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 VirusGlobal Issue

Approx. 33.3 million HIV Infections

Attacks Immune SystemAIDS (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 – NS2

New Gene Discovery – NS2Unknown Function and RoleCritical to Viral ReplicationLocalizes in Nucleus

NS2

Figure 4. HIV-1 Genome with NS2

Pastey Laboratory

GFP

DoubleStrandedDNA

RNA

RT

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 NS2

Perform 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

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 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 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

Confirm “positive” interactions via BiFCIsolate 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

Goal: Conduct BiFC experiment

Future Direction

Construct 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

Acknowledgements

Dr. Manoj PasteyMaciej MaselkoMeagan PrescottCasey Ward

Dr. Kevin Ahern

Howard Hughes Medical Institute

URISC: START – Oregon State