October 17, 2006 1
Molecular Components of HIV Protein RNA Lipid You heard in the first part of this course about the properties of two of the molecules of life - nucleic acids and proteins. In the next 3-4 lectures I would like to return to HIV and its life cycle. We will use the HIV lifecycle as a case study to learn about basic cell biology and cell membranes, which are made of the third molecule of life shown on this slide: lipids. 2
HIV Life Cycle and the Properties of Membranes 1. Difference between viruses and cells a. HIV anatomy and genome 2. Overview of HIV life cycle and targets for therapeutics a. Host cell recognition b. Entry into host cell 3. Membrane structure and properties a. Phospholipids b. Lipid properties and behavior in aqueous solution c. Membrane fluidity d. How we know: FRAP and green fluorescence protein e. Influence of fatty acid structure on fluidity f. Influence of cholesterol on membrane properties Lecture Readings Alberts 221-222, 365-388; McMurry 744-767, 808-810 3
The Anatomy of HIV capsid HIV viral particle viral RNA molecule coated with structural protein membrane glycoproteins Let s begin by taking a look at part of the anatomy of the HIV viral particle. Working our way from the outside of the virus to the inside: (1) There are two glycoproteins on the outside of the virus (green and black). These proteins are modified by sugars - this is what glycoprotein means. One of the two glycoproteins is anchored in the viral membrane. (2) The viral membrane (yellow) provides a barrier to separate the inside of the virus from the outside world. (3) A capsid (orange) comprised of structural proteins encases the genetic material of HIV. (4) Each HIV particle contains 2 copies of the viral RNA genome coated with a structural protein (purple). 4
Cells Can Replicate Autonomously But Viruses Cannot WHY? As you heard from Rob and Dan, the major difference betweens cell & viruses is that cells can replicate to make more cells, without the help of any other cells, but viruses cannot. The term autonomous is used to refer to this property. Why can t viruses replicate without help? 5
HIV Genome and Proteins capsid HIV viral particle viral RNA molecule coated with structural protein HIV genome A look at the genome of HIV gives us a clue to the answer! The virus is incredibly simple; it encodes: 1) structural proteins - these serve as scaffolds which organize other viral proteins and the viral genome (green, purple, orange and blue) 2) regulatory proteins - these proteins are involved in regulation of viral gene expression (gray; present inside the capsid but not drawn for simplicity) 3) cell surface proteins - gp41 & gp120 are displayed on the cell surface; gp stands for glycoprotein, which is a term used to refer to proteins that are modified by sugars (carbohydrates) (black, green) 4) enzymes (bright blue and red; all present in the capsid but not all drawn for simplicity) 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 polyproteins into individual proteins as part of the normal viral life cycle; David will tell you much more about the biochemistry of the protease and protease inhibitors used as therapeutics reverse transcriptase - involved in copying the viral RNA genome into DNA; Rob will talk to you more about this viral integrase - helps insert a copy of the viral genome into the host cell chromosome Now we see why viruses need host cells to replicate. The virus is missing many crucial parts found in living systems, including ribosomes for translation, metabolic proteins involved in energy generation, etc. These missing components are borrowed from cells so that the virus can make more viral particles. 6
HIV Life Cycle recognition of host cell viral amplification/ replication 1 2 3 4 HIV entry into cell viral assembly and exit from cell nucleus host cell There are 4 steps in the HIV lifecycle: 1) Host cell recognition The virus must recognize the correct host cell in which to replicate. In the blood HIV will encounter many cell types, only 2 of which are appropriate hosts - two types of immune cells called macrophages and T cells. 2) Entry into the host cell The virus must enter the host cell in order to replicate and co-opt the host cell machinery. This process requires fusion of the viral and host membranes, releasing the viral genome into the host cell. 3) Viral amplification and replication The virus must produce viral proteins and copy the viral genome. This is a challenge because, as you have heard, HIV uses RNA as its genetic material. Rob will talk to you about the process of viral amplification. 4) Viral assembly and exit from the cell The virus must assemble new copies of its genome, proteins, and membrane coat into viral particles and exit the cell. We will talk in the next four lectures about processes 1, 2, and 4. Rob will talk to you about process 3 when he lectures next. 7
HIV Targets for Therapeutics reverse transcriptase inhibitors recognition of host cell viral amplification/ replication 1 2 3 4 HIV entry into cell viral assembly and exit from cell host cell fusion inhibitors protease inhibitors Current HIV therapeutics target all 4 stages of the HIV lifecycle. In Life Sciences 1a we will talk about drugs that target three steps: 1) fusion inhibition - these drugs prevent entry of HIV into host cells 2) reverse transcriptase inhibition - these drugs prevent HIV from making a DNA copy of its RNA genome by inhibiting reverse transcriptase 3) protease inhibition - by inhibiting HIV protease these drugs prevent processing of the viral proteins into active forms required to make infectious viral particles To understand how these how these drugs work and why they are effective AIDS treatments, we need to know more about the HIV lifecycle and cell biology. 8
Recognition of Appropriate Host Cell HIV CD4 chemokine receptor nucleus chemokine membrane macrophage T-cell To begin its life cycle, HIV must find its appropriate host cells. As we mentioned, host cells for HIV are macrophages and T cells - 2 types of immune cells in your blood. HIV has to be able to recognize these cells among the dozens of cell types it might encounter; it does so by binding to molecules displayed only on the surface of these cells - proteins called CD4 and chemokine receptors. The CD4 protein and chemokine receptor each have a role within normal cells - both are involved in the functioning of the immune response (described below). HIV is exploiting these proteins to identify and get into its host cells. Extra information on CD4 and chemokine receptors: CD4 is a protein that plays an important role in the normal function of T cells. CD4 helps T cells recognize antigens - peptides derived from proteins recognized as foreign. CD4 accomplishes this task by binding to a complex of MHC protein (major histocompatibility complex is a complex of 2 proteins) and an antigenic peptide (an antigenic peptide is a fragment of a protein thought by the immune system to be foreign ). This binding triggers a signal transduction pathway inside the T cell that results in production of cytokines - soluble, secreted proteins that influence the behavior of other immune cells. Chemokine receptors are transmembrane proteins found on the surface of several types of immune cells that bind proteins called chemokines. Chemokines are secreted proteins involved in luring (attracting) immune cells to specific sites within the body, particularly sites of inflammation. Chemokines work by binding to the chemokine receptor and triggering a signal transduction pathway which causes changes in the ability of the cell to migrate and adhere to other cells. 9
Recognition of Appropriate Host Cell membrane HIV gp41 CD4 gp120 host cell membrane FREE VIRUS chemokine receptor CD4 ATTACHMENT CHEMOKINE RECEPTOR BINDING The HIV protein Gp120, displayed on the surface of the virus, plays an important role in the recognition of host cells; it first binds to CD4 on the macrophage or T cell. Favorable interactions between CD4 and Gp120 stabilize this binding (you heard about these forces from Dan and will hear more about them when David lectures again). The CD4-Gp120 interaction causes a change in Gp120 (represented by the change in the shape of Gp120, shown in purple) that allows it to bind to the chemokine receptor protein (green). The chemokine receptor is sometimes referred to as a co-receptor for HIV. At this point the HIV virus is bound stably to the outside of the host cell. Note: This drawing is NOT to scale. Gp41 and Gp120 are drawn much larger than they should be. This is done simply for clarity. 10
Entry into Host Cell membrane diameter ~100 nm glycoproteins gp120 gp41 membrane host cell diameter >10 µm To take advantage of its host, HIV must inject its genome into the host cell cytoplasm, together with some viral proteins. However, the viral and host cell membranes present a barrier to entry of the viral genome. As we will see, the virus solves the problem of entry into the host cell by catalyzing fusion of its membrane with that of the host. Note the scale of the virus and the host cell; the virus is <1/100th the diameter of the host cell! Before we can understand how virus solves the problem of entry into host cell we need to understand more about the properties of biological membranes. 11
Cell Membranes electron micrograph outside of cell cytoplasm plasma membrane ~10 nm long (covalent bond ~ 0.1 nm) Membranes are essential for life - they enclose the cell and create a boundary that allows the cell to maintain differences between the cytosol and the extracellular environment. The plasma membrane acts as a barrier between the inside and outside of the cell. The image on the left is an electron micrograph showing a section through a cell. You can see the cytoplasm of the cell as electron dense material (darker shading; this is mostly proteins), and also see the less electron dense outside of the cell. The structure separating the inside and the outside is the plasma membrane, which is a bilayer comprised of proteins (green) and lipids (red). In the plasma membrane lipids are arranged as a double layer. Biological membranes are relatively impermeable to water-soluble molecules. Different types of protein molecules spanning the bilayer mediate many cellular functions, including transport of molecules across the membrane. Other proteins serve as receptors to detect and transduce chemical signals in the environment (you will hear much more about these signaling proteins in Dan s lectures). To give you a feel for the scale of membranes, proteins, and bonds: (1) The lipid bilayer is ~ 5 nm think. (2) A typical membrane protein is ~ 10 nm long. (3) A covalent bond is ~ 0.1 nm. Extra information on electron microscopy: The image shown above on the left is taken using a method called transmission electron microscopy. In this technique, scientists make a very thin section of a specimen of interest, stain it with salts of heavy metals such as uranium and lead, and then image it using a microscope which focuses a beam of electrons. Electrons are absorbed or scattered by the heavy metals, removing them from the beam and resulting in dark sections in the image. The resolution that can be accomplished with this method is much greater than with the light microscope - details as small as 2 nm can be visualized. 12
Phospholipids: Phosphatidylcholine O O O O O O P O O N cis double bond saturated unsaturated The major lipid components of cell membranes are phospholipids which comprise ~50% of animal cell membranes (by mass). Phospholipids are amphipathic molecules; this means that they have both hydrophobic and hydrophilic parts. Shown here is phosphatidylcholine (PC), the predominant phospholipid in cell membranes. It has a hydrophilic head group comprised of choline linked to glycerol via a phosphate group. The choline group of PC is positively charged at neutral ph, and the phosphate group is negatively charged. This makes PC electrostatically neutral. In contrast, some phospholipids have net negative charge. Phospholipids also have a hydrophobic tail with 2 fatty acid chains (from 14-24 carbons). Often one chain is saturated (no double bonds) and the other is unsaturated (one or more cis double bonds). Each cis double bond creates a kink in the fatty acid tail. Other phospholipids include sphingomyelin and various aminophospholipids. These lipids differ in their head groups and the length of their fatty acid tails. As we will see, the chemical composition of the phospholipids (degree of saturation, type of head group, length of fatty acid chain) can influence the physical properties of the membrane. 13
Hydrophobic Effect Much of the basis for the unique properties of membranes resides in the hydrophobic effect. As you learned from David and Dan, hydrophilic molecules (like acetone; top) dissolve readily in water because their polar groups interact favorably with water. Hydrophobic molecules (like 2-methyl propane; bottom) are insoluble in water because they cannot form favorable interactions with water; they force water molecules to arrange in ice-like cages, decreasing the entropy of the water molecules and therefore increasing the free energy of this process. The free energy of putting hydrophobic molecules in water is minimized if hydrophobic molecules cluster to minimize their surface area in contact with solvent so that the smallest number of water molecules are affected. 14
Packing of Lipids in Aqueous Solutions conical cylindrical The shapes and packing of lipids can influence the structures they form in water, or waterbased solutions (referred to as aqueous, or water-containing). The lipid molecules spontaneously aggregate to minimize the amount of hydrophobic surface in contact with water. They bury their hydrophobic tails in the interior of the aggregate and expose their hydrophilic heads to water. Depending on their shape, lipids can aggregate to either form spherical micelles, or bilayers. Lipid molecules with one fatty acid chain and a large polar head group have an overall shape that is conical, so when these types of lipids pack together they form a micelle in which the tails are inside and the polar head groups are in contact with water. In contrast, lipids with two fatty acid tails (like those found in most biological membranes), have a cylindrical shape; the packing arrangement that minimizes contact between their fatty acid tails and water results in a bilayer. 15
Spontaneous Closure to Form a Sealed Compartment A lipid bilayer has free edges in which the hydrophobic lipid tails are exposed to water. Because the contact of hydrophobic molecules with water is energetically unfavorable, synthetic bilayers spontaneously rearrange to eliminate free edges. The free edges can be eliminated by forming a sealed compartment, which places the polar head groups in contact with water and buries the hydrophobic fatty acid tails inside the compartment and away from water. This spontaneous formation of sealed compartments is remarkable behavior that is fundamental to living cells; it follows from the shape and amphipathic nature of phospholipid molecules. 16
Phospholipid Mobility leaflet leaflet Biological membranes are dynamic structures because individual phospholids in bilayers are highly mobile. The two halves of the bilayer are referred to as leaflets. Individual phospholipids can rotate, their fatty acid tails can flex, and they can diffuse laterally. Lateral diffusion is so fast that a phospholipid will diffuse through the membrane from from one end of a bacterial cell to the other (~2 um) in 1 second. By contrast, exchange of phospholipids between the two leaflets - called flip-flop - is rare, occurring less than once per month for a single phospholipid. 17
Animation: Membranes are Dynamic! Animation of dynamics of lipid and protein movement in cell membranes 18
How Do We Know?: An Experiment fluorescence recovery after photobleaching (FRAP) fluorescent molecule protein in membrane cell Cell membranes are two dimensional fluids or liquid crystals in which hydrophobic components in the membrane - lipids and membrane proteins - are constrained within the plane of the membrane, but are free to diffuse laterally. How do we know that membranes are so dynamic? A technique called photobleaching is commonly used to measure lipid and protein mobility in membranes. Photobleaching is light-induced inactivation or fading of a fluorescent molecule, resulting from chemical damage and covalent modification. Fluorescent molecules can be photobleached by illuminating them with very intense light, typically from a laser beam. Photobleaching is typically combined with fluorescence microscopy to measure the rate of diffusion of a membrane protein or lipid in the plane of the membrane. A protein of interest can be labeled with a fluorescent antibody or a fluorescent protein. Fluorescent molecules are bleached in a small area of the membrane with a laser beam, extinguishing the fluorescence of the molecules in that area. The fluorescence intensity recovers as the bleached molecules (which are not fluorescent) move away and unbleached, fluorescent molecules move into the area. The diffusion coefficient is calculated from the rate of recovery of fluorescence; the faster the rate of diffusion the greater the rate of recovery. 19
Green Fluorescent Protein (GFP) Fusions jellyfish nucleus fluorescent proteins CFP GFP YFP cell Studies of the movement of proteins within cells have been enabled by the development of fluorescent proteins that can be visualized inside of single, living cells. The green fluorescent protein (GFP), derived from the jellyfish Aequoria victoria, can assemble a fluorophore through modification of its constituent amino acids. Therefore, the fluorescent protein is completely encoded in its DNA sequence - all that is needed to make fluorescent GFP is the encoded GFP polypeptide sequence. These properties of GFP can be exploited to follow the movement of a protein of interest within living cells. In a strategy referred to as tagging or generating fusion proteins, the gene encoding a protein of interest (skin color within gray chromosome) is joined to the DNA encoding GFP (red). When the chimeric (hybrid) gene is transcribed and translated inside the cell, a fusion protein is produced consisting of the protein of interest joined to GFP (GFP is shown in green). In this manner the protein of interest is tagged with a fluorescent molecule that can be visualized by fluorescence microscopy. By changing a small number of amino acids in the sequence of the GFP protein, scientists have created versions with different fluorescent properties. Two examples of this are CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein), which give off blue and yellow light, respectively. Multiple proteins within a cell can be tagged with different color fluorescent proteins, allowing them to be visualized simultaneously. 20
FRAP With Membrane Protein-GFP Fusion Alberts FRAP movie; mobile protein in membrane of endoplasmic reticulum and immobile protein in membrane of nuclear envelope 21
Measures of Membrane Fluidity: Melting Temperature T m (melting temperature) is a phase transition, a change from a more rigid solid-like state to a fluid-like state The fluidity - ease with which lipids move in the plane of the bilayer - of cell membranes has to be precisely regulated because many biological processes (e.g. membrane transport and some enzyme activities) cease when the bilayer fluidity is reduced too much. The fluidity of cell membranes depends on their chemical composition and also on temperature. As the temperature is raised, a synthetic bilayer made of one type of phospholipid undergoes a phase transition from a solid-like state to a more fluid-like state at a characteristic melting point temperature (abbreviated as Tm; this is the temperature at which the fluidity is half way in between the solidlike and fluidlike states). At low temperatures the lipids within the bilayer are well-ordered, packed into a crystal-like arrangement in which the lipids are not very mobile and there are many stabilizing interactions. As the temperature is raised, these interactions are weakened, and the lipids are in a less ordered, liquid-like state. Lipids with longer fatty acid chains have more interactions between the hydrophobic fatty acid tails (predominantly van der Waals interactions), stabilizing the crystal-like state and therefore increasing the melting temperature and making the membrane less fluid. 22
Membrane Composition Influences Membrane Fluidity: Fatty Acid Structure unsaturated fatty acid chains saturated fatty acid chains lower T m higher T m O OH O OH 17 carbons cis double bond olive oil candle wax oleic acid stearic acid The degree of saturation of the fatty acid chains also affects the melting temperature and membrane fluidity. Fatty acids that are saturated, containing no double bonds, are straight and can pack more tightly than those that have double bonds. The kinks in the unsaturated chains simply make it more difficult to pack them in an orderly manner. As a consequence, the melting temperature of bilayers containing lipids with saturated fatty acids is higher than the melting temperature of bilayers containing lipids with unsaturated fatty acids. Additionally, the fluidity of bilayers containing lipids rich in unsaturated fatty acids is greater than the fluidity of bilayers containing lipids rich in saturated fatty acids. An example of this difference in fluidity and melting temperature is oleic and stearic acid. These two fatty acids have the same number of carbons (17), but they differ dramatically in their fluidity and melting temperature. At room temperature oleic acid is fluid-like (olive oil) and stearic acid is solid-like (candle wax); the melting temperature of oleic acid is lower than that of stearic acid because oleic acid is unsaturated and cannot pack in as orderly a manner as saturated stearic acid. 23
Membrane Composition Influences Membrane Fluidity: Cholesterol Content HO H H H H H Cholesterol is a major component of eukaryotic cells. The ratio of cholesterol to lipid molecules in a membrane can be as high as 1:1. Cholesterol is a member of a class of natural products called steroids, characterized by a 4 ring structure. Although all steroids are based on the same scaffold, they can affect different biological processes, ranging from the development of secondary sex characteristics (testosterone and estrogen) to inflammation (corticosteroids). Like phospholipids, cholesterol is an amphipathic molecule; it has a polar head that contains a hydroxyl group. The rest of cholesterol is hydrophobic, consisting of a rigid 4 ring steroid structure and a hydrocarbon tail. The 4 ring structure makes most of the cholesterol molecule very rigid because bonds between atoms in a ring are not free to rotate as a result of the geometric constraints of being in a ring. 24
Cholesterol Influences Membrane Fluidity Cholesterol interacts with phospholipids by orienting its polar hydroxyl head group close to the polar lipid head group. The rigid rings of cholesterol interact with and partly immobilize the fatty acid chains closest to the polar phospholipid head group. As a consequence, lipid molecules adjacent to cholesterol are less free to adopt different conformations than those in a cholesterol-free membrane region. By decreasing the mobility of a few methylene groups (CH2) in the fatty acids tails, cholesterol makes lipid bilayers less deformable and lessens their permeability to small water-soluble molecules. Therefore, cholesterol makes membranes less fluid. Although cholesterol makes bilayers less fluid, at the high concentrations of cholesterol found in eukaryotic cells, it also prevents fatty acid hydrocarbon chains from coming together and crystallizing. Therefore, cholesterol prevents fatty acid chains from ordering into a crystallike state. Cholesterol inhibits phase transitions in lipids. At low temperatures it increases membrane fluidity by preventing fatty acid hydrocarbon chains from coming together and crystallizing. Under these conditions cholesterol inhibits the transition from liquid to solid (decreases the membrane freezing point). At high temperatures cholesterol decreases membrane fluidity by immobilizing a few methylene groups in the fatty acid tails of the lipids. Therefore, under these conditions cholesterol increases the melting point. Therefore, cholesterol acts like antifreeze - the temperature of your car engine is modulated by water circulating with antifreeze/coolant which lowers the freezing point of the antifreeze/coolant so it does not freeze in the winter. The antifreeze/coolant also raises the boiling point in the summer so that your engine does not overheat. The influence of cholesterol on membrane properties is critical for the normal functioning of eukaryotic cells. 25
Summary of Main Points HIV needs host cells to replicate because its genome does not encode all of the proteins required for living systems HIV recognizes host cells by interacting with specific protein receptors found on the surface of those cell types Cell membranes are bilayers composed of amphipathic phospholipids containing charged head groups and hydrophobic tails The hydrophobic effect drives the packing of lipids into structures which minimize exposed hydrophobic groups Membranes are fluid because phospholipids and proteins can move in the plane of the bilayer; fatty acid structure and cholesterol content influence fluidity 26