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Herpesviral Protein Networks and Their
Interaction with the Human Proteome
Presentation by:
Kyle Borge, David Byon, & Jim Hall
reconstruction of a Herpes Virus capsid
Presentation by:
Kyle Borge, David Byon, & Jim Hall
Introduction to the Herpesvirus
• Large double-stranded DNA genomes
• Eight different strains
• Causes diseases ranging from cold sores to
shingles
• Vaccine available for Varicella-Zoster Virus (VZV)
• Little known about protein interactions
Types of Herpesviruses Investigated
• Kaposi’s Sarcoma-associated Herpesvirus (KSHV)
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In the gamma (γ) herpes virus phylogenetic class
Causes cancerous tumors
Mostly associated with HIV patients
Sequenced in 1996
Genome is roughly 165 kbs
89 open reading frames (ORFs)
• 113 ORFs used in experiment (included 15 cytoplasmic and 5 external domains
derived from transmembrane proteins)
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Varicella-Zoster Virus (VZV) , in the alpha (α) herpesvirus phylogenetic class
– Causes chicken pox in children and shingles in adults
– Sequenced in 1986
– Genome is roughly 125 kbs
– 69 open reading frames (ORFs)
• 96 ORFs used in experiment (Included 13 cytoplasmic
and 10 external domains derived from transmembrane proteins)
Methods of Investigating Protein
Protein Interactions (PPI)
• Many Methods
• The Y2H technique is one of the top techniques
for detecting protein-protein interactions
• This article used Y2H to investigate proteinprotein interactions
Y2H Advantages
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• Relatively simple (automated)
• Quick
• Inexpensive
http://www.dnatube.com/video/993/Plasmid• Only need the sequenced genome (or sequence of
Cloning
interest)
• Scalable, its possible to screen for interactions among
many proteins creating a more high-throughput screen (ex.
viral genome)
• Protein/polypeptides can be from various sources;
eukaryotes, prokaryotes, viruses and even artificial
sequences…allows the comparison of interactomes w/in
and between different species…in this paper, eukaryote
(human) interactome vs. viral interactome
Y2H Limitations
• The Y2H system cant analyze some classes of proteins
• Transmembrane proteins, specifically their hydrophobic regions
which may prevent the protein from reaching the nucleus
• http://www.dnatube.com/video/993/Plasmid• Transcriptional activators; may activate transcription w/out any
Cloning
interaction
• False-negatives
• Y2H screen fails to detect a protein-protein interactions
• False-positives
• Y2H screen produces a positive result (characterized by reporter
gene activity) where no protein-protein interaction took place
Ex. bait proteins activate, transcribing the reporter gene, w/out the
binding of the AD (bait proteins act as transcriptional activators)
Yeast’s GAL4 transcriptional activator
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GAL4 transcriptional activator which splits into two separate fragments; a binding
domain (BD) and an activating domain (AD)
Y2H Method
• ORFs selected from published sequences
• Amplified by nested PCR
– Made primer sets of ends of ORFs
• Y2H bait and prey vectors
• Vectors transformed into Y187 and AH109
haploid yeast cells creating pools; a bait pool and
a prey pool
• Bait and prey mated in quadruplicates
• Positive diploid yeasts are selected
Open Reading Frames (ORFs)
• Every ORFs of both KSHV & VZV were cloned & ligated into both a bait and
prey GAL4 vector
• Bait
– protein of interest
– the protein is fused to the yeast Gal4 DNA-binding domain (DBD)
• Prey
– a protein/ORF fused to the Gal4 transcriptional activation domain (AD)
– interacting protein
• Physical interaction between the bait and prey brings the DNA-BD and an
AD of Gal4 together, thus re-creating a transcriptionally active Gal4 hybrid
• Gal4 activity can be assayed by the expression of reporter genes and
selectable markers
(1-2) ORFs cloned into vectors via Nested PCR
• KSHV
– 113 full-length and partial ORFs
• including 15 cytoplasmic and 5 external domains
derived from transmembrane proteins
• VZV
– 96 full-length and partial ORFs
• including 13 cytoplasmic and 10 external domains
derived from transmembrane proteins
Yeast-Two-Hybrid
Prey pool: (target)
•Each individual ORF sequence is
cloned into the ‘prey’ vector
(down stream of the GAL4 AD
gene) and is essentially fused to
the GAL4 AD gene
•Ampr for selection
•Hemagglutinin
Bait pool:
•Each individual ORF
sequence is also cloned
into the ‘bait’ vector (down
stream of the GAL4 DBD
gene) and is essentially
fused to the GAL4 DBD
gene
•vector conveys Kanr for
selection
Yeast-Two-Hybrid Background
…in a diploid cell.
Viral Protein Interactions in KSHV
•12,000 Viral Protein Interactions tested
•Identified 123 nonredundant interacting protein pairs
•118/123 were novel
•7/123 were previously reported
•Screen captures 5/7 (71%) of previously reported
interactions
•50% of Y2H interactions confirmed by
coimmunoprecipitation (CoIP)
Viral Protein Interactions in KSHV
Previously Reported Protein
Interactions of KSHV
Coimmunoprecipitation
Verification of Predicted Interactions in
other Herpesvirus Species
Correlation Between Viral Protein
Interaction and Expression Profile
•Average expression correlation [AEC]was calculated
•For random pairs of ORFs: 0.804
•For interacting pairs of ORFs: 0.839
•Correlation between AEC and clustering coefficient
•Used to propose static or dynamic interaction for
viral hubs
Protein Interaction Networks
Network Terminology
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Node – represents a protein
Edge – represents interaction between two nodes
Average (node) degree – the average number of neighbors or connections that any given
node has
Power coefficient (g) – derived from an approximate power law degree distribution plotted
on a bilogarithmic scale and fitted by linear regression
P value - (significance under linear regression) as fitted by a power-law degree distribution
(‘‘scale-free’’ property)
Characteristic path length – the distance between two nodes
Diameter (d) - describes the interconnectedness of a network; defined as the average length
of the shortest paths between any two nodes in the network
Clustering coefficient – A value given to depict the number of fold enrichment over
comparable random networks (‘‘small-world’’ property)
Small world property/network – Any network that has characteristics of a relatively short
path and dense cluster (high cluster coefficient)
Topology of KSHV and VZV Interaction
Network
KSHV protein
interaction network
VZV protein
interaction network
Comparison of Protein Interaction
Networks
Power Law Distribution Comparison
• http://www.dnatube.com/video/993/PlasmidCloning
Removal of Nodes in KSHV Network
Protein Interaction KSHV & Sequence
Conservation to EBV
Correlation Between Functional and
Phylogenetic Herpesviral Classes
Viral protein interactions between
functional classes
• http://www.dnatube.com/video/993/PlasmidCloning
Viral Protein Interactions Between
Phylogenetic Classes
View of the Human-Herpesviral
Networks
Varicella-Zoster Virus
Kaposi Sarcoma-associated Herpesvirus
Power Coefficient of KSHV-Human
Network
Interplay between KSHV and Human
Network
Viral Host Network / Random
Network Comparison
Conclusions
• Virus and host interactomes possess distinct
network topologies
• Integration of viral and host protein network
may lead to better understanding of viral
pathogenicity
• Future interactome data from other viruses
may improve understanding of functions of
viral proteins and their phylogeny
• Understanding networks may help to develop
future therapies