West Chester University’s Department of Biology Presents…

This Presentation Is Based On the Experimental Research Taken From

the BMC Biochemistry Research Article:

“Gene Expression Profile of HIV-I TAT Expressing Cells: A Close Interplay Between Proliferative

and Differentiation Signals”

Cynthia de la Fuente2, Francisco Santiago2, Longwen Deng2, Carolyne Eadie2, Irene Zilberman2, Kylene Kehn2, Anil Maddukuri2, Shanese Baylor2, Kaili Wu2,

Chee Gun Lee1, Anne Pumfery2 and Fatah Kashanchi2

Human Immunodefiency Virus

Shows two H.I.V. virus particles

Central Dogma of Molecular Biology

IMPORTANT!!!H.I.V. does not follow the rules of the Central Dogma of Molecular Biology

because it is a…..RETROVIRUS

What are these scientists trying to find?

• They want to explain the effect that Tat (Trans-Activator of Transcription) has on HIV-1 infected cells and Tat expressing cells.

• Cellular changes associated with this gene

The Culprit: Tat• HIV gene• Down regulate mannose receptor- spread of virus• Has been known to repress host cellular genes and involve itself in immunosuppression

– Example: MHC -1 (Major Histocompatibility Complex Type 1) - displays proteins that are present within the cell to Cytotoxic cells and if there are foreign peptides present, these cells will recognize them and kill them

• Increases levels of HIV RNA• Past research has focused on Tat’s ability to activate HIV-1 LTR (HIV-1 Long Terminal Repeat)• In-vivo effects of Tat

– Example: Xenopus embryo – delay of gastrulation, suppression of two early genes important for gastrulation (Xbra,gsc)

• In-vitro effects of Tat– Example: EDF-1 (Endothelial-related Factor-1) – regulates endothelial cell differentiation. Addition of Tat during

transcription resulted in the inhibition endothelial growth

• Contains protein transfer domain– Allows Tat to enter cells across cell membrane– Mechanism of Transfer: UNKNOWN

Important Information About Cell Culture Methods

• ACH2 cells : HIV-1 infected CD4 lymphocytic cells (plays a role in cellular immunity) containing wild type DNA*Cell Lines have a proviral sequence

• CEM T Cell: Parental cell for ACH2 cell

• TAR: Point mutation on Chromosome 37, which causes it to not respond to Tat. Although it does not respond to Tat, it is capable of making infectious viruses when certain stimuli are present. (TNF, PHA,PMA,etc)

• H9 Cells: CD4+ lymphocytes ; control integrated vector without Tat open reading frame

• H9/Tat Cells: CD4+ lymphocytes; integrated Tat expression vector• U1: monocytic clone; has two copies of viral genome from parent U973 cells• All cells were cultured at 37˚C up to 105 cells per ml in RPMI-1640 media

*Contained 10% Fetal Bovine Serum treated with mixture of 1% streptomycin and penicillin antibiotics and 1% L-glutamine

How the Cell Cycle was Analyzed for Experimental Purposes

• Blockage of HeLa cells with Hydroxyurea (prevents proliferation of HeLa cells) for 14 h

• Cells were then released by washing (2x) with phosphate-buffered saline (PBS; helps maintain a constant Ph) and adding complete medium.

• Suspended cells were treated with 1% serum for 48 hrs prior to addition of hydroxyurea.

• Collection of supernatants and analyzed by usage of an IL-8 ELISA

• Cells were washed with PBS and fixed by adding 50 ml of 70% ethanol

• FACS analysis : Fluorescence – Activated Cell Sorting– Cells Strained with Cocktail of Propidium Iodide Buffer (PL)- helps to determine cell cycle– PBS – NP-40 – can be used to determine cytoplasm content– PI

Purification of RNA

• 1.Cells grown to mid-log phase • 2.Pelleted• 3.Washed (2x) with cold D-PBS (maintains cell

culture media)• 4. Total RNA extraction on ice using Trizol

Reagent • 5.Purified RNA was analyzed on 1% agarose

gel (Quality and Quantity Purposes)

Glass Slide Microarray Method

• 1.2400 known human genes were arrayed onto a microarray glass slide into four separate grids (A,B,C and D). Each contained 600 genes respectively

• All genes were 2200 bp cDNAs

• 3 plant control genes were used to balance Cy-3 and Cy-5 fluorescence signals

Why Use DNA Microarray Analysis?

• 1. Price

• 2. Ability to study many genes simultaneously

• 3. Speed

• Information about the DNA Sequence is not required

Uninfected HIV-1 Cells Latently Infected HIV-1 CellsFigure 1

Shows all genes that were activated above 1 fold. (139 genes)

Shows all genes that were expressed below 1 fold. (449 genes)

Controls

Brief Description of Northern Blot Procedure

1. Gather sample that you wish to extract RNA from and isolate it by treating it with formaldehyde2. Electrophoresis with agarose gel. At this point, the RNA will separated by its size.3. Transfer of RNA to membrane, which is also termed as Northern Blotting4. Fix RNA to the membrane by using either Ultraviolet Light or Heat (IMMOBOLIZE IT!!)5. Soak the membrane in a hybridizing buffer. The usage of a hybridizing buffer will prevent the fluorescence of un-

reactive binding groups. Also, add labeled probes (antibodies specific to protein) to the membrane and incubate.6. Wash off the excess hybridizing buffer7. Detection of labeled RNA

Note: In this experiment, instead of X-Ray film, they used a Phosphorlmager cassette. Although a X-Ray film can be used, the detection time of Phosphorlmager is quicker.

Figure 2

1

2

1. Shows 695 genes that were up-regulated above 1 fold2. Shows 1705 genes that were down-regulated below 1

fold

Summary of Tables 1, 2 and 3 = Genes that Were Up-Regulated

Receptor Genes

Translation Genes

Signal Trans-

duction Genes

Genes in Cytoskeleto

n

Cell Cycle Genes

DNA Repair/Repl

ication Genes

Transcription Genes

Chromatin Remodeling

Genes

DNA Binding Genes

8 46 1 7 8 5 5 4 6

53 0 6 0 5 6 9 3 8

Total 61 46 7 7 13 11 14 7 14

= Genes that Were Down-Regulated

What is the significance of the box highlighted in yellow?

Figure 3

Figure 4

Figure 5

Figure 6

Conclusions

• More than 2/3 of cellular genes were down-regulated by Tat

• Genes belonged to receptor,co-receptor, and co-activator pathways that are part of serine/threonine receptor tyrosine kinase, Ras/Raf/MEK/ERK (MAPK)cascade, which play a role in proliferative and differentiation signals

• HIV-1 accessory spliced doubly spliced messages (TAT), may control host genome in latently infected cells and determine both viral transcription and possibly the fate of post-transcriptional events

BBibliography