb 5’ cap, 3’ poly(A) tail Stop codon between gag and pol, suppressed by readthrough or frameshifting Looks like mRNA, 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, Pol and Env • Lentiviral genomes encode a number of additional auxiliary proteins, Tat, Rev, Nef, Vif, Vpr and Vpu

HIV integration:

Genome of Simple vs. Complex Retroviruses 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

Genome of Simple vs. Complex Retroviruses

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) - 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

Enzymatic Proteins: 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

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

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 make vaccines. SU (gp120) Lipid Bilayer (derived from cell) TM (gp 41)

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

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, the transcriptional complexes become competent to carry out transcription

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)

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

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

HIV accessory proteins Vif Protein: • 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, 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

Retrovirus replication cycle Attachment of the virion to a specific cell surface receptor Penetration of the virion core into the cell Reverse transcription within the core structure to copy the genome RNA into DNA Transit of the DNA to the nucleus Integration of the viral DNA into random sites in cellular DNA to form the provirus Synthesis of viral RNA by cellular RNA polymerase II using the integrated provirus as a template Processing of the transcripts to genome and mRNAs Synthesis of virion proteins Assembly and budding of virions Proteolytic processing of capsid proteins

Retrovirus life cycle:

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 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

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 binding and entry • The use of each coreceptor corresponds to viruses with different biological properties and pathogenicity. • Viruses isolated at the beginning of infection use the CCr5 co-receptor, which is the major coreceptor for macrophage-tropic strains, these viruses are not cytophatic • In full-blown AIDS cases, new viral species appear with high level of replication, cytophatic effects and they use the coreceptor CXCr4, 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, which enables it to insert into the cellular phospholipid bilayer

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)

Reverse transcription cont. 2) RNA portion of the RNA-DNA hybrid is digested by the RNase H activity of RT, resulting in a single-stranded 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

Reverse transcription cont. 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)

Reverse transcription cont. 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)

Reverse transcription cont. 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.

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).