Confirmation of Interaction of a
Novel HIV NS2 Protein with Host
Proteins in Living Mammalian Cells
By: Minhazur Sarker
Mentor: Dr. Manoj Pastey
HHMI Summer 2011 Internship
Oregon State University
What is HIV?
 Human Immunodeficiency Virus
 Global Issue
 Approx. 33.3 million HIV Infections
 Attacks Immune System
 AIDS (Acquired Immune Deficiency Syndrome)
 “Greatest pandemic of the second half of the twentieth
century”
HIV Classification
 Group VI Retrovirus
 (+) ssRNA Genome
 Reverse Transcriptase
 Integrase
Figure 1. HIV Enveloped Virus
http://www.niaid.nih.gov/topics/HIVAIDS/Understanding/B
iology/Pages/organization_hiv1virion_fig.aspx
HIV Life Cycle
Figure 2. HIV Life Cycle
Rambaut 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
gag
LTR
vif
rev
pol
nef
tat
vpr
vpu
Figure 3. HIV-1 Genome
Pastey Laboratory
LTR
env
HIV-1 Genome – NS2
 New Gene Discovery – NS2
 Unknown Function and Role
 Critical to Viral Replication
 Localizes in Nucleus
gag
LTR
NS2
GFP
vif
rev
pol
nef
tat
vpr
vpu
Figure 4. HIV-1 Genome with NS2
Pastey Laboratory
LTR
env
Possible Function of NS2
HIV
RNA
RT
Cell
Figure 5. NS2 Hypothesis
Pastey Laboratory
Double
Stranded
DNA
NS2
Integration
Host Protein?
Blocks HIV
Replication
If NS2 is absent
Yeast Two-Hybrid Screening
 Tests entire library for protein interactions
 False 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 reform shortly after and the full YFP protein
fluoresces
Figure 6. BiFC Outline
http://sitemaker.umich.edu/kerppola.lab/kerppola_lab_bifc
Methodology
 Determine localization of NS2
 Perform a Y2H on NS2
Previous
Projects
 Confirm “positive” interactions via BiFC
 Isolate sequence of interest then insert into
plasmid with YFP2
 Co-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
HHMI
2011
Future
Project
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 apoptosis
 After 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 apoptosis
 Consistent 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 protein
 Interaction 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 nucleus
 Binding complex may initiate viral gene expression
Source: 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 cells
 ABA plays a fundamental role in response to abiotic stress
 LANCL2 interaction with white blood cells may hint at a
role in HIV infections
 Also localizes in the nucleus and thus may have an NS2
interaction
Source: 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 Progress
 Homo 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
Goal: Conduct BiFC experiment
 Confirm “positive” interactions via BiFC
 Isolate sequence of interest then insert into plasmid
with YFP2
 Co-transfect mammalian cells with experimental group
(NS2-YFP1 and protein of interest-YFP2) and control
groups (positive and negative control)
 Screen for fluorescence
Future Direction
 Construct NS2-YFP1 plasmid
 Conduct BiFC experiment on PDCD5 and DNAJB1
 Research further if interaction is positive
 Isolate LANCL2 sequence and construct LANCL2-YFP2
plasmid
 Conduct BiFC experiment and research further if results
are positive
Acknowledgements
 Dr. Manoj Pastey
 Maciej Maselko
 Meagan Prescott
 Casey Ward
 Dr. Kevin Ahern
 Howard Hughes Medical Institute
 URISC: START – Oregon State