Retroviruses and Human T Cell Lymphotropic Virus Flashcards
What was the first retrovirus discovered?
Rous Sarcoma virus
What enzyme is required reverse transcription?
RNA-dependent DNA polymerase (Reverse transcriptase)
What was the first human retrovirus discovered?
Human T cell leukaemia
How were retroviruses first classified?
- Morphology/assembly
- Biological properties
What are the basic properties and morphology of retroviruses?
- Lentivirus is slow growing
- Spherical enveloped viruses
- Approximately 100 nm diameter
- Prominent peplomers - spikes
- Contain dense core 70-80 nm - shape variable
- Positive stranded, diploid RNA genome - 2 identical copies
How are the RNA genome copies held together?
By non-covalent interaction base-pairing at the 5’ end
What does the surface glycoprotein, SU, aid?
Helps recognition of target cells
What does transmembrane protein, TM, aid?
Helps fusion with target cells
What are the 3 common retrovirus genes?
- GAG
- POL
- ENV
What does GAG encode?
- Group-specific antigens
- Internal proteins - includes matrix, nucleocapsid and in some viruses the protease
What does POL encode?
- Reverse transcriptase
- Integrase
- For specific viruses it encodes the protease
What does ENV encode?
Glycoprotein spikes
What ends of the genome have regulatory sequences?
Both 5’ and 3’
What is found in the regulatory sequences?
- Primer binding site
- Untranslated region in 5’ and 3’
- Polypurine tract in the 3’
- Repeat region found on both ends
What is the function of the tRNA bound to the PBS?
Acts as a primer for reverse transcription
Describe the retrovirus life cycle
- First have to have recognition between surface envelope proteins and specific receptors on the host cell
- Following this entry proceeds via fusion - fusion-mediated entry
- Fusion between the viral envelope and the cellular envelope
- Once in the cytoplasm the capsid disassociates and releases the RNA genome
- Reverse transcription produces a DNA copy of the genome
- Proviral DNA is transported into the nucleus where it’s integrated into the host chromosome by integrase
- Viral DNA can be transcribed and used to make viral proteins and multiple copies of viral RNA
- New infectious particles are assembled and bud out of the infected cell
- Particle that buds out isn’t a mature virion - processed by retroviral protease to adopt the final structure
What are the properties of reverse transcriptase?
- RNA-dependent DNA polymerase
- Able to synthesise DNA remnant that is complementary to the RNA
- Has RNase H activity - able to degrade RNA molecule hybridised to the DNA fragment
Describe the reverse transcription process
- Once the viral capsid it releases the single stranded RNA genome
- The negative strand is primed by tRNA bound at tb
- Reverse transcriptase produces a DNA copy of the 5’ sequence
- RNase H exposes the DNA copy of R and U5
- As the RNA genome has identical sequences at the 5’ and 3’ ends, the newly extended DNA fragment is able to jump from the 5’ end to the 3’ end and anneal onto the repeat segment
- This promotes elongation of the full stretch of the DNA strand
- Template RNA degradation by RNase H
- Polypurine oligo (polyP) segment on the RNA is specifically spared degradation to prime positive strand synthesis - acts as a primer
- Positive strand uses the new DNA as a template and continues until a modified base in tRNA blocks extension
- tRNA is degraded by RNase H
- tb and tb’ at 3’ ends of the positive and negative strands base pair - second template jump
- Extension of both strands produces a complete double stranded DNA copy of the RNA genome
Describe the integration of viral DNA into the host’s genome
- Integrase recognises the 5’ and 3’ end of the proviral double stranded DNA
- Integrase creates random nicks in the host chromosome
- Integrase repairs these nicks with the viral DNA - this is reversible
- Repair occurs by the host cell machinery - this is irreversible
- End result is the viral genome integrated in random places within the host chromosome
What levels are the retrovirus genes regulated at?
- Transcriptional level
- Translational level
Which retrovirus genes are needed to be expressed in a large quantity and what genes are needed in a smaller quantity?
- Gag-pro and Env are structural genes needed in large amount
- Pol is needed in a smaller amount
How are retrovirus genes regulated at the transcriptional level?
ENV genes are made from a separate spliced mRNA in large amount to generate ENV proteins separately from the others
How are retrovirus genes regulated at the translational level?
- Full-length mRNA has to be used as mRNA for Gag (large quantity) and Pol (smaller quantity)
- Retroviruses have 2 methods of translational control depending on the reading frame
- Gag and Pol in the same reading frame or in different reading frames
Describe translational control when Gag and Pol are in the same reading frame
- The coding regions are separated by a stop codon
- Readthrough is by suppression of termination
- 90% of ribosomes translate Gag-pro and terminate at the stop - don’t translate Pol
- 10% insert GIn residue (CAG codon) and continue translating - called a readthrough event
- Therefore, Pol is translated less than Gag-pro
Give an example of a virus where Gag and Pol are in the same reading frame
Murine Leukaemia virus
Describe translational control when Gag and Pol are in different reading frames
- Gag and Pol are in different reading frames so ribosomes must change reading frame
- Regulated by frameshifting
- Ribosomes are stalled on heptamer sequence by RNA structure and can slide from 0 to +1 reading frame
- Depends on structural end sequence elements of the viral genome
- Around the stop codon that the ribosome normally recognises, there are strong secondary structural elements that are formed slow down the progress of the ribosome - ribosome will slow down onto a slippery surface where the ribosome can slide back and forth one nucleotide in order to change its reading frame
- Makes the stop codon out of frame and leads to Pol being produced
How do retroviruses assemble and mature?
- Full-length mRNA transcripts act as genomes to associate with virus proteins, forming cores
- These cores acquire spikes and envelope by budding
- Core formation occurs in 2 ways - preformed structures in the cytoplasm or simultaneous budding and core formation
Describe the incidence of ATL and HTLV-1 in Japan
- ATL is clustered in South and West Japan - relatively rare in the North
- HTLV is clustered in South and West Japan - relatively rare in the North
- Found that where the virus was prevalent there was increased incidence of ATL
- Still not causal link but has good correlation
- Then investigated the population that each disease affects
- ATL occurs in adults and is rare in children
- HTLV seropositivity increases in adults and is low in children
- Virus distribution matches geographical prevalence of disease
- Virus infections show similar familial clustering
- All ATL patients are seropositive for HTLV-1
`What are the clinical features of adult T cell leukaemia (ATL)?
- Lymphadenopathy - results in reduced T cell numbers
- Enlargement of the liver and spleen
- Calcium lost from bone, elevated in blood, and deposited in skin
- T cell leukaemia of CD4+ cells
- Deformed T cells with convoluted nuclei
What is the lifetime risk of ATL for HTLV positive persons?
2-4%
Describe the epidemiology of ATL
- All cancerous cells have integrated HTLV DNA - non-cancerous cells in the patient don’t
- DNA integrated at the same site in any one patient, but different sites between patients even if from the same family
- Most ATL patients produce no active virus
What are the additional regulatory genes expressed by HTLV?
- Tax
- Rex
What does Tax do?
Activates and increases RNA synthesis
What does Rex do?
Increases RNA export from nucleus to cytoplasm
Describe how Rex functions
- Poly(A) signal and poly(A) site are usually 15 nucleotides apart in HTLV-1 mRNA so physical interaction needed between polyadenylation site and 3’ end of the genome doesn’t usually happen
- Rex-responsive element folding brings the Poly(A) signal close to the cleavage/polyadenylation site
- Rex protein binding causes 3’ UTR to structure itself into a specific scaffold that brings the polyadenylation signal and 3’ end close together
- Rex binds the Rex-responsive element through the N-terminal domain and mediates mRNA export by binding Exportin 1 via the C-terminal domain - scaffold formed by Rex is recognised by Exportin 1
- In the early phase, there is no Rex protein so only doubly spliced RNA is synthesised - leads to Rex translation
- In the late phase Rex binds to unspliced or singly spliced RNA to trigger their export
What is Tax activity mediated by?
cAMP-responsive sequence elements (Tax responsive element)
How long is the TRE?
21 basepair repeats
How does TRE activate transcription?
- TRE shares homology with cAMP response element (CRE) and recruits CRE binding protein that also binds Tax
- CREB-binding protein (CBP) is subsequently recruited
- CBP is part of the RNA polymerase II complex - remodels chromatin allowing RNA polymerase to bind the DNA
- CBP acetylates histone and the chromatin remodelling allows TFIID and RNA polymerase II to bind DNA and activate transcription
How does Tax activate transcription factors?
- ATFs target control elements for different genes allowing gene expression in a flei le coordinated response to stimuli
- Tax binds cellular ATFs and promotes their activation - indirect activation of factors that promote cellular transcription
- Activated ATFs bind to virus DNA at TRE in a sequence specific manner
- Complex then activates transcription of genes around the ATF binding site
How does Tax activate cellular RNA transcription?
- Tax binds IKK
- Activated IKK phosphorylates IkappaBalpha/NFkappaB - normally NFKappaB is sequestered by IkappaBalpha
- Phosphorylated IkappaBalpha is targeted for ubiquitination
- Proteasome degradation releases NFkappaB
- NFkappaB binds to target sequence to activate transcription of cell growth and apoptosis genes
- Tax removes regulation of NFkappaB activity
How does HTLV cause leukaemia?
- HTLV infection expresses Tax
- Tax increases frequency of T cell division by activating cell cycle and growth control genes
- Over a lifetime excess divisions increases chance of somatic mutation in T cells
- Somatic mutation when it occurs permits continuous cell division, uncontrolled growth and tumour development
How is HTLV transmitted?
- Sex - virus in semen and vaginal secretions
- Drug use/needle sharing
- Vertical transmission
- Neonatal transmission via milk
- Blood transfusion
What CNS diseases does HTLV cause?
- Tropical spastic paraparesis
- HTLV-associated myelopathy
