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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
• Diseases they cause: AIDS, leukemia, cancers
• A cellular tRNA behaves as primer fro viral
genome replication
Kaposi’s Sarcoma
Kaposis Sarcoma is cancer
It is rare
More common in AIDS
patients
Structure
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)
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
Viral Entry
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
Conversion of ssRNA Viral
Genome Into dsDNA
Conversion of ssRNA Viral
Genome Into dsDNA
DNA Genome Is Integrated Into
Cellular Genome
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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
DNA Genome Is Integrated Into
Cellular Genome
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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
Proviral DNA Will Be Expressed At
Any Time In The Future
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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
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In similar way polyadenylation only occurs to the right
– AAUAAA (poly A signal sequence) is also present on the left
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
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
Retrovirus Based Gene Therapy
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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
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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
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Target cells are mixed with virus
– A selection gene (neomycin resistant gene) is used to eliminate cells that are not
infected
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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