Bill Sugden (McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, USA) “How EBV Prevails During Productive Infection”
- Hybrid of P3HR1 and D98. (Endogenous P3HR1 cells)
- EBV’s lyticv phase commandeers the cell much as do a-herpesviruses
- Determining when vEBV DNA amplifies following induction off the productive cycle (treated cells with tamoxifen —> BZLF 1 expression)
- Tamoxifen was added before G2
- Virus waits until S phase
- Imaging of single cells for 45 house show that cells begin amplifying their amplicon DNA form 5-22 hours following induction
- Once viral DNA is made, it runs fast
- How much is viral (and cellular) DNA made
- Real-time PCR snows that both endogenous EBV and its amplicon DNA increase by 1 100-1000 fold during the lytic cycle
- Endogenous EBV 3000-5000 / Cell. (In 45 hours). == 10^8
- Amplicon ~ 1000 /cell (in 45 hours)
- What is limiting factors that regulate EBV DNA replication
- (A)Label Lamine A/C (nuclear laminitis) so that the volume of the nuclei can be estimated.
- (B) regarding encapsidated viral DNA (measuring EBV virus parties for their DNase-resistent DNA shows surprisingly little EBV DNA is encapsdidated. Extra cellular visions contain 0.5-1% of amplified DNA (= very insufficient encapsidateion)
- (C) Meselson-Stahl or density shit experiments measure changes in the density of DNAs resulting from the incorporation of heavy nucleotide during DNA synthesis
- — 8h MEselson-Stahl experiments show that EBV DNA is synthesized rapidly (_90 min / duplication of the pool)
- — 20 h.
- — 30h the rate of duplication of the pool of EBV DNA has declined dramatically
- EBV initiated the amplification of its genomes asychronously during the lytic cycle
- EBV genomes increase ~ 1000 fold in induced cells
- This amplification is expotentional for ~ 13 hours and then declines
- This decline results from the rate of duplication per se of the pool of EBV DNNA declining dramatically
- Only ~ 1% of EBV’s amplified genomes are encapsidated, indicating that encapsidation does not contribute to the decline of genome amplification