HPV16 Papers

Question 1

Evidence against CME (entry paper)

Answer 1

2x siRNA AP2, 2x siRNA CHC -> max 20% red with AP-2 (+ SFV);
Chlorpromazine blocks almost 100% at 10µg/ml (controls: + SFV, - SV40)
no cointernalisation in TIRF, different timing
dynamin is dispensable

Question 2

Evidence against CavME

Answer 2

Cholesterol independent: Nyst/Prog 1, 5, 10µg/ml no effect (blocks SFV, SV40)
2x siRNA -> max 25% (con: - VSV)
Cav-KO MEFs -> no effect
coloc index below 0.1

Question 3

Internalization kinetics

Answer 3

Low MOI: 50% after 12h, 100% after 24h
High MOI: 50% after 4h, 100% after 10-12h
slightly slower in HaCaT than HeLa

Question 4

Internalization of virus particles

Answer 4

120s (100-190s)

CME to compare: 50-60s

Question 5

HPV16 + Flotillin

Answer 5

siRNA Flot 1+2, single and combined -> no effect (con: SV40, enhanced)
coloc index below 0.1 (2h) to 0.15 (8h)

Question 6

HPV16 + dynamin

Answer 6

entry paper:
K44A vs. wt: no difference (con. SFV, SV40 +)
dynasore: 20, 80, 100µM -> up to 150% inf. (SFV, SV40 +)

Question 7

HPV16 + actin

Answer 7

entry paper:
Cyto D: 1, 10, 50µg/ml -> 40- ~5% (SFV -, SV40 +)
Jasplakinolide: 0.2, 1, 5µM -> blocks (SFV -, SV40 +)

Question 8

HPV16 + RhoGTPases

Answer 8

C3 toxin (blocks Rho) -> no effect (SFV -, SV40 +)
Toxin B (blocks Rho,Rac,Cdc42) -> no effect (SFV -, SV40 +)
Cdc42: T17N (DN), Q61L (CA) -> no effect (VV +)
Rac1: T17N (DN), Q61L (CA) -> no effect
RhoA: T19N (DN), G14V (CA) -> enhanced with high wt
Arf6: T27N (DN), Q67L (CA) -> no effect

Question 9

HPV16 + Na+/H+ exchangers

Answer 9

EIPA: 0.2, 1, 5µM -> 40% with 1µM, blocked with 5µM (SFV, SV40 -, up to 30% reduced)

Question 10

entry paper signaling results

Answer 10

Genistein (RTK): 10, 50, 200µM -> blocked by 200µM (SFV -, SV40 +)
H-7 (Ser/Thr Kin): 1, 10, 100µM -> max 30% (SFV -, SV40 +)
Iressa (RTK): 5, 25, 50µM -> 25%-5% (SFV -, VV +)
Calphostin C (PKC): 0.05, 0.5, 5 -> 10% (SFV -, SV40 (+) )
Wortmannin (PI3K): 10, 100, 1000µM -> ~50% (SFV - reduced like HPV, SV40 +)
PI-103 (PI3K): 5, 25, 50µM -> 40% (SFV -, SV40 +)
IPA-3 (PAK): 10, 50. 150µM -> blocked (SV40 -, VV +)
2x siRNA PAK1 -> 40-50% (VSV -, VV +)

Question 11

inhibitors that block internalization

Answer 11

Chlorate, Chlorpromazine, Cyto D, Japlakinolide, EIPA, Genistein, IPA-3, PI-103, H-7

Question 12

intracellular kinetics of HPV16

Answer 12

low MOI: 12h in endosomes
high MOI: ~6h in endosomes
(slower in HaCaT!)

Question 13

intracellular requirements (entry paper)

Answer 13

Nocodazole (MT): 0.3, 3, 30µM -> blocks (SFV, SV40 +)
Rab5: S34N (DN), Q79L (CA) -> 50%
Rab7: S22N (DN) -> no effect
coloc with LAMP1 after 8h
no coloc with EEA1, Giantin

Question 14

HPV16 release by MMPs (Surviladze, Ozbun)

Answer 14

• blots show increase of HPV16 in supernatant from 1-4h
• release is reduced by Batimastat (10µM), blocked by Marimastat (62µM)
• infection is blocked by both inhibitors

Question 15

Results of donor/recipient assay (Surviladze, Ozbun)

Answer 15

• pgsd donors cannot be infected, CHO can
  pgsd -> CHO => 30% (why not more?? -> should just pass pgsd donors)
  CHO -> CHO => 100%
  pgsd -> pgsd => nix
  CHO -> pgsd => 130%

CAVE: CHO have no EGFR -> WTF??

Question 16

EGFR activation by HPV16 (Surviladze, Ozbun)

Answer 16

• 10min HPV16 -> phosphorylation of EGFR (Y1173), ERK1/2
• blocked by PD168393, genistein, cetuximab (all EGFR)
• increase in nuclear p-ERK at 15min, 30min, gone at 60min
• dose dependent reduction in infection after EGFR KD