By: Minhazur Sarker Mentor: Dr. Manoj Pastey HHMI Summer 2011 Internship Oregon State University
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Transcript of By: Minhazur Sarker Mentor: Dr. Manoj Pastey HHMI Summer 2011 Internship Oregon State University
Confirmation of Interaction of a Novel HIV NS2 Protein with Host
Proteins in Living Mammalian Cells
By: Minhazur SarkerMentor: Dr. Manoj Pastey
HHMI Summer 2011 InternshipOregon State University
What is HIV?
Human Immunodeficiency VirusGlobal Issue
Approx. 33.3 million HIV InfectionsAttacks Immune System
AIDS (Acquired Immune Deficiency Syndrome)“Greatest pandemic of the second half of the
twentieth century”
HIV Classification
Group VI Retrovirus(+) ssRNA GenomeReverse TranscriptaseIntegrase
Figure 1. HIV Enveloped Virushttp://www.niaid.nih.gov/topics/HIVAIDS/Understanding/Biology/Pages/organization_hiv1virion_fig.aspx
HIV Life Cycle
Figure 2. HIV Life CycleRambaut A. et al. Nat. Rev. Genet. 5:52-61, 2004
HIV-1 Genome• Structural Genes: Gag, Pol, Env• Regulatory Genes: Tat, Rev• Accessory Genes: Nef, Vif, Vpr, Vpu
LTRpol
nefgag vif
vpr envvpu
rev
tatLTR
Figure 3. HIV-1 GenomePastey Laboratory
HIV-1 Genome – NS2New Gene Discovery – NS2
Unknown Function and RoleCritical to Viral ReplicationLocalizes in Nucleus
NS2
Figure 4. HIV-1 Genome with NS2
Pastey Laboratory
GFP
DoubleStrandedDNA
RNART
Integration
Host Protein?
NS2
Blocks HIVReplicationIf NS2 is absent
HIV
Cell
Possible Function of NS2
Figure 5. NS2 HypothesisPastey Laboratory
Yeast Two-Hybrid Screening
Tests entire library for protein interactionsFalse positives pose major problem
NS2 Y2H yielded 19 “positive” interactions
Bimolecular Fluorescence Complementation (BiFC)
Protein A tagged with YFP-1 and Protein B tagged with YFP-2
Interaction between protein A and protein B must occur first
If A and B interact, YFP-1 and YFP-2 re-form shortly after and the full YFP protein fluoresces
Figure 6. BiFC Outlinehttp://sitemaker.umich.edu/kerppola.lab/kerppola_lab_bifc
MethodologyDetermine localization of NS2Perform a Y2H on NS2
Confirm “positive” interactions via BiFC Isolate sequence of interest then insert into
plasmid with YFP2Co-transfect mammalian cells with
experimental group (NS2-YFP1 and protein of interest-YFP2) and control groups (positive and negative control)
Screen for fluorescence
Determine function/role of NS2
PreviousProjects
HHMI2011
FutureProject
Proteins of Interest (HHMI)
Programmed Cell Death 5 (PDCD5)
Homo sapiens DnaJ (Hsp40) homolog, subfamily B, member 1 (DNAJB1)
Lantibiotic Synthetase C-like Protein 2 (LANCL2)
Programmed Cell Death 5 (PDCD5)
Plays a critical role in apoptosisAfter apoptosis is induced, PDCD5 relocates to
the nucleus from the cytoplasm and programs for cell death
NS2 may inhibit PDCD5 and its ability to carry out apoptosisConsistent with out proposed NS2 mechanism
Source: http://www.ncbi.nlm.nih.gov/gene/9141
DnaJ Homolog, Heat Shock Protein 40 (Hsp40) (DNAJB1)Hsp40 interacts with HIV-1 infected cells
via HIV Nef proteinInteraction leads to increased Hsp40
translocation into nucleus, facilitating viral gene expression
Nef signals for Hsp40 translocation and NS2 may bind with Hsp40 once in the nucleusBinding complex may initiate viral gene
expressionSource: http://www.ncbi.nlm.nih.gov/pubmed/16179353
Lantibiotic Synthetase C-Like Protein 2 (LANCL2)
Required for abscisic acid (ABA) binding and signaling pathway in mammalian granulocytes cellsABA plays a fundamental role in response to abiotic
stress
LANCL2 interaction with white blood cells may hint at a role in HIV infectionsAlso localizes in the nucleus and thus may have an
NS2 interactionSource: http://www.ncbi.nlm.nih.gov/pubmed/19667068/
HHMI Project Progress
Programmed Cell Death 5 (PDCD5):PDCD5-YFP Plasmid constructed
Analyzed by OSU CGRB to confirm sequence is correct and in frame with YFP
Figure 7. Sequencing Results of PDCD5 (CGRB)
HHMI Project ProgressHomo sapiens DnaJ (Hsp40) homolog,
subfamily B, member 1 (DNAJB1):DNAJB1-YFP Plasmid constructed
Analyzed by OSU CGRB to confirm correct sequence and ensure sequence is in frame with YFP
Figure 8. Sequencing Results of DNAJB1 (CGRB)
HHMI Project Progress
Lantibiotic Synthetase C-like Protein 2 (LANCL2):Complication with
isolating gene sequence due to issues with restriction site Figure 9. pBiFC Plasmid
Sequence
HHMI Project Progress
Confirm “positive” interactions via BiFCIsolate sequence of interest then insert into
plasmid with YFP2Co-transfect mammalian cells with experimental
group (NS2-YFP1 and protein of interest-YFP2) and control groups (positive and negative control)
Screen for fluorescence
Goal: Conduct BiFC experiment
Future DirectionConstruct NS2-YFP1 plasmid
Conduct BiFC experiment on PDCD5 and DNAJB1Research further if interaction is positive
Isolate LANCL2 sequence and construct LANCL2-YFP2 plasmidConduct BiFC experiment and research further if
results are positive
AcknowledgementsDr. Manoj Pastey
Maciej MaselkoMeagan PrescottCasey Ward
Dr. Kevin AhernHoward Hughes Medical InstituteURISC: START – Oregon State