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RETROVIRUSES

RETROVIRUSES. Characteristics. Name originates from the fact that they use reverse transcriptase (retroviruses) Enveloped virion, 100 nm diameter Linear +ssRNA genome 2 identical genomes are packaged in each virion 7-10 Kb 7 genera are part of this family including HIV

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RETROVIRUSES

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  1. RETROVIRUSES

  2. Characteristics • Name originates from the fact that they use reverse transcriptase (retroviruses) • Enveloped virion, 100 nm diameter • Linear +ssRNA genome • 2 identical genomes are packaged in each virion • 7-10 Kb • 7 genera are part of this family including HIV • Diseases they cause: AIDS, leukemia, cancers • A cellular tRNA behaves as primer fro viral genome replication

  3. Kaposi’s Sarcoma • Kaposis Sarcoma is cancer • It is rare • More common in AIDS patients

  4. Structure

  5. Viral Genome • Viral genome exhibits characteristics of cellular mRNA • R sequences-repeated sequences found both at 5’ and 3’ end (~150-200 nt) • U5 region is what keeps the 2 ssRNAs together • PBS-primer binding sequence • In reality it is cellular tRNA that binds this sequence • Downstream the PBS • 3 genes encoding 3 types of proteins • Gag (group specific antigen), pol (polymerase), env (envelope)

  6. Viral Entry • Mediated by SU protein • SU interacts with cell surface proteins • In HIV case the CD4 and CCR5 or CXCR4 • Receptor interaction allows for viral entry into cell in 2 ways • Receptor mediated endocytosis followed by virion release via a pH decrease release mechanism • Fusion at plasma membrane, capsid is released into cytosol

  7. Viral Entry

  8. Conversion of ssRNA Viral Genome Into dsDNA • Reverse transcriptase has 2 distinct activities • 1st is to synthesize DNA • 2nd is to degrade RNA from DNA/RNA molecule • This activity is referred to as ribonuclease H activity • Does not degrade ssRNA

  9. Conversion of ssRNA Viral Genome Into dsDNA

  10. Conversion of ssRNA Viral Genome Into dsDNA

  11. DNA Genome Is Integrated Into Cellular Genome • Insertion sites are random • Enzyme responsible for insertion is Integrase • Integrase is found in the core of the virion • Integrase binds the 2 ends of the viral dsDNA genome and brings them together

  12. DNA Genome Is Integrated Into Cellular Genome • Enzyme targets phosphodiester bonds for cleavage/insertion • 2 hanging nucleotides are removed • 4-6 nt of host ssDNA is matched and ligation site is fixed entirely • Loss of 2 nt from viral DNA is insignificant

  13. Proviral DNA Will Be Expressed At Any Time In The Future • The appropriate transcription factors are needed for expression of inserted genome to begin • U3 region is the binding site for a number of cellular transcription factors • A TATA box is present upstream (U3/R segments) allowing transcription initiation to begin by RNA Pol II • Transcription begins at the junction of U3/R and proceeds through the whole genome • A Poly(A) signal directs cleavage of transcript at R/U5 junction • RNA is polyadenylated by cellular enzymes • RNA transcript generated is identical to initial infecting RNA genome • Despite the fact that 2 LTR exist at the ends of proviral DNA, transcription begins only at left side • It is thought to be due to Promoter occlusion • RNA Pol II displaces transcription factors on the right • In similar way polyadenylation only occurs to the right • AAUAAA (poly A signal sequence) is also present on the left

  14. Differential Splicing Generates Multiple mRNAs • Transcription produces genome length mRNA • The different viral proteins are produced from this mRNA after it is spliced • At least 2 types of mRNAs are produced in retroviruses • 1 unspliced (whole genome) • Used for gag and gag/pol proteins • 1 spliced (gag and gag/pol is removed) • Only env proteins are produced from this mRNA • Some retroviruses have more elaborate splicing schemes • Ex. Lentiviruses (HIV), Rous Sarcoma virus

  15. Differential Splicing Generates Multiple mRNAs • HIV overcomes stop codon resulting in translation of gag/pol protein • It achieves that by shifting ribosome at a precise position prior to termination codon • This way it avoids stop codon and addresses the fact that pol protein has a different reading frame • HIV and some other retroviruses achieve this SHIFTING by making use of heptamers such as UUUUUUA (HIV-1) • Why such a scheme? • To ensure right ratio of gag to gag/pol proteins for generating virions

  16. Retrovirus Based Gene Therapy • The ability of retroviruses to permanently introduce genes into host genome make them good candidates for gene therapy to fix mutated genes or introduce new genes • One major issue is to ensure that no tumors are created • Engineered retroviruses are missing gag/pol/env genes • This ensures no viral replication • Provides space for new gene • Engineered virus is prepared by packaging recombinant RNA into packaging cell lines (they are missing RNA packaging signals) • Vector is expressed in packaging cell line and gets packaged into virions • Target cells are mixed with virus • A selection gene (neomycin resistant gene) is used to eliminate cells that are not infected • In the past the limitation was that they could only infect dividing cells • Now lentiviruses (HIV) are used which can infect non-dividing differentiated cells • One limitation is the size of the gene • Up to 10 Kb gene size can be inserted in the genome

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