TRIM28 Represses Transcription of Endogenous Retroviruses ...
RETROVIRUSES. Characteristics Name originates from the fact that they use reverse transcriptase...
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Transcript of RETROVIRUSES. Characteristics Name originates from the fact that they use reverse transcriptase...
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
Viral Genome
• 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 genome exhibits characteristics of cellular mRNA
• 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
• 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
• 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
• 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
DNA Genome Is Integrated Into Cellular Genome
• 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
Proviral DNA Will Be Expressed At Any Time In The Future
• 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
Differential Splicing Generates Multiple mRNAs
• 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
Retrovirus Based Gene Therapy