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  • The Molecular Biology of HIV/AIDS

    The Nature of Viruses

    The biosphere contains organisms that are, like ourselves, based upon cells. However, there are other forms of life around us. Among these are the viruses, defined as a cellular, obligate intracellular parasites.

    I call them “acellular” because during their replication, they do not obey the cell theory. That is viruses do not arise from preexisting viruses by binary fission. I won’t go into the details of a viral replication cycle for the purposes of this course, except to tell you that the world of the viruses is quite a different place from our world.

    And yet, the virus depends completely upon its host cell for its growth, replication and propagation. This is the “obligate intracellular parasite” part of the definition. Because of this viruses obey the same molecular rules that cells do. In fact, they must, in order to grow within the host cell environment.

    Viruses have nucleic acid as genetic material, but, depending upon the virus, it may be RNA or DNA, single or double stranded. In fact, most viruses (by number) have single stranded RNA as their genetic material.

    Among this group are found the retroviruses. We encountered some of these in the last section when we talked about oncogenes. Here again is the general genomic organization of members of this family:

    Recall that some members of this family can cause transformation in cell culture or tumors in certain animals. However, there is another part of the family, the lentiviruses. This group of retroviruses causes a chronic, slow infection.

    HIV: A Different Kind of Retrovirus

    The current pandemic of acquired immune deficiency syndrome (AIDS) is caused by a member of this family, human immunodeficiency virus (HIV). By pandemic we mean that the infection is found literally word wide. Here is a world map showing the status of the infection as documented by the World Health Organization as of the end of 1998:

    For a more current estimate of the extent of this pandemic, check this site maintained by the University of California at San Francisco Medical School.

    HIV is a virus that has leapt from its normal host and has invaded human hosts quite recently. The origin of the virus seems to be chimpanzee populations in West Africa, according to a recent publication (Feng Gao, Paul Sharp and Beatrice Hahn [Nature (1999) 397, 436-441] )

    Consequences of HIV Infection

    To see an electron micrograph at high resolution of an HIV particle, go to this site, maintained by the University of Utah. There you can also see a diagram of the components of the virus particle.

    The genome of HIV is similar to and yet different from other retroviruses, as shown in this figure:

    One very striking difference is the presence of a number of small proteins, all of which play a role in the infectious process.

    Molecular Consequences

    When HIV attaches to its host cell, it does so by virtue of an interaction between the viral protein gp120 and proteins on the surface of the cell. The principle cellular protein is the CD4 molecule, present on the surface of the T-helper cells and certain macrophage in the immune system. There are co-receptors, CCR5 and CXCR4, shown in this figure:

    After the viral RNA genome enters the cytoplasm of the host cell, it is immediately converted into double stranded DNA by reverse transcriptase, an enzyme carried as a part of the virus structure. This DNA enters the nucleus where it is integrated into the host cell genome by a site-specific recombination event, catalyzed by the viral integrase.

    Notice that the regions called the long terminal repeats (LTRs) contain the control sequences for viral gene expression. For instance, on the left, the LTR contains the transcription start signals, the TATA and CAT boxes, as well as enhancer sequences. On the right, the LTR has the polyadenylation signal, AATAAA.

    HIV has its own set of sequences:

    In this case, in addition to the TATA and Sp1 (instead of CAT) sequences, the enhancer sequences (K and B) respond to a particular cellular activator, NK-kB. This protein is produced only under certain circumstances. It turns out that when the host T-helper cell is activated to do its job in the immune system, this protein is made. As a result, when the CD4+ helper cell starts to divide, the virus starts to be expressed and the cell is killed.

    Clinical Consequences

    A patient who is infected with HIV has, at first, a normal immune response to the presence of the virus. The patient produces antibody and within 4 to 10 weeks can be found to be “HIV positive” meaning that these antibodies are present in the circulation. However, as time passes, things begin to change, as shown in this figure:

    As the virus continues to replicate over the years after the initial infection (remember, genomes have become stabily integrated into chromosomes of host cells), the patient begins to loose CD4+ cells. After the level of these cells drops below 200 cells per mm3, the patient is said, according to the Centers for Disease Control and the World Health Organization, to have clinical or “full-blown” AIDS.

    Without an immune system, the AIDS patient is no longer able to fend of infections that are not normally threatening. Opportunistic infections such as cytomegalovirus, pneumocystis pneumonia and cryptosporidium become severe illnesses. A tumor, called Kaposi’s sarcoma, is caused by a herpes virus (HHV-7 or KHV) that is normally innocuous. In addition to this, HIV invades the central nervous system, causing AIDS related dementia.

    If left untreated AIDS has a greater than 95% case fatality rate. This means that more than 95 out of 100 people infected will eventually die from their illness.

    The Mutation Problem

    One major problem in contending with the HIV pandemic is the incredible mutation rate of this virus. Remember that only DNA polymerases have editing and only DNA is repaired. It turns out that a combination of the reverse transcriptase and the fact the new genomes are made by RNA polymerase means that this virus is very error prone. In fact, HIV has the highest mutation rate.

    During the replication of the virus, about one error is made in every 105 to 104 bases. Since this is about the size of the genome, this means that one error is made, on average, every time a genome is replicated.

    John Coffin at Tufts Medical School has estimated the rate of replication of HIV during the lifetime of an infected patient (“HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy,” J. M. Coffin, Science (1995), 267, 483-489). He estimates that about 109 new cells are infected every day. Given the replication rates of the virus in his model, this means that, on average, every possible point mutation in the viral genome occurs in the patient about 104 to 105 times each day. There are two consequences to this that he mentions:

    • Over a 10 year clinical latency period, the virus has gone through more than 3000 generations
    • Virus transmitted by an infected person to an uninfected person is, on average, 1000 generations removed from the virus causing the initial infection

    The implications of this mutation rate of of this continually changing population of virus particles will become apparent when we talk about treatment.

    Treatment and Prevention

    Vaccine

    As soon as it was realized that AIDS was caused by a virus, the race began to produce a vaccine. Remember that if a vaccine were available tomorrow and distributed world wide immediately, it would do nothing for the millions already infected. Vaccines are prophylactic, not therapeutic. Nonetheless, there are greater problems. The mutation rate means that the virus is able to escape the immune system by simply allowing for the selection of immunological variants, which it does quite successfully. This has been the biggest issue in the development of any kind of vaccine therapy to prevent the spread of AIDS.

    Inhibitors

    The first chemotherapeutic inhibitor of HIV was AZT (Zidovudine) shown here along with another similar inhibitor, dideoxycytidine (ddC):

    Both of these act in the same way: they are chain terminating inhibitors of DNA replication. Notice that in each case there is no 3′ hydroxyl. As a result, when these analogues are phosphorylated to triphosphates, reverse transcriptase will incorporate them in place of T (in the case of AZT) or C (in the case of ddc). This results in a termination of chain growth since there is no 3′ end on which to add the next base.

    An excellent strategy with two drawbacks. First, the inhibitor also works on the host cell DNA polymerase. Therefore, the dosage has to be carefully adjusted to account for the toxicity. More importantly, the problem of the mutation rate comes to play again. The presence of the drug selects for AZT-resistant mutants at a high rate.

    The Three Drug Cocktail

    The most recent advance has been the derivation of an inhibitor that works against the viral protease. When HIV is maturing (when new virus particles are being made) a series of proteolytic cleavages are necessary to form the final structure. Inhibitors of this viral protease were developed. The idea then took hold that, rather than giving this alone, it would be given in combination with other inhibitors. The plan is to make it much less likely that resistant mutants to all three drugs would develop.

    Therefore, the three-drug cocktail was developed. Here is a link to a Web paper that describes this therapy. Patients are given a combination of a protease inhibitor (Invirase and Saquinovir are two of the trade names) and two reverse transcriptase inhibitors, usually AZT and ddc or dideoxyinosine (ddI).

    The results so far with this have been astounding. Patients have been returned from the brink of death. Viral loads in near terminal patients have dropped to manageable levels and their immune systems have rebounded.

    The news is not all good, however. First, the regimen for taking this cocktail is extreme: many pills a day, some with meals, some between meals. No pills can be missed, since the mutation rate of the virus means the chance of resistant mutants rises dramatically. In addition, the cocktail does not eliminate the virus and therefore AIDS patients must stay on this for life. The cost is typically $10,000 to $20,000 per year for the medications. Finally, recent evidence points to the existence of HIV mutants that are resistant to all three in some patients.

    The high cost of the medication means that HIV infected people in third world countries will have little hope of every seeing this cocktail. In my opinion this is a sad state of affairs for the world in that entire populations in Africa and Southeast Asia will be lost to this pandemic.

    Prevention

    AIDS is preventable. It is a disease of choices for most patients. Except for those unfortunate individuals infected by laboratory accidents or by contaminated blood (no longer a real danger), AIDS is acquired by either unprotected sexual behavior or sharing of needles during drug use. We live in an age where choice is a very important thing.

    The term “safe sex” has been popularized. Realize that there is no such thing as “safe” sex — there is only “safer” sex. That is, every sexual partner whose history is unknown to you is a potential risk. That risk is lowered by using protection (e.g., condoms) but I emphasize that the risk is lowered, not eliminated. There are only two perfectly safe sexual practices: abstinence or solitary masturbation. Other than that, everything carries an inherent risk in this time of pandemic.

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