Week 5 Section. Junaid Malek, M.D.

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1 Week 5 Section Junaid Malek, M.D.

2 HIV: Anatomy Membrane (partiallystolen from host cell) 2 Glycoproteins (proteins modified by added sugar) 2 copies of RNA Capsid

3 HIV Genome Encodes: Structural Proteins - scaffolds which organize other viral proteins and viral genome Regulatory Proteins - involved in regulation of viral gene expression Cell-surface proteins - gp41 & gp 120 are displayed on the cell surface (gp = glycoprotein) Enzymes

4 Encoded Enzymes Protease - involved in processing the viral polyproteins into active forms; many viral proteins are made as polyproteins, where one polypeptide is joined to another; the viral protease processes these into individual proteins as part of the normal viral life cycle Reverse transcriptase - involved in copying the viral RNA genome into DNA Viral integrase - helps insert a copy of the viral genome into the host cell chromosome

5 Let s Play Doctor... A patient comes to you with complaints of a sore throat. You take a swab of the inside of the inflamed throat and culture the material from the swab in media plus all necessary nutriets. Any of the patient s cells transferred with the swab do not survive.

6 Q: If the sore throat is caused by a bacteria (such as streptococcus), will anything grow in the media? A: Yes! Bacterial replicate autonomously. Q: If the sore throat is caused by a virus (such as influenza), will anything grow in the media? A: No! Viruses need host cells to replicate.

7 The Cell Membrane

8 Phospholipids Essential to cell membrane formation Amphipathic molecule What are some chemical properties of amphipathic molecules we can exploit to make a cell membrane?

9 Phospholipids Are made of one glycerol, two fatty acids and one phosphate group Different phospholipids will vary based on the fatty acids that are attached to the glycerol O OH H 2 C OH HC OH H 2 C OH Glycerol Oleic Acid - O O P O - O - Phosphate

10 Phospholipids

11 Phospholipid Modification Phospolipids can be modified, for example, by attaching choline, inositol or other functional groups (like amino acids) to the phosphate N O O P O O O O O O Phosphatidyl Choline

12 Question Q: How would phospholipids be oriented in a hydrophobic environment? A: The amphipathic phospholipids have a columnar shape. The hydrophobic effect would drive the phospholipids to back their hydrophobic fatty acid tails together and leave the charged part of the molecule exposed to water. This would cause them to form a bilayer. Finally, forming a sealed compartment can eliminate the free edges of the bilayer

13 Membrane Composition and Fluidity Cell membrane is a dynamic structure Phospholipids can move via lateral diffusion, rotation, flexion and flip-flop Hydrophilic interactions at the polar heads can H-bonds with water molecules Van der Waals interactions of the hydrocarbon tails

14 Membrane Composition and Fluidity Kinks (unsaturated fatty acids) prevent tight packing and thus increase membrane fluidity Saturated fats (straight chains) have more VDW interactions and are thus less mobile and decrease fluidity Temperature (at low temps a lipid bilayer is in a two-dimensional rigid crystalline (or gel) state. At a characteristic melting point called Tm, this gel state melts into the liquid state normally seen in membranes

15 Question Q: How do length and saturation of fatty acid tails affect the Tm of the membrane? A: T m is lower (e.g. membrane more difficult to freeze) if the hydrocarbon chains are short or unsaturated. Shorter chains

16 Cholesterol Amphipathic molecule with hydrophilic -OH group and hydrophobic rings/tail Present to some degree in cell membranes Orients self within bilayer so that the -OH groups are close to the polar heads of the phospholipid

17 Cholesterol Rigid steroid ring Interacts with and decreases the mobility of the first few - CH2- groups of the hydrocarbon chains on phospholipid molecules Makes lipid bilayer less deformable and decreases the permeability of the bilayer to small water-soluble molecules Thus, it plugs holes between phospholipids

18 Cholesterol At high temperatures, cholesterol interacts with the fatty acid side chains and decreases the mobility of individual phospholipids At low temperatures, cholesterol prevents the phospholipid molecules from packing in as tightly as the could otherwise and thus helps increase membrane fluidity

19 The red line on the following graph shows the fluidity of the fatty acids of a phospholipid bilayer as a fuction of temperature. The blue line shows the fluidity in the presence of cholesterol

20 Q: What is the effect of cholesterol? A: At lower temps, cholesterol prevents the fatty acid tails from closely packing together. Since the tails form fewer interactions, the membrane becomes more fluid. At higher temps, cholesterol decreases the mobility of individual phospholipids, which reduces the fluidity of the membrane

21 Q: Why might this effect be biologically important? A: Cholesterol inhibits phase transitions in lipids. That is, it acts like antifreeze for cell membranes. Since protein function depends on the proper fluidity of the membrane, cholesterol helps maintain the proper environment for membrane-protein function.

22 Proteins Functions Transporters, anchors, receptors, enzymes Association Transmembrane, membrane-associated, lipid-linked, protein attached

23 Q: What are some strategies transmembrane proteins can use to span the cell membrane? A: Use charged amino acids at the extracellular/intracellular domains and hydrophobic residues in the transmembrane domain (e.g. use α-helices and β-sheets)

24 FRAP NOT THIS!!!

25 FRAP

26 Let s Play Scientist... You discover a new phospholipid in a rare cell type. To learn whether it is freely mobile you decide to use a fluorescent tag to label the phospholipids on the membrane surface of these cells. When you examine the membrane using a microscope, you find that the tag is distributed diffusely across the cell surface. To determine the mobility of this phospholipid in the membrane, you use FRAP!

27 Question: You notice that after bleaching an area of the cell membrane that 95% of the fluorescence is recovered in this area within 5 minutes. Draw the recovery graph:

28 Q: What is the rate of recovery a measure of? A: The rate of recovery of fluorescence in the bleached area is a measure of the rate of the mobility (lateral diffusion) of the labeled phospholipid Q: Why does the level of fluorescence never reach the pre-photobleaching level? A: Some of the phospholipids will have permanently lost their fluorescence so the recovery is not 100%

29 NOTE: In the previous experiment the phospholipids were labeled ex vivo (outside the cell). The movement of proteins inside of cells can be visualized by using Green Fluorescent Protein (GFP) Fusions and fluorescent microscopy

30 GFP Fusion Proteins The gene encoding a protein of interest is joined to the DNA encoding GFP When the chimeric gene is transcribed and translated inside the cell, a fusion protein is produced consisting of the protein of interest joined to GFP Using this technique, proteins of interest can be tagged and visualized by fluorescence microscopy

31

32 HIV Entry into Host Cells Let s first review HIV life cycle...

33 Initial Binding of HIV to Host Cells HIV utilizes 2 host cell receptors which, with 2 viral proteins, make up 4 critical players in the process of viral entry Both viral proteins have attached carbohydrate (sugar) groups and are thus called glycoproteins - gp41 and gp120 HIV gp41 is a single pass transmembrane protein (goes through lipid bilayer once) while gp120 is non-covalently attached to gp41 on the external side of the viral membrane

34 Initial Binding of HIV to Host Cells On the host side, there are 2 important human receptor proteins: CD4 and a chemokine receptor CD4 is expressed on T helper lymphocytes Remember the chemokine receptor? (CCR5 and CCR5Δ32)

35 The Players

36 First HIV gp120 binds to CD4 on Th Lymphocyte This binding changes the conformation of gp120 such that it will now interact with and bind to the chemokine receptor

37 The Story Continues Next Lecture...

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