By: Minhazur Sarker Mentor: Dr. Manoj Pastey HHMI Summer 2011 Internship Oregon State University

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. 33.3 million HIV Infections • Attacks Immune System • AIDS (Acquired Immune Deficiency Syndrome) • “Greatest pandemic of the second half of the twentieth century”

3. HIV Classification • Group VI Retrovirus • (+) ssRNA Genome • Reverse Transcriptase • Integrase Figure 1. HIV Enveloped Virus http://www.niaid.nih.gov/topics/HIVAIDS/Understanding/Biology/Pages/organization_hiv1virion_fig.aspx

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

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

6. gag vif rev nef L TR pol tat L TR vpr vpu env HIV-1 Genome – NS2 • New Gene Discovery – NS2 • Unknown Function and Role • Critical to Viral Replication • Localizes in Nucleus GFP NS2 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 http://sitemaker.umich.edu/kerppola.lab/kerppola_lab_bifc

10. Methodology Previous Projects • 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 HHMI 2011 Future Project

11. Proteins of Interest (HHMI) • Programmed Cell Death 5 (PDCD5) • Homo sapiensDnaJ (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: http://www.ncbi.nlm.nih.gov/gene/9141

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: http://www.ncbi.nlm.nih.gov/pubmed/16179353

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: http://www.ncbi.nlm.nih.gov/pubmed/19667068/

15. 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. 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. 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. 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 • MaciejMaselko • Meagan Prescott • Casey Ward • Dr. Kevin Ahern • Howard Hughes Medical Institute • URISC: START – Oregon State