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

• 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

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

Conversion of ssRNA Viral Genome Into dsDNA

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