1. 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

2. What is HIV? Human Immunodeficiency Virus Global Issue
Approx million HIV Infections
Attacks Immune System
AIDS (Acquired Immune Deficiency Syndrome)
“Greatest pandemic of the second half of the twentieth century”

3. Figure 1. HIV Enveloped Virus
HIV Classification
Group VI Retrovirus
(+) ssRNA Genome
Reverse Transcriptase
Integrase
Figure 1. HIV Enveloped Virus

4. Rambaut A. et al. Nat. Rev. Genet. 5:52-61, 2004
HIV Life Cycle
Figure 2. HIV Life Cycle
Rambaut A. et al. Nat. Rev. Genet. 5:52-61, 2004

5. HIV-1 Genome Structural Genes: Gag, Pol, Env
Regulatory Genes: Tat, Rev
Accessory Genes: Nef, Vif, Vpr, Vpu L TR
pol
nef
gag
vif
vpr
env
vpu
rev
tat
Figure 3. HIV-1 Genome
Pastey Laboratory

6. Figure 4. HIV-1 Genome with NS2
HIV-1 Genome – NS2
New Gene Discovery – NS2
Unknown Function and Role
Critical to Viral Replication
Localizes in Nucleus L TR
pol
nef
gag
vif
vpr
env
vpu
rev
tat
NS2
GFP
Figure 4. HIV-1 Genome with NS2
Pastey Laboratory

7. Possible Function of NS2
HIV
NS2
RNA RT
Double
Stranded
DNA
Cell
Integration
Host Protein?
Blocks HIV
Replication
If NS2 is absent
Figure 5. NS2 Hypothesis
Pastey Laboratory

8. Yeast Two-Hybrid Screening
Tests entire library for protein interactions
False positives pose major problem
NS2 Y2H yielded 19 “positive” interactions

9. 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 Outline

10. Methodology Determine localization of NS2 Perform a Y2H on NS2
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
Previous
Projects
HHMI
2011
Future
Project

11. 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)

12. 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:

13. 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:

14. 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:

15. Figure 7. Sequencing Results of PDCD5 (CGRB)
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)

16. Figure 8. Sequencing Results of DNAJB1 (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)

17. Figure 9. pBiFC Plasmid Sequence
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

18. Goal: Conduct BiFC experiment
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

19. 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

20. Acknowledgements Dr. Manoj Pastey Dr. Kevin Ahern
Maciej Maselko
Meagan Prescott
Casey Ward
Dr. Kevin Ahern
Howard Hughes Medical Institute
URISC: START – Oregon State