The Deadly HIV Virus
HIV (human immunodeficiency virus), the virus that causes AIDS, is one of the hottest areas of medical research today. It is estimated that 30.6 million people throughout the world were infected with HIV by the end of 1997. After a person has been infected with HIV, the virus usually remains dormant for long periods of time. Then the virus begins a cycle of attacking cells of the immune system by incorporating its genetic material into the cells, using the immune cells' machinery to make more viruses from the incorporated genetic material, and then breaking the cells apart (killing them) so that the new viruses can infect more cells. In this manner, the immune system is weakened, so that the body can no longer defend itself against the pathogens that it encounters every day. Sadly, patients typically die within a few years of showing symptoms of AIDS (i.e., signs of drastically decreased immunity due to the virus's attack on the cells of the immune system). Because so many people are affected by HIV, and because the virus is so deadly, much research has been devoted to finding ways to fight this epidemic. Researchers are seeking treatments for HIV-infected individuals, cures, and vaccines. So far, the most promising findings have been treatments to lessen the damage to the immune system from HIV; several treatments that have been developed have dramatically improved the outlook for many HIV patients. However, these treatments have many severe side effects, and in most cases are too costly to be administered widely in many parts of the world. Thus, more research is needed to improve the available treatments, making them more tolerable to patients and more accessible, and to continue the search for a cure for HIV and a vaccine, which would prevent the infection in the first place.
This tutorial describes two of the most successful treatment options available today: reverse-transcriptase inhibitors (e.g., AZT) and protease inhibitors. These treatments interfere with enzymes that are needed for HIV to make copies of itself, a key step in the virus's attack on the cells of the immune system. In order to understand how these treatments help, we must first discuss the immune cells that come under attack, and the mechanism by which the virus kills these cells if left untreated.
HIV Attacks Helper T Cells
Our body's immune system contains many different types of cells, but only one of these cell types, known as helper T cells, is attacked by HIV. Helper T cells are necessary to stimulate the activation of other immune cells that attack infectious particles (antigens) in the body. When these cells come under attack by HIV, the immune system can no longer function effectively, and the body is incapable of combating many types of foreign infections and cancers. A more detailed description of normal immune function, and why helper T cells are critical to the immune system, is given in the peach box, below.
Overview of HIV's Attack on the Immune System
How does HIV invade and kill helper T cells, thereby depleting our immune system? To answer this question, we must first clarify what we mean by the term virus. Then we shall outline the major steps in the life cycle of HIV, and how these steps lead to the destruction of helper T cells. As you read this description, note that many of these steps are made possible by particular enzymes, biological catalysts that change the mechanism (and therefore the kinetics) of a biochemical reaction in order to enable the reaction to proceed with a smaller activation energy. (A more in-depth explanation of how enzymes work is given in the section below, "Enzymes as Biological Catalysts".)
A virus is often classified as a living thing, although it is not made up of cells, like other organisms. Viruses consist of proteins surrounding genetic material. Depending on the virus, this genetic material may be either DNA (the form in which our cells' chromosomes contain genetic material) or RNA (the form used for the expression of genetic information in cells, and the form in which some viruses carry their genetic information). Of the two types of viruses, DNA viruses and RNA viruses, HIV represents the second. A virus containing RNA is known as a retrovirus. In order to reproduce, a retrovirus must attach to a cell of the infected organism, insert its RNA into the cell, and make a DNA copy of the RNA. This DNA copy then incorporates into the cell's own chromosomes (which are made of DNA), and uses the cell's biochemical machinery to replicate the viral DNA (along with the host cell's DNA), make viral proteins from the DNA that is replicated, and assemble new viral particles from these proteins.
The steps by which HIV infects and kills helper T cells are described below, and can be viewed in Figure 3.
HIV Infection Cycle
As you can see from the steps outlined above, enzymes play a vital role in HIV's attack on helper T cells. The generation of a DNA copy of the viral genome by reverse transcriptase (Step 3) and the cleavage of viral proteins (Step 7) by protease are two important processes catalyzed by enzymes. Therefore, the enzymes reverse transcriptase and protease are major target sites for HIV-fighting drugs. Before we can explain how these drug treatments work to combat HIV, we must first discuss how enzymes work to catalyze biochemical reactions.
Questions on HIV's Attack on the Immune System
Enzymes as Biological Catalysts
Many biological reactions, such as most normal reactions of the cells, and the reactions employed by HIV to replicate itself, are thermodynamically favorable (DG<0) or can be made thermodynamically favorable by coupling with other reactions. Recall that thermodynamics tells us whether a reaction (or process) is spontaneous under a specified set of conditions. However, thermodynamics does not tell us how fast (or the rate with which) a reaction will proceed. Rates of reactions are the realm of chemical kinetics and are determined experimentally. From Arrhenius Theory, the rate of a reaction is dependent on the activation energy (Ea), which is the minimum amount of energy needed for a reaction to occur (See Figure 4, Path A). Hence, even though a reaction is thermodynamically favorable, it still can not occur unless there is enough energy available (an amount greater than or equal to the activation energy) to initiate the reaction. If sufficient activation energy cannot be supplied (i.e., the rate of the reaction is too slow), then a catalyst may be used. Recall from the introduction to the Experiment that a catalyst is a substance that increases the rate of a reaction without being consumed. A catalyst influences the mechanism (pathway) of a reaction, but does not affect the thermodynamics (DG). A catalyst allows the reaction to proceed by an alternate mechanism that has a lower activation barrier (Figure 4, Path B). Often biological reactions require catalysts called enzymes to change the pathway (mechanism) of the reaction, and thus lower the activation energy. When a catalyst changes the reaction pathway to lower the activation energy, the reaction rate is increased, but the thermodynamics of the reaction are not changed. That is, a catalyst cannot form products that are not allowed by thermodynamics (DG); it does increase the rate of forming the products that are allowed by thermodynamics. Enzymes are molecular catalysts in biological systems, and are usually proteins. Virtually all biological reactions are catalyzed by enzymes.
How Enzymes Work
Enzymes are very specific to the substrates (reactants) and reactions that they can catalyze. Enzymes work by binding the substrate into a favorable orientation in an enzyme-substrate complex (an intermediate in the reaction), which promotes the making and breaking of chemical bonds. The substrate is bound to the active site (a specific region of the enzyme), is converted to the product, and then released from the active site. The active site is a three-dimensional crevice, and is usually only a small portion of the total volume of the enzyme (Figure 5).
The substrate is "bound" to amino acids in the active site of the enzyme by multiple intermolecular interactions, including charge-charge interactions (electrostatics), hydrogen bonding, and van der Waals forces. Enzymes are often very sensitive to changes in pH and temperature, in part because these changes can affect the shape and charge of the active site, thus changing the interaction with a substrate. If the optimal conformation of the enzyme is lost, the enzyme becomes nonfunctional (i.e., the substrate can no longer bind to active site).
The study of the kinetics of a reaction that is catalyzed by an enzyme is essentially like the kinetics studies that you perform in lab, but involves a few additional, specialized concepts. The model for understanding the kinetic properties of most enzymes is known as the Michaelis-Menten model. This model proposes the following mechanism for enzyme catalysis. First, the enzyme (E) and substrate (S) come together to form an enzyme-substrate complex (ES), as shown in Equation 1, below. The reaction occurs, and the substrate is converted to the product of the reaction. Then, the enzyme-substrate complex is broken apart, yielding enzyme (E) plus product (P), as shown in Equation 2, below.
The Michaelis-Menten model assumes that only a negligible amount of enzyme-substrate complex reverts to reactants (i.e., k-1 << k1 in Equation 1). The rate of formation of product (shown below in Equation 4) can be determined from Equation 2 in the mechanism written above
Rate of formation of Product = k2[ES](4)
and the rate of formation of the intermediate ES (shown in Equation 5) can be determined from Equations 1 and 2 in the mechanism written above
Rate of formation of ES = k1[E][S] - (k2 + k-1)[ES](5)
Using the steady-state approximation (i.e., the assumption that the concentrations of intermediates (in this example, ES) stay constant while the concentrations of reactants and products change), and making several substitutions (which are not shown here), we can form an equation for the rate of formation of the product (Equation 6).
where [E]o is the initial concentration of free enzyme, [S] is the substrate concentration, and Km is a constant specific to a given enzyme known as the Michaelis-Menten constant. The value of Km relates to the rate constants shown in Equations 1 and 2, as given by Equation 7:
The Michaelis-Menten constant (Km) is very important, because it can be determined experimentally and describes the catalytic power of an enzyme. Km can also be used to predict the rate of a reaction catalyzed by an enzyme, given the starting conditions.
Enzyme function may be hampered by the addition of molecules or ions called inhibitors. Many drugs work by inhibiting (slowing or stopping) various enzymes. Inhibitors function by forming an enzyme-inhibitor complex, which impedes the ability of the enzyme to convert substrate to product.
Inhibitors can be reversible or irreversible. Irreversible inhibitors are tightly bound to the enzyme, often through covalent bonds. Hence, the enzyme-inhibitor complex does not dissociate (or it dissociates very slowly). Reversible inhibitors bind through electrostatic interactions (e.g., dipole-dipole interactions). These weaker interactions form an enzyme-inhibitor complex that dissociates very quickly. Reversible inhibitors are generally classified as competitive or noncompetitive. (Irreversible inhibitors do not dissociate; therefore, they cannot be classified as competitive or noncompetitive.)
A competitive inhibitor often binds to an enzyme's active site (i.e., competes with the normal substrate for the enzyme's active site), thus preventing substrate molecules from binding to the enzyme and reacting. Note that both the substrate and the inhibitor bind loosely enough to the enzyme that the enzyme-substrate and enzyme-inhibitor complex dissociate rapidly. In a typical scenario, a single molecule of enzyme collides with a substrate molecule and binds it for a short time. Either the substrate is rapidly converted to product, or it quickly "falls off" the enzyme unchanged. The enzyme will then collide with and bind another molecule, perhaps an inhibitor this time. While the inhibitor is bound, the enzyme does not convert any substrate to product, because the competitive inhibitor and substrate do not bind simultaneously. After a short period, the inhibitor dissociates, and another collision between the enzyme and either substrate or inhibitor results in a new binding event. Note that competitive inhibitors prevent the substrate from binding.
Noncompetitive inhibitors usually bind to a different site on the enzyme (i.e., not the enzyme's active site for the normal substrate). When a noncompetitive inhibitor binds to an enzyme, the shape of the enzyme may be changed. This changes the mechanism of the enzyme reaction and slows the rate at which the substrate is converted to product. Note that the presence of a noncompetitive inhibitor does not completely prevent the substrate from binding, it just slows down the rate at which the substrate is converted to product.
The most common method of determining whether a reversible inhibitor is competitive or noncompetitive is to see what effect the inhibitor has on the rate at which product is formed using different concentrations of the substrate. Recall, the enzyme randomly binds either a competitive inhibitor or substrate molecule, but not both, depending on which it collides with first. A competitive inhibitor will be more effective at low substrate concentrations because the enzyme is more likely to collide with and bind the inhibitor if there are fewer substrate molecules in the vicinity. A noncompetitive inhibitor will be equally effective at low and high substrate concentrations. Recall, noncompetitive inhibitors bind along with substrate, not instead of it. Hence, adding more substrate does not influence whether the enzyme-inhibitor complex forms. In other words, to determine whether an inhibitor is competitive or noncompetitive, one compares the measured value of Km in the presence and absence of inhibitor. For a competitive inhibitor, the measured value of Km increases in the presence of inhibitor. For a noncompetitive inhibitor, the measured value of Km remains the same. As will be discussed below, the most successful treatments for HIV have resulted from using inhibitors for the two enzymes reverse-transcriptase and protease.
Questions on Enzymes as Biological Catalysts
Enzyme-Targeting Drugs to Fight HIV
We have described the role of two critical enzymes, reverse transcriptase and protease, in HIV's ability to infect helper T cells, make copies of itself, and ultimately destroy the cells, depleting the body's immune system. We have also seen how enzymes catalyze biochemical reactions. Now, we turn our focus to two of the most successful treatments for HIV, reverse-transcriptase inhibitors and protease inhibitors. As their names imply, these drugs inhibit enzymes that are necessary for HIV to replicate itself inside helper T cells and deplete the body's immune system.
Reverse-Transcriptase Inhibitors: AZT
Recall from Figure 3, above, that reverse transcriptase catalyzes the formation of a DNA copy of the virus's RNA genetic information. Without the reverse transcriptase enzyme, the virus HIV cannot make DNA copies of its genetic material (i.e., the RNA), because the activation energy for the uncatalyzed reaction is too large. The DNA copy is essential for the virus to take over the infected cell's machinery and produce new copies of itself. Therefore, reverse transcriptase has long been an obvious target in the scientific battle against HIV and AIDS.
How does reverse transcriptase lower the activation energy to enable the virus to make the DNA copy of its RNA? The reverse-transcriptase enzyme (Figure 6) is a protein consisting of two peptide subunits (amino-acid chains). At the interface of the two subunits is an active site where a strand of viral RNA, together with the newly-emerging strand of DNA formed from the RNA template, can fit. A portion of the larger subunit functions as a ribonuclease, digesting the RNA once the DNA copy has been made. Except for the ribonuclease region, the two subunits are identical.
To more fully understand how reverse transcriptase enables the virus to make DNA copies of its RNA, we must know something about the structure of DNA, and the mechanism by which a strand of DNA is generated. The concepts are described in the blue box, below.
How does reverse transcriptase catalyze the formation of a DNA copy of HIV's RNA genetic material (the reaction described in the blue box, above)? To synthesize DNA, reverse transcriptase first positions the RNA strand (called the "template") into the active site, together with an additional, special piece of viral RNA that serves as the primer, or first piece, of the new strand. The primer RNA lines up opposite the template RNA strand, forming hydrogen bonds between the complementary purines and pyrimidines, as shown in Figure 8, above. Then, reverse transcriptase positions a new nucleotide such that a covalent bond can form between the 3' -OH group of the last nucleotide in the primer and the phosphate group of the new nucleotide (see Figures 7 and 8). Reverse transcriptase lowers the activation energy for this reaction by bringing the molecules in close proximity to one another in the active site.
It is possible to inhibit the action of reverse transcriptase using drugs. Currently, six drugs that act as inhibitors of reverse transcriptase are on the market for treating HIV-infected patients. Zidovudine, or AZT, is one of the earliest and best known of these drugs; it is marketed under the brand name Retrovir. AZT (Figure 9) functions as an analog for thymidine (Figure 10), one of the nucleotide building blocks of DNA. This means that AZT has the same shape as thymidine, and therefore it can be incorporated into the developing nucleic acid in place of a thymidine molecule. The phosphate group attached to thymidine or AZT forms a bond with the 3' -OH group of the preceding nucleotide in the developing DNA chain. When thymidine is incorporated into the DNA chain, its 3' -OH becomes the binding site for the next nucleotide's phosphate group. However, AZT lacks the -OH functional group that is necessary to form a bond with the next nucleotide; in its place is an azido (-N3) group. Because the azido group cannot form a bond with a phosphate group, no additional nucleotides can be added once AZT is incorporated into the DNA chain. Hence, reverse transcription stops after AZT is incorporated.
A major problem with AZT is that the HIV virus quickly mutates, and strains that are resistant to the drug may arise in patients who have been taking AZT for extended periods of time. One strategy that doctors use to get around this problem is "multiple-drug therapy." AZT is administered in combination with other reverse transcriptase inhibitors or, increasingly, with one or more protease inhibitors (see next section below). Thus, mutants that evolve with resistance to any one of the drugs are still likely to be killed by the other drugs in the therapeutic regimen.
Protease Inhibitors: A New Line of Attack
In December 1995, the FDA approved a new type of drug for combating HIV. This class of drug acts by inhibiting protease, the enzyme required by HIV to cut its protein into the proper segments to assemble new viral particles. Protease inhibitors, used in combination with two reverse transcriptase inhibitors, have proven to be quite successful. In 80 to 90 percent of patients, this combination treatment reduces the amount of HIV in the blood to an undetectable level.
As shown in Figure 3, the proteins produced from HIV's genetic material are larger than the proteins needed to form new viral particles. In fact, a large "polyprotein" is formed that contains several viral proteins joined together. The polyprotein must be cleaved into the individual functional proteins. This cleavage is catalyzed by protease (Figures 11 and 12). (Cells also contain proteases of their own; the protease described in this section, however, refers to the protease that is made from HIV's genetic information and is used to cleave HIV proteins.) This enzyme is a symmetric homodimer, or a protein consisting of two identical peptide subunits. Like the active site of reverse transcriptase, the protease active site lies at the interface of its two subunits. The mechanism by which protease cleaves the HIV polyprotein will not be discussed in this tutorial. Protease inhibitors reversibly bind to the protease enzyme and, while bound, prevent the enzyme from cutting the viral protein molecules down to their proper sizes.
Questions on Enzyme-Targeting Drugs to Fight HIV
Thymidine Kinase + Substrate + Phosphate ---> Thymidine Kinase + Substrate-Monophosphate
where Substrate = Thymidine, ADRT, or AZT
To reproduce, HIV infects an immune cell called the helper T cell. (T cells help control the body's response to many types of infections.) The HIV then uses the machinery of the helper T cell to make copies of the HIV virus. The major reproductive steps in HIV's infection of the helper T cell are (1) attaching to the cell, (2) injecting RNA into the cell, (3) making a DNA copy of the genetic information contained in HIV's RNA, (4) incorporating the viral DNA into the cell's chromosomes, (5) replication of the viral DNA using the cell's machinery, (6) producing proteins from the viral genetic information, using the cells machinery, (7) cutting the proteins to proper size, (8) assembling new viral particles, and (9) bursting out of the host cell to continue the infection.
As discussed in the tutorial, most of these steps are accomplished by using enzymes, which, in biological systems, are proteins that catalyze reactions. Enzymes form an enzyme-substrate complex with the normal reaction substrates. The formation of the enzyme-substrate complex helps to lower the activation energy of the reaction. The treatment to help HIV-infected people has been based on developing drugs that inhibit these major reproductive steps. Inhibitors are drugs that work by forming an enzyme-inhibitor complex, which impedes the ability of the enzyme-substrate complex to form. The most successful treatments for HIV have resulted from using inhibitors for the two enzymes reverse transcriptase (step 3 above) and protease (step 7 above). These two types of inhibitors are described in the tutorial.
Since the discovery of HIV virus in 1984, much research has been conducted and resulted in an increased understanding of the virus, and the development of drugs that have been successful in the treatment (but not cure) of HIV. However, more research is needed in order to effectively combat the global epidemic of HIV. Research is continuing to develop new drug treatments for the disease. In addition to the inhibitors described in the tutorial, current research is focusing on inhibitors that will block the attachment of HIV to helper T cells (step 1 above), the integration of HIV's DNA into the cell's genetic material (step 4 above), and the assembly of new viral particles (step 8 above). An inhibitor for the enzyme integrase (used to incorporate viral DNA into the cell's chromosomes in step 4 above) has been developed and is currently in clinical trials. The development of the other new inhibitors is still in its infancy.
Of course, researchers would ultimately like to find a cure and a vaccine for the virus, rather than rely forever on treatments that only limit the extent of the infection. Researchers are focusing on the attachment of HIV (in step 1) for developing vaccines, and many researchers are hopeful that an effective vaccine will be found within the next ten years.
To view the molecules interactively, please use Jmol. To download the pdb files for viewing and rotating the molecules shown above, please click on the appropriate name below. (Note: if you want to view multiple molecules simultaneously, please download each file (.pdb) and save on your computer. Then open Jmol outside of your browser.)
Chen, M.S. et al. "Metabolism of 4'-azidothymidine," (1992) J. Biol. Chem., 267, 257-260.
"Good news about AIDS," National Institute for Science Education. The Why Files. 26 Mar. 1998.
Guex, N. and Peitsch, M.C. Electrophoresis, 1997, 18, 2714-2723.
(SwissPDB Viewer) URL: http://www.expasy.ch/spdbv/mainpage.htm.
"La zidovudina (AZT, ZDV, Retrovir)," National AIDS Treatment Information Project. 1 June 1998. URL: http://www.kff.org/hivaids/index.cfm.
Persistence of Vision Ray Tracer (POV-Ray). URL: http://www.povray.org.
Stryer, Lupert. Biochemistry. 4th ed., W.H. Freeman and Co., New York, 1995, 229-230, 229-230, 835.
"Adults and children estimated to be living with HIV/AIDS as of end of 1997," UNAIDS 20 July 1999.
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Weber, I.T. et al. "Molecular modeling of the HIV-1 protease and its substrate binding site," (1989)Science, 243, 928. PDB coordinates as "HIV-1 protease complex with substrate (theoretical model)," Brookhaven Protein Data Bank.
Yamazaki, T. et al. "Three-dimensional solution structure of the HIV-1 protease complexed with DMP323, a novel cyclic urea-type inhibitor, determined by nuclear magnetic resonance spectroscopy," (1996) To be published. PDB coordinates as "HIV-1 protease-DMP323 complex in solution, NMR minimized average structure," Brookhaven Protein Data Bank.
The authors thank Dewey Holten, Michelle Gilbertson, and Jody Proctor for many helpful suggestions in the writing of this tutorial.
The development of this tutorial was supported by a grant from the Howard Hughes Medical Institute, through the Undergraduate Biological Sciences Education program, Grant HHMI# 71192-502004 to Washington University.
Copyright 1998, Washington University, All Rights Reserved.
Revised November, 2011.