9. PURPOSE OF THESIS
• Two strategies:
• 1) Stable cell lines expressing encapsidation proteins
• 2) Encapsidation protein transposon libraries for use in
functional studies
• Hypothesis: specific regions of encapsidation
proteins will be essential for DNA encapsidation
since it is known that any one deletion of HSV-1
homologs results in accumulation of empty capsids
and un-cleaved DNA in the nucleus
10. STRATEGY 1: CELL LINE
COMPLEMENTATION
• Cell line complementation as a tool to study
encapsidation gene mutants
• HCMV, HSV-1, PRV
• Hypothesis: deletion of each putative DNA
encapsidation gene will yield a phenotype
consistent with a defect in cleavage and/or
packaging
• Essential to this process is the isolation of
stable, DNA encapsidation protein expressing cell
lines
11. CELL LINE COMPLEMENTATION
Deleted gene
VZV Mutant
Cell expressing deleted gene
• VZV genome without one encapsidation gene
• VZV requires each encapsidation gene for replication
12. MEWO CELL LINE
• Previously, each encapsidation ORF was transiently
expressed in MeWos (Visalli, 2007)
• Stable cell line production
• Geneticin sensitivity assay for LC75
• Transfection of ORF30 & ORF54 vectors
14. MEWO CELL LINE
• Transfection of ORF30 & ORF54
• Indirect immunofluorescence (IF)
• 6, 15, & 27 d
• Western Blot at 1 month
• confirmed IF results
15. FLP-IN CV-1 CELL LINE
• Flp-In two plasmid system
• Designed for rapid generation of stable cell lines
• Utilizes an FRT site & has reporter genes
• Successful integration of ORF means cells are:
• zeocin sensitive
• hygromycin resistant
• lacks B-gal activity
• expresses ORF
http://products.invitrogen.com/ivgn/product/K601001
16. FLP-IN CV-1 CELL LINE
• Complementation is
dependent on VZV
replication
• Can VZV grow in
CV-1?
• Plaque assay from 7 d
infection
• IF from 10 d infection
17. FLP-IN CV-1 CELL LINE
• Transient expression of ORF25, ORF54, & ORF30
• IF at 3 to 4 d post transfection
18. ORF30 TRANSFECTION
• 3 variables:
DNA, Lipofectamin
e, Hygromycin
• Only 6 colonies
survived
19. ORF 30: B-GAL ASSAY
• 6 ORF30 colonies
were assayed for
B-gal activity at 1
and 3 months
20. ORF30: PCR
• Presence of ORF30 gene in ORF30 cell lines were
tested by PCR
• 770 bp or 0.7 kbp
28. DISCUSSION
• VZV ORF expressing FlpIn cell lines isolated?
• Hygromycin resistant
• Lacked B-gal activity
• PCR
• Indirect immunofluorescence
• Western blot
• Immunoprecipitation followed by Western blot
• Complementation
29. DISCUSSION
• Stable cell lines must have ORF incorporated in
genome
• A rare event
• Promotors of ORF versus reporter genes
• Cellular toxicity
• Inducible promotor
• Improper ORF orientation
• Using complementation as selection
30. DISCUSSION
• Isolate encapsidation gene expressing cell lines
• Grow VZV mutants for functional studies
• Experimentally prove that the ORF is essential
• Deficient in DNA cleavage versus DNA translocation
31. STRATEGY 2: ORF54 TRANSPOSON
LIBRARY
• Little is known about the role of specific functional
regions within the pORF 54 portal
• ORF54 transposon library as a tool to study portal
function (pORF54)
• Portal isolation in HCMV, HSV-1, VZV
• Hypothesis: specific regions of pORF54 will be essential
for interaction with the viral capsid, terminase complex,
and viral DNA.
• Essential to this is a ORF54 vector prepared for
mutagenesis for the use in functional studies
32. CONSTRUCTION OF
ORF54 VECTOR
• 3165 bp
23130
9416
6557
4361
2322
2027
564
http://tools.invitrogen.com/content/sfs/vectors/pcr4blunttopo_map.pdf
33. CONSTRUCTION OF
ORF54 VECTOR
• Transformation of ORF54 vector into E. coli
• EcoR1 digestion
Not to scale
34. MANIPULATION OF
ORF54 VECTOR
• Removal of Not1 site
• Digested with Not1 Restriction
Enzyme
• Termination End Reaction
• Repair sticky ends to blunt
ends
• Resulted in the deletion of
Not1
• Ligation
• Transformation of ORF54
minus Not1
Transformation ORF54 without Not 1 site
35. VERIFICATION OF
ORF54 VECTOR
• Selection for Not1 Deletion
Undigested versus Not1 digested ORF54 vector
39. MUTAGENESIS
Epicentre EZ-Tn5 In-Frame
Linker Insertion Kit was used
to randomly insert mutations
on our ORF54 vector via
transposons
http://www.epibio.com/item.asp?id=289
41. MUTAGENESIS
Plasmid DNA was isolated from individual E.coli transformants
Digestion with Not1 to remove transposons
Removal of Transposon with Not1 digsetion Gel of Not1 digested mutated ORF54
http://www.epibio.com/item.asp?id=289
42. DISCUSSION
• Prepared the foundation for ORF54 transposon
library
• Isolated our ORF54 fragment
• Prepared the ORF54 vector by deleting Not1 and Kan
• Vector is ready for mutagenesis to make transposon library
for future functional analysis
• pORF54 regions can then be characterized by
complementing ORF54 VZV BAC
• ORF54 versus mutated ORF54
• Map essential and nonessential regions
43. THESIS CONCLUSION
• Characterizing functional regions will reveal the
mechanisms of DNA cleavage and packaging
• Both strategies would prove encapsidation proteins are
essential for VZV replication
• Hopefully these studies will get us closer to
understanding encapsidation
44. THANK YOU
• Dr. Visalli
• Vi Tran
• Rick Covington
• Dr. Hua Zhu (Rutgers)
• Funding from NIH R15 AI062713-02
Editor's Notes
1) Dissociation of tegument (pink bubbles), 3)Concatameric DNA, 5)Virion picks up glycoproteins from Golgi body before exiting
Spp1
Complementation of an encapsidation mutant virus depends on whether VZV replication can be supported by CV-1 cells
9 hour transfection. 3 days cells were diluted and 17 days post transfection, counted. At 22 days, transferred colonies. 200 ug/ml was decreased to 100.
The lack of protein expression was perplexing. it was difficultto find GFP expressing CV-1 cells from the ORF30BAC virus. There were only single cells expressing GFP and no obvious clusters of GFP cells, which would be an indication of plaque formation and spread, were observed.
25: 22 kda54: 91 kda30: 92.1
There were single cells expressing GFP (Figure 14D) and some larger clusters of GFP expressing cells (potential plaque formation; Figure 14C) suggesting that the mutant ORF54 genome was complemented.
