Lecture 13 Flashcards
(24 cards)
Importance of Parvovirus in virology
- Known for being most successful in gene therapy for humans
- Causes some human diseases
- Associated with new acute hepatitis pathology in children, although not known exactly if it causes it or not
- Smallest known virus to infect animals
Parvovirus morphology
Small naked icosahedral, 18-26 nm diameter
Parvovirus genome
ssDNA (odd to infect humans), 4-6kbp, linear, only two genes
Parvovirus name origin
Parvis => latin => small
Subfamilies of parvoviridae
Parvovirinae and Densovirinae (infect insects, only one important economically Bombyx More densovirus which infects silkworms)
What are the two types of parvoviruses?
- Autonomous: don’t need other viruses for successful infection and replication but do require dividing cells, so will often go for like erythroblasts (precursors of RBCs and such) or simply small creatures that have way more dividing cells than grown creatures
- Require helper virus (dependovirus): require other virus to already be infecting a target cell (often an adenovirus so named often adeno-associated virus) because they may stimulate the production of oncogenic proteins necessary for THEIR own replication (because cell division is stimulated)
How did parvoviruses and AAVs get discovered?
They were observed as a contaminant with cells infected by an adenovirus.
What are some parvovirus diseases in animals?
- Canine parvoviruses (CPV) infect puppies and can result in death
- Feline panleukopenia (FPV) virus affects blood formation in cats and can result in death
- Only treatment really is vaccination, if not death, symptoms include diarrhea, anemia and severe dehydration
Human B19 infection of erythroid progenitors? fetus? Children?
- Erythroid progenitors: result in anemia due to decreased red blood cells in circulation
- Fetus: Hydrops fetalis resulting in spontaneous abortion or birth defects (especially if pregnant person has not already been exposed to it, so no antibodies)
- Children: Erythema Infectosium (Fifth disease) (rashes)
Other four infections that cause rash, fever, cold like disease aka slapped cheeks disease, looks like it due to lysed RBCs are scarlet fever (streptococcus), measles (measles virus), German measles (rubella virus), pseudoscarlatina (similar to scarlet fever, streptococcus)
B19 discovery and histology?
- Discovered in the sera of a patient who was severely anemic
- B19 infected RBC precursor cells which still retain their nuclei and are rapidly proliferating. Infection induces the formation of vacuoles.
We see replication centres and inclusion bodies in the nucleus. Vascularisation of the cytoplasm
Structure of the parvovirus in more detail
So small only 60 copies of VP1 (only one main structural protein) (3/facet). T = 1. Smaller number of VP2 and VP3 which are on the inside simply to stabilize the capsid. There are spike proteins though they are not involved in attachment. It is flat red highly negatively charged spots that are actually doing the attachment.
Secondary structure of the genome of parvovirus
There are only two genes that are either named NS and VP for non-structural (replication) and viral protein (structure) (for MVM parvovirus, B19 erythrovirus, GmDNV densovirus) or rep and cap for replicase and capsid (for AAV2 dependovirus). The secondary structures at the ends of the genome are important for recognition for replication and assembly. There are often two or more transcriptional start sites for early vs late transcription and different proteins.
Explain the attachment step of the parvovirus.
There are different receptors for the different parvoviruses which is an advantage of using this virus for gene therapy as different receptors can target different tissues, so we can select the right AAV with the desired tropism. If cells do not express the receptor, no infection is possible.
B19: Erythrocyte P antigen
CPV/FPV: Transferin receptor (iron receptor)
AAV: Heparin sulphate, EGFR integrins
Enter through clathrin-mediated intake and moves to early endosome, it can then escape when the endosome acidifies causing conformational changes in the capsid and activation of phospholipase that can cut the endosome membrane and the virus can enter into the nucleus through nuclear pore complexes using microtubules. There are no known NLS and the mechanism of entry into the nucleus is unknown.
Explain the replication step of the parvovirus.
The genome is ss, must become ds to be compatible for transcription, only then will early genes be turned on. There is a free 3’ hydroxyl group thanks to the inverted terminal repeats, no need for a primer. DNA pol can just extend there and then the other terminal repeat can unfold. The NS1 or rep protein can then be transcribed and then it will come back to make a nick in one of the strands with its endonuclease activity. The hairpin is unwound by NS1/Rep and then DNA pol can come and extend the 3’ end. The hairpin will refold and DNA pol will continue to extend causing ssDNA displacement which will fold its second hairpin and can restart the process or get packaged. The other component is a dsDNA which can also continue its replication at another step.
How does transcription work with parvovirus in terms of possible protein mRNAs produced?
MVM: NS1, NS2 (early ones promoter p4) VP1, VP2 (late ones, structural, promoter p38), only two genes but Dif proteins thanks to alternative splicing
AAV: Rep 78, 68, 52 and 40 (early) AND VP1 (major product), VP2 and VP3 (late) (again alternative splicing)
What are some transcription factor binding sites in MVM DNA that regulate transcription?
Autonomous virus
Early promoter p4: E2F also Ets (TF turned on in dividing cells, so it’s a way for the virus to wait and make sure the cell is dividing) binding site to drive transcription of NS1, then when NS1 is made, it can bind to the promoter region (NF-Y) to activate transcription of early genes even more (feed-forward mechanism)
Late promoter p38: NS1 has a higher affinity for its own promoter but when enough is made, will start also binding to the promoter region of late protein, temporally controlling transcription
What step in the cell cycle does the cell have to be in to be infected by a parvovirus?
Must be in S phase, no RB activity, E2F is free. NS1/Rep78 lengthens S phase. Cellular genes involved in DNA synthesis/cell replication are on for parvovirus genome transcription. (E2F is important sign that cell is in S phase)
What are some transcription factor binding sites in AAV DNA that regulate transcription?
Early promoter p5: MLTF (major late TF) is a cellular TF but its transcription is ramped up by adenovirus (oncogenic, drives cell division, E1A protein) and binds to the early promoter of AAV when cell also infected by Adenovirus. NS1/Rep78 will also activate transcription at promoter once MLTF activates the first transcription of NS1/rep78
Late promoter p40: E1A of adenovirus also activates ATF (activating transcription factor) which can bind to late promoter.
How is E1A involved in S phase activation?
E1A can bind to RB, releasing E2F and activating S phase and E2 genes (required for AAV early transcription from p4) (from adenovirus). This happens so we don’t have to wait for Rb to be phosphorylated by Cdk.
How does AAV infect at the same time as Adenovirus?
Well simultaneous infection is nearly impossible, a thing of chance. So, when AAV infects, a bit of replicase can mediate genome insertion into host genome where it exists as a latent provirus until Adenovirus infects and triggers cell division so more Rep78 is made and mediates release of AAV genome. The virus particles exit through cell lysis caused by adenovirus mechanisms.
Parvovirus assembly
VP1 follows a self-assembly process. And other proteins or VP1 will recognize the secondary structure of the ITR of the genome to assemble it. If the ITRs are identical, 50% of the viral particles will contain the + strand and the other 50% will contain the - strand. If the ITRs are asymmetrical, we can more readily select the + vs the - strand. (ex: MVM 99% (-) while 1% (+))
What are the different possible functions of NS1/rep78?
- Binding viral DNA to induce late gene expression of capsid encoding genes and for endonuclease activity during replication
- ATPase dependent helices to unwind viral DNA (prevent genome from intense torsional strain)
- Sequence specific endonuclease
- Also appears to retain cells in S phase (god knows how though)
SELF-SUFFICIENT BADDIE, DOES EVERYTHING NEEDED FOR REPLICATION!
Why can AAV be used as a gene therapy vector?
- Does not cause disease in humans (except for that AAV2 involved in Hep in children, kinda awkward lmao)
- Different AAV serotypes use a variety of receptors so there is potential to target a specific tissue
- Low toxicity
- Stable expression (has been observed for up to one year since it CAN integrate as a provirus and just chill there)
- Easy to genetically manipulate (small genome) which is also kind of an issue since it must be a small gene to work and fit in capsid one such disease that they’re trying to make work is muscular dystrophy (use for monogenetic disease)
How can AAV be used as a gene vector?
- The rep and cap are removed
- Still need ITR and region where nick is made, and strong promoter, and of course gene we want to deliver
- The cloned transgenre is packaged into a viral particle with an AAV helper plasmid containing Rep and cap in trans because this way it cannot actually be incorporated into the viral genome, but also need Adenovirus helper plasmid containing E1A, E1B, E4, E24, VARNA. E1 genes are expressed in 293 immortalized cells which then produce AAV virions which will be our cute vectors.
