HIV Infection Dysregulates MicroRNA Expression by Human Macrophages. This seminar was presented in partial fulfillment of the requirements of the State of NY for obtaining my Ph.D. in the Biomedical Sciences.

1. HIV Infection Dysregulates MicroRNA Expression by Human Macrophages Aim ée Joanne Luers Laboratory of Dr. Joan W. Berman

2. HIV/AIDS: A Global Health Crisis Total: 33.4 million (31.1 – 35.8 million) 2009 http://www.unaids.org/en/KnowledgeCentre/HIVData/EpiUpdate/EpiUpdArchive/2009/default.asp Western & Central Europe 850,000 [710 000 – 970 000] Middle East & North Africa 310,000 [250 000 – 380 000] Sub-Saharan Africa 22.4 million [20.8 – 24.1 million] Eastern Europe & Central Asia 1.5 million [1.4 – 1.7 million] South & South-East Asia 3.8 million [3.4 – 4.3 million] Oceania 59,000 [51 000 – 68 000] North America 1.4 million [1.2 – 1.6 million] Latin America 2.0 million [1.8 – 2.2 million] East Asia 850,000 [700 000 – 1.0 million] Caribbean 240,000 [220 000 – 260 000]

7. Macrophages (Mɸ)

10. MicroRNA Maturation and Machinery

12. Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. Aims: Specific Aim 1: To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile macrophage expression of MicroRNAs.

13. Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. Aims: Specific Aim 1: To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile macrophage expression of MicroRNAs. Specific Aim 2: To identify MicroRNA expression alterations in HIV-infected human primary macrophages.

14. Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. Aims: Specific Aim 1: To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile macrophage expression of MicroRNAs. Specific Aim 2: To identify MicroRNA expression alterations in HIV-infected human primary macrophages. Specific Aim 3: To determine whether differentially expressed MicroRNAs have an effect on HIV production and/or host cell protein production in HIV-infected human macrophages.

15. Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. Aims: Specific Aim 1: To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile macrophage expression of MicroRNAs.

16. Whole Blood or Leukopak Separate by Ficoll Gradient PBMC Incubate with Magnetized CD14 Immuno- Beads Magnet CD14 - CD14 + Column Culture CD14 + Cells with MCSF for 6 days Apply to Magnetized Column Isolation of CD14+ Monocytes and Culture of Monocyte-Derived Macrophages CD14 + , CD68 + , CD11b + Migrate, Phagocytic Y Y Y Y

18. Aims: Specific Aim 1: To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile macrophage expression of MicroRNAs. Do macrophages express miRNA machinery proteins? Yes. Do macrophages express miRNAs?

20. Fractionation, Purification, and Analysis of Macrophage RNA Total RNA Small RNA Fraction < 40 nt Fractionation Purification Bioanalyzer Analysis

24. 119 MicroRNAs Expressed by Human Primary M acrophages Luers et al, Cellular Immunology, 2010 MicroRNA Microarray

30. Aim 1 Summary: Do Macrophages express miRNA machinery proteins? Yes. Do Macrophages express miRNAs? Yes.

31. Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. Aims: Specific Aim 1: To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile macrophage expression of MicroRNAs. Specific Aim 2: To identify MicroRNA expression alterations in HIV-infected human primary macrophages. Specific Aim 3: To determine whether differentially expressed MicroRNAs have an effect on HIV production and/or host cell protein production in HIV-infected human macrophages.

36. Specific Aim 2: To identify MicroRNA expression alterations in HIV-infected human primary macrophages. Does HIV-1 infection alter the production of miRNAs? Yes, some are increased, some are decreased.

42. qRT-PCR Indicates that Expression of MicroRNA-99b Is Increased during HIV Production

43. qRT-PCR Indicates that Expression of MicroRNA-125a Is Increased during HIV Production

44. qRT-PCR Indicates that Expression of MicroRNA-146a Is Decreased during HIV Production

45. Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. Aim 2 Summary: Specific Aim 2: To identify MicroRNA expression alterations in HIV-infected human primary macrophages. MicroRNA-99b and -125a are increased and MicroRNA-146a is decreased during HIV production in OM10.1 cells

46. Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. Aims: Specific Aim 1: To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile macrophage expression of MicroRNAs. Specific Aim 2: To identify MicroRNA expression alterations in HIV-infected human primary macrophages. Specific Aim 3: To determine whether differentially expressed MicroRNAs have an effect on HIV production and/or host cell protein production in HIV-infected human macrophages.

51. OM10.1 p24 Production is Increased by Mimic-146a

57. OM10.1 p24 Production is Increased by AntagomiR-99b

61. Chromatin Immunoprecipitation (ChIP) Performed in collaboration with Loreto Carvallo C/EBP β

62. AntagomiR to miR-99b Increases the Presence of C/EBP β at the HIV LTR in Transfected OM10.1 Cells

63. AntagomiR to miR-99b May Increase and/or Stabilize the Occupancy of C/EBP β at the Viral Promoter