HIV Replication Cycle, Text The following is a draft only. The HIV Replication Cycle Everyone should have some familiarity with the steps that occur when HIV infects a cell. Knowing this cycle helps to better understand how therapies are intended to interfere with the process of HIV replication. Most of the antiretroviral drugs are aimed at interfering with some aspect of the viral life cycle. The short version is that HIV gets into a cell, hijacks the cell's reproductive machinery, makes baby HIV which then break out. Of course, there are a lot more steps to this process. To review, the steps are: attachment to the cell, fusion, injecting the genetic material (RNA), the RNA goes to the nucleus where it is turned into DNA, it is integrated into the cell's DNA, the cell is activated and produces the proteins needed for a new virion which are then assembled. The new virus buds out of the cell. (The term "life cycle" may be inappropriate. "Life" is perhaps not the best word for a virus. The term "replication" avoids this argument. If viruses indeed are "non-living," they certainly undergo have a great deal of activity and change!) Human immunodeficiency virus (HIV) is a retrovirus, meaning that its genetic material is RNA. "Regular" viruses contain DNA. DNA is found in every cell's nucleus and it makes new proteins. Different types of RNA are used by cells to help DNA make proteins. HIV is shaped like a sphere with studs projecting out. These studs are glycoproteins, a sugar and protein blend, known as gp120 and gp41. There are many other proteins on the surface. Inside, there is a cone-shaped object that contains the HIV-RNA and other proteins like p24 and reverse transcriptase. RNA is the retrovirus's genetic material it uses to make more of itself. There are several genes (each which makes its own protein) that make up HIV, including gag, pol, env, tat, rev, nef, vpr, and vpu. Their activities and functions are summarized in Table 1 at the end of this section. The replication cycle begins when HIV meets with a cell that expresses a "doorway" protein, usually CD4, that is the gateway for getting it inside the cell. HIV then binds to this cell surface receptor. Since HIV's outer coat is very similar to the cell's membrane, the two then wind up fusing. This fusion process is aided (on T cells) by a molecule called fusin. This allows HIV to spew its RNA guts into the cell along with various enzymes. One of these enzymes is the reverse transcriptase enzyme. At this point, the RNA of the virus is transformed into DNA by the reverse transcriptase (RT), in the process called, logically enough, reverse transcription. RT is the target of therapies like the nucleoside analogues (like AZT, ddI, ddC, d4T, 3TC and so forth, also known as "nukes"). These interfere with the virus at this early stage before HIV's genetic material is incorporated into the host cell's genetic material (genome), or in other words, its own DNA. The nucleoside analogue is, in essence, a DNA building block that is faulty and, therefore, stops the reverse transcriptase from producing a complete DNA copy of HIV's genes within a newly infected cell. Without this step, HIV's genes can't be inserted into the cell's own genetic machinery in the cell nucleus. Thus, nucleoside analogues can only work to prevent new (or acute) infection of cells. For a cell that is already infected (chronically infected), the nukes are useless. Many, many other substances have been shown to inhibit reverse transcriptase's activity. Aside from toxicities, a problem with many of these (including the nukes) is that HIV changes (mutates) and new HIV variants emerge that are not affected by these drugs. This is called resistance. The next step is integration when another enzyme, integrase, helps to incorporate the newly-formed HIV DNA into the chromosome of the cell. At this point, the HIV DNA has been incorporated into the host cell's own genetic material. The three-dimensional structure of integrase has been discovered and some integrase inhibitors have been discovered. Among these are 15 derivatives of flavones, chemicals found in many plants (see the Information Sheet on Bioflavonoids). Such integrase inhibitors would stop the virus from inserting its genes into the cell's genes, thus preventing the virus from reproducing. Cells may be then activated by a variety of stimuli, including cytokines like TNF. (See the Immunology Primer section for a discussion of these). When the cell is activated, signals are sent into the cell's nucleus telling it to get busy and produce other proteins, split and create new cells or any of a number of functions. Here's where the hijacking comes in. When the cell gets these signals, it also gives HIV the opportunity to make new HIV (virions). Various protein factors like nuclear factor kappa B (NF-kB) bind to the DNA to help transcription. Unfortunately, they also bind to part of the integrated HIV known as the long terminal repeat (LTR), turning on production of proteins that make new HIV. There are other transcription factors (AP-1, SP1 and TFIID) that also play a role. Such binding may be inhibited by a number of different substances, including antioxidants and n-acetyl-cysteine (NAC). (The Table at the end of this section describes HIV's genes and the protein products). The rate and quantity of production of new HIV-RNA and proteins is influenced by several regulatory genes. One important one is the Long Terminal Repeat (LTR). The LTR acts like an "on/off" switch for transcription of new HIV. This process is aided by other regulatory proteins known as tat, nef, rev and others. These also help in creating new strands of HIV-RNA for the newly forming virions. The first of these is the target of the tat (transactivator of transcription) gene inhibitors and tat protein inhibitors. The tat gene protein is a strong activator of the LTR sequence to which it attaches. A drug made by Hoffmann-La Roche called the tat inhibitor has unfortunately failed to show any benefit in clinical trials. A smaller biotechnology company called Allelix currently has a candidate tat inhibitor. Other natural products available from DAAIR have some test tube evidence of being tat inhibitors. The tat protein is also a critical component for the transcribing of new HIV proteins. The tat binds to an element known as TAR which basically puts HIV protein production on to full throttle. Inhibiting tat is yet another target for intervention, as has been mentioned. Since the cytokine tumor necrosis factor (TNF) is elevated in many people, especially those with wasting, and this elevation in turn induces more HIV production in the body, a number of interventions to lower TNF are under investigation. These include thalidomide (Synovir), pentoxifylline (Trental) and an antihistamine, ketotifen. In addition, there is evidence showing that the amino acid carnitine may reduce elevated levels of TNF back to normal (see the Information Sheet on Carnitine). (In addition, carnitine lowers triglyceride levels which may in turn help to lower TNF levels that are too high). This is an indirect way to simultaneously limit viral replication and correct pathological changes in immune function, since sustained, elevated levels of TNF are like having a high, unchecked fever. A little bit is necessary, but too much can be really bad. Other HIV proteins produced during this activation include internal proteins that will be incorporated into the whole virus's structure. Once the HIV proteins are expressed, they appear as larger units (long, fused-together strings of proteins) that need to be "cut up" into functional subunits by molecular shears called protease enzymes. The protease inhibitors, like Crixivan (Indinavir), Saquinavir (Invirase), Ritonavir (Norvir), Viracept (Nelfinavir) and so forth, interfere with this cleaving process. These subunit proteins form the base material of the virions which are assembled in the cell and on its surface and eventually bud out of the cell. This replication cycle may result in the death of the cell. For many years, most scientists believed that this was how people's T-cell counts dropped to nothing, through the direct influence of HIV infection of the cell, replication and budding. This is known as the cytopathic model. However, it is inadequate to describe the full extent of T-cell depletion, especially since many uninfected CD4 cells are known to die. Many mechanisms have been discussed for this death of uninfected cells. Unfortunately, even though the evidence is strong that the body's own responses may be implicated in the disease process, inadequate attention has been paid to this area of research. Still, there is compelling evidence that an oxidative stress model may play a crucial role in the death of uninfected CD4 cells. This involves a vicious cycle where HIV infection results in stimulation of the immune system with subsequent inflammatory response which causes damage and further activates HIV replication. Understanding the way AIDS develops can have a direct and meaningful impact on the types of therapeutic strategies that will work to best defeat the virus while helping the body not only in its ability to restrain the virus but also to restore balance and health.

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