Functional Microbial Genomics…HIV

1. Functional Microbial Genomics…HIV Shainn-Wei Wang, Ph.D. NCKU, College of Medicine Institute of Molecular Medicine

2. HIV exhibits tremendous genetic diversity Garber D. A., et al., Lancet Infec. Dis., 2004

3. HIV Data Base • http://www.hiv.lanl.gov/content/index

4. HIV/HCV Database http://lasp.cpqgm.fiocruz.br/LaspIngles/ferramentas.html

7. http://www.flu.lanl.gov/

11. Human Immunodeficiency Virus (HIV)

13. The nature of HIV infection Persistent and chronic infection - venereal spread (mucosal immunity) - hematogenous spread (systemic immunity) - poor immunogenicity and viral escape - progressive destruction (impaired regeneration of CD4 T help) Latent infection Acute infection

14. Infection and reservoir

15. Functional genomics of HIV infection • Host gene re-programming to viral infection • Host cells or Immune cells related • Immune suppression or activation • Invasion or evasion • Transcriptional networks in response to a viral protein • Cellular protein networks in response to assembly, replication, and latency • Functional search for MHC epitopes that are related to disease protection or progression • Epitope based vaccines

16. Mock control PBMC Target cells Non-Infection: gene sets Infection: signature gene sets comparison Change in disease or functional Status

17. The Journal of Infectious Diseases    2004;189:572-582

18. Functional genomics of HIV infection • Host gene re-programming to viral infection • Host cells or Immune cells related • Immune suppression or activation • Invasion or evasion • Transcriptional networks in response to aviral protein • Cellular protein networks in response to assembly, replication, and latency • Functional search for MHC epitopes that are related to disease protection or progression • Epitope based vaccines

20. Reprogramming the iDC • As the major activator of HIV transcription, Tat drives viral gene expression. • Tat regulates the expression of chemokines that promote lymphocyte and monocyte migration. • By recruiting susceptible host cells to infected dendritic cells,Tat may facilitate HIV dissemination

21. Expression analysis of primary iDC infected with HIV-1BAL and adeno-Tat • Expression profiles of iDC genes whose RNA levels were affected similarly by adeno-Tat and • HIV-1 infection. • Genes are divided into functional groups; the fold change in expression levels relative to the • 0 time point is displayed in red (increased expression) or green (decreased expression). • - Asterisk (*) marks IFN-inducible genes.

22. What they found in the array: • HIV-1 Tat induces expression of interferon-inducible genes • HIV-1 Tat mediates chemokine upregulation in iDC

23. RT-PCR analysis of selected immature dendritic cell genes whose expression is affected byTat. • Identical amounts of total RNA from iDC infected with adeno-LacZ and adeno-Tat were used. • Control β-actin mRNA was not affected by HIV-1 or adeno-Tat, confirming the microarray analysis.

24. Rt-PCR gene expression analysis in iDC obtained from donors A, B, C,D: donors

25. MCP-2 expression and SIV infection in axillary lymph nodes a, SIV Nef–expressing cells (red) in the paracortex. b, Digital overlay of images from the same field labeled for MCP-2 (green) and SIV Nef (red). Arrows indicate double-labeled cells (yellow) that are positive for both markers in the digital overlay of images. c–e, High-powered fields of lymph nodes showing SIV Nef (c),MCP-2 (d) and DC-SIGN (e, blue) expression by a single cell (indicated by arrows). f, Digital overlay of c–e shows a single SIV-infected dendritic cell (indicated by arrow) expressing all three markers (original magnification: a,b, ×200, c–f, ×400).

26. Chemotaxis of Monocytes and activated T cells

27. None of the typical dendritic cell maturation markers (such as CD40, CD80, CD83, CD86 and CD25) were expressed at increased levels during the time course of adeno-Tat, adeno-LacZ or HIV-1 infection

28. Summary and Discussion of the results • Genes encoding four different chemokines were induced in iDC by both Tat expression and HIV-1 infection • (interferon inducible protein-10 (IP-10), • human monokine induced by interferon-γ (HuMIG), • monocyte chemoattractant protein-2 (MCP-2) • monocyte chemoattractant protein-3 (MCP-3) • IP-10 and HuMIG are CXC chemokines whose production is normally IFN-γ dependent. These two chemokines attract activated T cells, whose chemotaxis is mediated by the CXCR3 receptor. • MCP-2 and MCP-3 are CC chemokines that attract monocytes and are produced by a variety of cells, including fibroblasts, endothelial cells, monocytes and macrophages

29. HIV-1 infection and Tat expression do not result in activation and maturation of iDC • No production of cytokines that are a hallmark of dendritic cell activation, • No phenotypic changes typical of dendritic cell maturation • the induction of IP-10, HuMIG, MCP-2 and MCP-3 in iDC by Tat and HIV-1 seems especially important for the spreading of HIV • The lack of iDC maturation with respect to cell surface markers paralleled the lack of induction of proinflammatory cytokines such as TNF-α, IL-1, IL-6, IL-10, IL-12) • whether the lack of iDC maturation after HIV-1 infection adversely affects the adaptive immune response require further exploration confirmation • Therapies designed to affect Tat function may produce the combined benefit of limiting viral transcription and reducing the interactions between infected dendritic cells and T cells that contribute to the expansion of viral infection

30. Genomic Database for HIV Infection

32. Functional genomics of HIV infection • Host gene re-programming to viral infection • Host cells or Immune cells related • Immune suppression or activation • Invasion or evasion • Transcriptional networks in response to a viral protein • Cellular protein networks in response to assembly, replication, and latency • Functional search for MHC epitopes that are related to disease protection or progression • Epitope based vaccines

33. Hepatocyte-growth factor-Regulated tyrosine kinase Substrate The Protein Network of HIV Budding, Cell, Vol. 114, 701–713, September 19, 2003

34. Proteome analysis • Protein-Protein interaction • Two Hybrid Sysytem • (Tandem) Affinity Tag • Protein complex purification requires expression of the target protein at, or close to, its natural expression level. • Among all kinds of protein complex purification method, protein A and CBP tags allowed efficient recovery of proteins • Proteome analysis, in particular using mass spectrometry (MS), requires fast and reliable methods of protein purification.

36. Tandem Affinity Purification Method Guillaume Rigaut, 1999 TEV cleavage site calmodulin-binding peptide protein A TAP tag

37. Tandem Affinity Purification Method Protein composition of TAP-purified U1 snRNP. Guillaume Rigaut, 1999

38. Tandem Affinity Purification Method Guillaume Rigaut, 1999

39. Tandem Affinity Purification Method Protein composition of TAP-purified U1 snRNP. Guillaume Rigaut, 1999

40. Tandem Affinity Purification Method Protein composition of TAP-purified U1 snRNP. Guillaume Rigaut, 1999

41. Protein identification and functional analysis MALDI-TOF Protein samples from GE - Online Data mining - Functional assay

42. Nature. 2002 Jan 10;415(6868):141-7.

43. Synopsis of the screen a. Schematic representation of the gene targeting procedureThe TAP cassette is inserted at the C terminus of a given yeast ORF by homologous recombination, generating the TAP-tagged fusion protein. b. Examples of TAP complexes purified from different subcellular compartments separated on denaturing protein gels and stained with Coomassie. Tagged proteins are indicated at the bottom. ER, endoplasmic reticulum. c, Schematic representation of the sequential steps used for the purification and identification of TAP complexes (left), and the number of experiments and success rate at each step of the procedure (right).

44. The polyadenylation machinery Primary validation of complex composition by `reverse' purification: a, A similar band pattern is observed when different components of the polyadenylation machinery complex are used as entry points for affinity purification. Underlined are new components of the polyadenylation machinery complex for which a physical association has not yet been described. The bands of the tagged proteins are indicated by arrowheads. b, Proposed model of the polyadenylation machinery.

45. The protein complex network, and grouping of connected complexes Links were established between complexes sharing at least one protein. For clarity, proteins found in more than nine complexes were omitted. The graphs were generated automatically by a relaxation algorithm that finds a local minimum in the distribution of nodes by minimizing the distance of connected nodes and maximizing distance of unconnected nodes. In the upper panel, cellular roles of the individual complexes are colour coded: red, cell cycle; dark green, signalling; dark blue, transcription, DNA maintenance, chromatin structure; pink, protein and RNA transport; orange, RNA metabolism; light green, protein synthesis and turnover; brown, cell polarity and structure; violet, intermediate and energy metabolism; light blue, membrane biogenesis and traffic. The lower panel is an example of a complex (yeast TAP-C212) linked to two other complexes (yeast TAP-C77 and TAP-C110) by shared components. It illustrates the connection between the protein and complex levels of organization. Red lines indicate physical interactions as listed in YPD22.

46. Functional genomics of HIV infection • Host gene re-programming to viral infection • Host cells or Immune cells related • Immune suppression or activation • Invasion or evasion • Transcriptional networks in response to a viral protein • Cellular protein networks in response to assembly, replication, and latency • Functional search for MHC epitopes that are related to disease protection or progression • Epitope based vaccines

48. CTL activity HIV encoded proteins MHC CD4 # or function HIV viral load • Viral genetic diversity • - individuals infected with different viral clades show different response patterns • MHC genetic variability in human populations Disease Protection or Disease progression Caucasian Africans Asians Hispanics South American Indians • these populations differ in their HLA allele • frequencies • the HIV-specific responses detected in • these ethnicities can also differ • significantly.

49. HLA molecules may be directly involved in restricting HIV replication • The human leukocyte antigens (HLAs) are also known as MHC (major histocompatiblity complex) or "self" molecules. • T and B cells recognize antigens only when "presented" to them next to an MHC ("self") molecule. • There are two main types of HLA. • Class I is divided into HLA A, B, C and are expressed by most human cells. • Class II is divided into HLA DP, DQ, DR and are expressed by B, macrophages and dendritic cells. Class II is involved in presenting antigen to CD4 cells, thus activating CD4 cells. When CD4 cells recognize antigen presented by HLA class II, they secrete cytokines (e.g., IL-2, IL-4) which in turn stimulate further immune responses

50. Nat Med. 2005 Dec;11(12):1290-2. Epub 2005 Nov 20