Lecture 5 - Parvovirus / Adeno-Associated Virus – Detailed Study Notes

“Perboviruses” (context indicates the parvovirus family; single-stranded DNA viruses)
- Classified as Class II in the Baltimore scheme (ssDNA ➜ dsDNA ➜ mRNA).
- Important in veterinary medicine – frequently isolated from tumor tissue, prompting early speculation about a possible role in oncogenesis.

Icosahedral, non-enveloped particle.
Capsid built from 60 capsomers (T=1 symmetry).
Diameter ≈ $26\,\text{nm}$ (implied although not explicitly verbalized; typical for parvoviruses).

Total length ≈ $5\,\text{kb}$ (the speaker says “five total base pairs in length”, clearly meaning kilobases).
Linear ssDNA with inverted terminal repeats (ITRs) that fold into hairpins (A, B, C arms) providing:
- A built-in primer (free $3'\text{-OH}$ ) for DNA polymerase.
- Resolution sites for rolling-hairpin replication.
Two major transcription units separated by two potential splice sites ⇒ multiple mRNAs.

P4 / P38 promoters in Minute Virus of Mice (MVM) – earlier reference made.
In AAV (adeno-associated virus) promoters are:
- P5 (rep78/68), P19 (rep52/40), P40 (capsid)
- “P40” name = located at 40 map units of the genome.

Unspliced transcript → Rep 78 (large Rep protein) – homologous to NS1 in MVM.
Differentially spliced transcripts generate:
- Rep 68 (shorter N-terminal truncation)
- Smaller Rep isoforms (e.g.
- $\text{60 kDa}$
- $\text{80 kDa}$ ) termed RBP1/2 in slide text.
- DP1 / E10: alternative splice that omits an internal exon – produces a protein initiating during S-phase.
Structural (capsid) proteins VP1/2/3 translated from P40-driven, polyadenylated mRNAs once dsDNA replicative form is established.

N-terminal origin-binding & nickase motif (endonuclease) – introduces site-specific nicks within ITRs.
Central ATPase/helicase domain (Walker A/B motif) – DNA unwinding & motor for genome processing.
C-terminal helix–turn–helix DNA-binding element (illustrated as successive α-helices in the slide) – sequence specificity for A, A’ sites.

Virion binds cell-surface carbohydrate/protein receptor via the capsid itself (no envelope glycoproteins).
Entry by receptor-mediated endocytosis ➜ intact capsid delivered to cytoplasm.
Nuclear import:
- Capsid + genome associates with Ran-GDP/importin complex (speaker pronounces “brand g d p”).
- Rep 68/78 bound to the $5'$ end plus importins guide the nucleoprotein complex through the nuclear pore.

SS genome folds back using terminal hairpins (A, B, C arms) ⇒ free $3'\text{-OH}$ primes complementary-strand synthesis by host DNA polymerase.
Resulting dsDNA (“RF I”) resembles the cartoon shown early in lecture; ONLY this form is transcription-competent from P5/P19/P40 (AAV) or P4/P38 (MVM).

Key hairpin intermediates written in lecture as:
- $C'\;A\;B$ → DNA synthesis generates $C'\;A\;B'$ then refolds to $B'\;A\;C'$ , recreating a new $3'\text{-OH}$ .
Each cycle duplicates one genome length; resolutions require Rep-mediated site-specific nicking at the terminal resolution sites (TRS).

AAV is dependovirus – needs adenovirus (or HSV, vaccinia, etc.) helper functions for productive replication.
In absence of helper, AAV establishes latency/integration; with helper, Rep‐directed activation of P19/P40 and genome amplification ensues.

Rep 78 ≅ NS1 (MVM) – identical roles: origin recognition, helicase, transcriptional regulation.
Eukaryotic chromosomes: multiple origins because they’re too long for single initiation site – virus solves this with terminal hairpins acting as built-in origins.
Clinical & Ethical angle: veterinary tumour association remains correlational; no proven causal oncogenesis to date.