Week 5 Section
Junaid Malek, M.D.
HIV: Anatomy
•
Membrane (partiallystolen from host cell)
•
2 Glycoproteins
(proteins modified by
added sugar)
•
•
2 copies of RNA
Capsid
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
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
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.
• 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.
The Cell Membrane
Phospholipids
•
Essential to cell
membrane formation
•
•
Amphipathic molecule
What are some chemical
properties of amphipathic
molecules we can exploit
to make a cell
membrane?
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
OH
O
H2C OH
HC OH
H2C OH
Glycerol
O
-O P OOOleic Acid
Phosphate
Phospholipids
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
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
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
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
Question
• Q: How do length and saturation of fatty
acid tails affect the Tm of the membrane?
• A: T
is lower (e.g. membrane more difficult
to freeze) if the hydrocarbon chains are
short or unsaturated. Shorter chains
m
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
Cholesterol
•
•
Rigid steroid ring
•
Makes lipid bilayer less
deformable and decreases the
permeability of the bilayer to
small water-soluble molecules
•
Thus, it “plugs holes” between
phospholipids
Interacts with and decreases
the mobility of the first few CH2- groups of the
hydrocarbon chains on
phospholipid molecules
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
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
• 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
• 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.
Proteins
• Functions
• Transporters, anchors, receptors, enzymes
• Association
• Transmembrane, membrane-associated,
lipid-linked, protein attached
• 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)
FRAP
NOT THIS!!!
FRAP
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!
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:
• 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%
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
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
HIV Entry into Host
Cells
Let’s first review HIV life cycle...
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
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)
The Players
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
The Story Continues
Next Lecture...