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 (~ 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