Presentation by:

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Presentation by Kyle Borge, David Byon, Jim
Hall
Herpesviral Protein Networks and Their
Interaction with the Human Proteome
reconstruction of a Herpes Virus capsid
Presentation by Kyle Borge, David Byon, Jim
Hall
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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

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Types of Herpesviruses Investigated

• Kaposis Sarcoma-associated Herpesvirus (KSHV)
• In the gamma (?) 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)
• Varicella-Zoster Virus (VZV) , in the alpha (a)
  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)

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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
  protein-protein interactions

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Y2H Advantages

• http//www.dnatube.com/video/993/Plasmid-Cloning

• Relatively simple (automated)
• Quick
• Inexpensive
• Only need the sequenced genome (or sequence of
  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 sequencesallows the comparison of
  interactomes w/in and between different
  speciesin this paper, eukaryote (human)
  interactome vs. viral interactome

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Y2H Limitations

• http//www.dnatube.com/video/993/Plasmid-Cloning

• The Y2H system cant analyze some classes of
  proteins
• Transmembrane proteins, specifically their
  hydrophobic regions which may prevent the protein
  from reaching the nucleus
• Transcriptional activators may activate
  transcription w/out any 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)

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Yeasts GAL4 transcriptional activator

• GAL4 transcriptional activator which splits into
  two separate fragments a binding domain (BD) and
  an activating domain (AD)

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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

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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

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(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

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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

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Yeast-Two-Hybrid Background
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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)

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Viral Protein Interactions in KSHV
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Previously Reported Protein Interactions of KSHV
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Coimmunoprecipitation
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Verification of Predicted Interactions in other
Herpesvirus Species
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Correlation Between Viral Protein Interaction and
Expression Profile

• Average expression correlation AECwas
  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

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Protein Interaction Networks
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Network Terminology

• 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)

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Topology of KSHV and VZV Interaction Network
KSHV protein interaction network
VZV protein interaction network
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Comparison of Protein Interaction Networks
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Power Law Distribution Comparison

• http//www.dnatube.com/video/993/Plasmid-Cloning

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Removal of Nodes in KSHV Network
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Protein Interaction KSHV Sequence Conservation
to EBV
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Correlation Between Functional and Phylogenetic
Herpesviral Classes
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Viral protein interactions between functional
classes

• http//www.dnatube.com/video/993/Plasmid-Cloning

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Viral Protein Interactions Between Phylogenetic
Classes
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View of the Human-Herpesviral Networks
Varicella-Zoster Virus
Kaposi Sarcoma-associated Herpesvirus
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Power Coefficient of KSHV-Human Network
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Interplay between KSHV and Human Network
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Viral Host Network / Random Network Comparison
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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