Scientists are exploring treatments and techniques aimed at blocking HIV replication by interfering with HIV proteins other than reverse transcriptase, protease and the envelope proteins. HIVs remaining proteins fall into two categories.

Firstly, there are the regulatory proteins Tat, Rev, Vpr (Vpx for HIV-2) and Nef. These proteins are produced by their corresponding genes (tat, rev, vpr and nef) and regulate the speed and efficiency with which HIV reproduces itself.

Secondly, there are the accessory proteins Vpu and Vif. At first these were thought not to be essential to the replication of HIV because even when their genes are removed from strains of HIV in the laboratory, the virus can still flourish in the test tube. However recent research has shown that these proteins do play an important, if not essential, role in the HIV life-cycle within the body. Scientists are just now beginning to understand the subtle and interconnected functions that they serve in the replication and survival of HIV within infected cells.

Integrase inhibitors

Integrase is essential for the integration of HIV's DNA into the human cell nucleus. Integration happens in two stages. The viral DNA is first prepared for integration, before it is transferred into the human cell's DNA. Drugs are being investigated which might inhibit the activity of integrase. Although early investigations focusing on the first stage of integration had disappointing results, more promising results have been shown with later studies.

Merck Pharmaceuticals has commenced human studies with a new agent called L-870,810, which acts at the second stage of the integration process. Developed from a class of compounds called diketones, L-870,810 has achieved adequate concentrations in animal studies and shown promising antiviral activity (Young 2002). L-870812, a second Merck product, has been tested in rhesus macaques. Animals treated ten days after infection with simian-HIV showed stabilisation of CD4 cell counts and ten- to 100-fold reductions in viral load (Hazuda 2004).

A new class of integrase inhibitors called pyranodipyrimidines has also been discovered. A compound dubbed V-165 has shown the greatest antiviral activity in test-tube studies to date (Debyser 2003). Resistance studies suggest that V-165 will be active against virus which has developed resistance to the diketone drugs (Fikkert 2003).

The first integrase inhibitor to be studied in humans was called AR-177, but researchers found that the primary mode of action of this drug appeared to be inhibition of viral attachment or fusion, not inhibition of integration of proviral DNA in the host genome.

Other integrase inhibitors currently under investigation are dicaffeoylquinic acids (DCQAs) isolated from medicinal plants from Bolivia. Test-tube research suggests that these compounds inhibit HIV's integrase enzyme but do not interfere with human cellular processes. However, these drugs only appear to have an anti-HIV effect when added to cells at the same time as the virus.

Merck is currently investigating a range of compounds that may prove to be effective integrase inhibitors in human studies, although none of these have yet completed test tube investigation. Gilead Sciences are also developing an integrase inhibitor called GS 9137. The development rights to this drug were purchased from Japan Tobacco in March 2005 and phase I / II trials started in June 2005.

Another group of researchers has announced that a human enzyme, DNA-dependent protein kinase (DNA-PK), may play an important role in the process of HIV replication. DNA-PK has the job of repairing damaged DNA. It appears that DNA-PK may be activated during the process of HIV replication, and it may facilitate the insertion of viral genetic material into the human DNA. Cells deficient in DNA-PK produce little viral DNA; instead they experience programmed cell death (Daniel 1999).

Several natural substances have also been proposed as integrase inhibitors of HIV: curcumin (from turmeric), caffeic acid-based chemicals, L-chicoric acid, and a number of fungal metabolites although there is no evidence of clinical benefit. Curcumin and its derivates have also been shown to block the activity of the HIV Tat protein in test-tube studies (Barthelemy 1998).

Tat inhibitors

Tat appears to be an important factor affecting whether or not HIV that has infected a cell becomes activated and begins to produce new viral particles. In test-tube studies, HIV-infected cells remain inactive and do not infect other cells if the tat gene is removed or inhibited. Tat is now known to be necessary for the production of new viral proteins from the virus' DNA that is incorporated into the human cell's genetic material. One line of research has therefore been to develop drugs to inhibit the Tat protein.

Only one Tat inhibitor, called Ro 24-7429, has progressed into human trials, but was abandoned after it showed little signs of efficacy and had toxic side-effects. The problem with this particular drug was its poor absorption into HIV-infected cells. Enthusiasm for Tat inhibitors in general has waned somewhat in the light of evidence suggesting that in some circumstances HIV may still be able to replicate efficiently even if Tat is inhibited.

More recently, researchers from the University of Michigan have found that a molecule that mimics the RNA strand to which Tat binds may be useful as a repressor of Tat. The HIV-2 Tat activation-response RNA (TAR) decoy inhibits HIV-1 and HIV-2 replication by binding to the Tat protein (Browning 1999).

A recent study has shown that Tat has an additional function of preventing human cells from carrying out RNA interference (RNAi), a primitive antiviral mechanism that targets the genetic material of invading organisms. Inhibiting this action of Tat may allow the human cell's RNAi mechanism to attack HIV and prevent it from replicating (Bennasser 2005). For more information, see Other anti-HIV strategies in Anti-HIV therapy: Ways of attacking HIV.

Nef inhibitors

Nef has been identified as a promising target for inhibition by a number of research groups, because the protein is involved in several processes critical to both virus entry and the budding of packaged virions from the surface of a cell. It is thought that inhibition of this protein or the pathways it governs could attack the very beginning and the very end of the viral life-cycle within a cell.

Nef down-regulates the expression of CD4 receptors on the surface of cells in order to allow virions to bud out from the cell wall more efficiently. If too many CD4 molecules are displayed on the surface of the cell, there is not enough room for the virions to bud out from beneath the cell surface. One study has shown that introducing incomplete CD4 receptors using gene therapy that are resistant to down-regulation by Nef can reduce the infectivity of the virus particles released from the cells (Pham 2005). For more information, see Inhibiting cellular factors required for HIV replication in Anti-HIV therapy: Ways of attacking HIV.

Nef also down-regulates the expression of major histocompatibility complex 1 (MHC-1) molecules. These molecules present viral antigens to the immune system so that infected cells may be cleared by natural killer (NK) cells. Down-regulation of MHC-1 may be partially responsible for maintaining the reservoir HIV-infected cells, by preventing their elimination by NK cells. However, NK cells also eliminate cells with no MHC-1 whatsoever on the surface, so cells will only escape elimination if they display very limited amounts of MHC-1 on the surface.

One strategy suggested in relation to Nef inhibition is to developing a compound which will stimulate a major up-regulation of MHC-1 expression, in order to encourage a much more potent immune response, while at the same time also encouraging neutralising antibody responses which can control viremia without the need for further or continuous treatment (Trono 2001). This form of therapy would probably need to be administered after viral replication had already been brought under control by other drugs, and might be the precursor to a treatment interruption to test the potency of natural killer responses.

Nef has also been linked to a decrease in CD4 T-cells. The protein somehow inhibits the production of these cells, possibly by increasing levels of cytokines including interferon gamma. In test-tube studies, blocking Nef with antibodies halts the destruction of cells that carry the CD4 receptor, notably T-helper cells. Nef also seems to accelerate viral production within already infected CD4 cells. It is therefore thought that Nef inhibitors could increase CD4 cell counts and reduce viral loads.

Nef also plays a role in HIV replication through its expression on the surface of HIV-infected macrophages. Nef induces the production of two inflammatory chemokines called MIP-1alpha and MIP-1beta. Test-tube experiments found that Nef-expressing macrophages could induce movement and activation of resting T-cells, leading to productive T-cell HIV infection (Swingler 1999).

Other regulatory proteins

Another important regulatory protein is Rev. One of its chief functions is to shuttle DNA from the outer part of the cell into the nucleus and then later back out again where it can be transcribed back into RNA for packaging into new virus. American researchers have developed a group of compounds known as guanidinoglycosides which target Rev. Preliminary laboratory studies show that these compounds are highly potent against HIV (Nathan 2000). Further trials are underway.

Vpr, another regulatory protein necessary for the spreading of HIV throughout the immune system seems to have no effect on viral reproduction within CD4 T-cells, but it does appear to be essential in viral reproduction within other HIV-infected cells such as macrophages. It may also play a role in the neurological damage associated with AIDS. United States scientists have found that glucocorticoid antagonists, such as mifepristone and mifepristone analogues, prevent Vpr-induced virus replication in the test tube, and further trials are planned (Schafer 2004).

Vpu is primarily associated with the final release of newly created virus from the host cell. Vpu seems to stimulate the proper release of these new viruses so that they can infect new cells. This occurs, at least in part, through down-regulation of CD4 receptors, in a similar manner to Nef. For more information, see Inhibiting cellular factors required for HIV replication in Anti-HIV therapy: Ways of attacking HIV.

Vif is clearly an important component of effective viral infection of HIV. Viral strains developed without Vif in the laboratory were 1000 times less infectious. It is not entirely clear why this is so, but the most current research indicates that Vif is essential for the proper packaging of the new virus within the host cell prior to release. HIV strains that do not have Vif seem to lack the necessary elements to infect new cells. Some studies of long-term survivors have suggested that being infected with Vif-deficient HIV strains may be one factor that reduces the risk of developing AIDS. Research into Vif inhibitors is ongoing.

Zinc finger inhibitors

Another potential target is HIVs zinc fingers. These are chains of amino acids found in HIVs nucleocapsid, a protein found in the virus's core. They are involved in binding and packaging viral RNA into new virus particles budding from an infected cell, and may also play a role in the process of reverse transcription.

Experiments have shown that viruses without zinc fingers are unable to infect new cells, and that any new virus particles that they produce do not incorporate the viral RNA and are thus non-infectious and dysfunctional. Potential drugs that might inhibit zinc fingers are in development.

Azodicarbonamide (ADA) is a zinc finger inhibitor. One study has shown that ADA treatment in a group of 15 individuals with advanced HIV disease and virological failure on highly active antiretroviral therapy (HAART) was associated with a significant viral load reduction when added to failing therapy. Although the study was not specifically designed to test efficacy, and treated a very small number of patients, a clear relationship between introduction and withdrawal of ADA and the suppression and subsequent rebound of viral load was demonstrated (Goebel 2000)

This was confirmed in a subsequent open label stage of the study in which viral load fell by between 0.5 and 1.2log₁₀ in three of five individuals who received ADA, and viral load rebounded when ADA was ceased. CD4 cell count improvements were also noted in the open label phase of the study, with an average rise of 120 cells/mm³ after three months.

A number of drawbacks have been identified with ADA including kidney pain and urine abnormalities as well as glucose intolerance and increases in fasting glucose levels. ADA needs to be re-formulated to improve absorption, so that lower doses may be given, thereby reducing the risk of some of these side effects. ADA is being developed by a Belgian company, Hubriphar, in collaboration with the Rega Institute, Leuven.

Maturation inhibitors

The HIV-1 capsid protein is a critical element in the maturation of viral particles, and work is being carried out by the University of Maryland and Achillion Pharmaceuticals to develop compounds that can inhibit the assembly of the capsid protein.

Another approach is to prevent the conversion of the HIV capsid precursor the mature capsid protein. This approach is known as maturation inhibition.

Panacos Pharmaceuticals is developing a derivative of betulinic acid, a cheap by-product of the paper industry, which has been shown to interfere with HIV maturation in the test tube (Li 2003). The drug is called PA-457 and it works by disrupting a late step in Gag processing, warping the shape and activity of the newly produced HIV capsid. Through this process, replication of infectious, healthy HIV is blocked.

Phase I data suggest that PA-457 is well tolerated in healthy volunteers and will be suitable for once-daily dosing. A phase II study in HIV-positive people has shown promising results so far. For more information, see PA-457 in Drugs used by people with HIV: Maturation inhibitors.

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