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Orthopoxvirus proteomics

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Hundreds of Cases in Africa, Dozens in USA. Is 66,000 times more lethal ... Carrie Nicora. Angela D. Norbeck. Jason Page. Samuel O. Purvine. Karin D. Rodland ... – PowerPoint PPT presentation

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Title: Orthopoxvirus proteomics


1
Orthopoxvirus proteomics
National Institute of Allergy and Infectious
Diseases Biodefense Proteomics Research Centers
2
Orthopoxviruses
Double-Stranded DNA Viruses Genome 200
kBp Proteome 220 Predicted Proteins 200nm
Diameter, 300nm Long Among the Largest and Most
Complex of Viruses
Unvaccinated Africans J Gen Virol 86266172
Vaccinia Used in Smallpox Vaccine Approx
0.0002 will die from vaccine-related
complications Monkeypox Emerging Threat? Poor
Human-to-Human Transmission? Hundreds of Cases
in Africa, Dozens in USA Is 66,000 times more
lethal than vaccinia Smallpox Eradicated
Worldwide in 1977 Possible Bioterrorism
Agent Yugoslavia 1972 1 Case ? Vaccination of
20,000,000
http//www-micro.msb.le.ac.uk/
3
Monkeypox virus (MPV)
  • MPV is an orthopoxvirus, approx 198kb genome,
    encoding at least 190 proteins
  • First identified in 1950s from a macaque and
    later found in human cases in the 1970s
  • MPV infection is essentially indistinguishable
    from smallpox
  • closely related to Variola major virus (smallpox)
    at the genetic level (94 identical)
  • MPV infection in the recent past has been
    associated with mortality.
  • MPV is a Category A select bioterrorism agent

4
MPV infection indistinguishable from smallpox
  • MPV outbreak in the Democratic Republic of Congo
    in 2001. Involved 31 individuals and caused 5
    deaths.
  • Evidence for person-to-person transmission

5
MPV infection not limited to Africa
  • Outbreak in the U.S. in 2003 affecting up to 71
    individuals 35 of these were confirmed. No
    fatalities associated with the outbreak, however
    there was significant morbidity observed.
  • Outbreak was concluded to be derived from an
    imported Gambian rat. The rat was housed with
    prairie dogs, and the prairie dogs passed MPV to
    humans.

6
Monkeypox virus genome comparisons
7
Orthopoxviral growth factor and immune evasion
genes
8
Objectives of the study
  • To characterize the comprehensive proteome of
    defined orthopoxvirus particles using Vaccinia
    Virus (VV) and Monkeypox virus (MPV).
  • To define host-pathogen interactions by
    quantitative measurements of the proteome of
    orthopoxvirus particles from infected epithelial
    (HeLa) and macrophage (TPA-differentiated THP-1)
    cells.
  • Implement technical advances to extend the global
    proteomic analysis capabilities based upon the
    use of AMT tags to enable quantitative proteomic
    analyses for studies of pathogen/host cell
    proteomes.

9
What have we done thus far?
  • Produce infectious IMV and EEV particles from VV
    and MPV-infected HeLa cells for proteomic
    analysis
  • Analyze the peptides and identify novel proteins
    found in the IMV and EEV particles that have been
    found to be associated with virulence in related
    pox viruses.
  • Identify low abundance viral proteins to confirm
    process

10
Vaccinia and Monkeypox Virus Workflow
11
Purity of IMV and EEV particles defined by TEM
12
Purity of IMV and EEV particles by Western blot
Silver stain
Western with anti-A35R
13
Shotgun or MuDPIT Proteomics
X!Tandem or SEQUEST w filtering Archive
Tandem MS spectra
Parent MS spectra
14
Accurate Mass and Time Tag Approach
  • SEQUEST and/or X!Tandem Results
  • Filtering
  • Calculate Exact Mass
  • Normalize Observed Elution Time

High-throughput LC-FTICR-MS Analysis (AMT) tag
?LC- FTICR-MS
Peak-Matched Results
Compare Abundances across Multiple Proteomes
15
Proteomics Samples Compared
Proteins not present in EEV
16
Monkeypox Vaccinia IMV Highly Abundant Proteins
17
Functional annotations of viral proteins
identified
Genome
Proteome
18
(No Transcript)
19
Intact Protein Analysis to Identify
Post-Translational Modifications
Population Protein of Interest with Peptides and
Some Dependent PTM







Top-Down (Intact Analysis)
Bottom-up (MudPIT)

















Results are an average and coverage is incomplete
Approach is still very challenging, But results
are complete
20
Some possible modifications
Delta M14 Da
Delta M14 Da
Delta M14 Da
21
Some possible modifications
Delta M42 Da
Delta M42 Da
22
Validation of novel targets found in virus
particles
  • MPV chemokine binding protein. This protein was
    detected in all virus preparations and
    consistently in low amounts. As such, this
    represents a good target to validate, and is
    biologically an important molecule.
  • Viral proteins involved in actin tail formation.

23
viral CC Chemokine Inhibitor (vCCI)
  • Also called viral chemokine binding protein (vCBP)
  • Encoded by and secreted from most pox viruses
  • - Human isolate of MPV-Zaire..ORF-J1L

? Present in IMV and EEV particles
  • Binds ?-chemokines (CC) with high affinity and
    disrupts
  • normal signaling function
  • - binding site blocks receptor binding with lt nM
    affinity
  • Wt infected mice show reduced cellular
    infiltrates and
  • chemokine expression in the lungs compared to
    vCCI k.o.
  • infected mice

24
Groups involved in the studies
  • PNNL Team
  • Richard D. Smith
  • Joshua N. Adkins
  • Kenneth J. Auberry
  • David Camp II
  • Therese Clauss
  • Penny Colton
  • Xiuxia Du
  • Navdeep Jaitly
  • Mary Lipton
  • Nathan P. Manes
  • Matthew Monroe

Wong Laboratory Scott Wong Ryan Estep John
Jones Bonnie Yen Mike Powers Ilhem
Messaoudi Michael Axthelm Alfred Legasse Shannon
Planer Randa Mills Jennifer Wilk Pat Jewett
Heather M. Mottaz Carrie Nicora Angela D.
Norbeck Jason Page Samuel O. Purvine Karin D.
Rodland Informatics, Instrumentation,
Production, and Applications teams for other
contributions
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