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(Please note: below is a text file of the Searchlight Spring 2002 "HIV Overview article". It therefore does not contain the figures and illustrations referred to in the text. For a complete copy of the article as it appeared in SEARCHLIGHT, including all images & charts, please download the complete PDF of this issue.)

HIV-1: Two Decades of Clinical and Scientific Advances

by Marjan Hezareh, Ph.D.

The worldwide AIDS pandemic has became not only a major cause of human suffering, but also a major cause of social, economic, and political instability. A look back over the past 20 years since the beginning of the AIDS epidemic shows that we have come a long way in understanding of HIV virus and the host immune response to it. Impressive and considerable progress has been made within each domain of HIV research, bringing us remarkable new anti-viral drugs leading to a dramatic success in fight against HIV/AIDS.

The field of antiretroviral therapy has become more complex due to the emergence of drug resistance and toxicities associated with therapy. Therefore, the need for new treatment strategies is increasing, and strategies to enhance the immune response and either to discontinue or decrease the length of prolonged highly active antiretroviral therapy (HAART) are under investigation. It is worth noting that problems with therapy shouldnÕt outweigh the benefits of treatment, since untreated HIV infection is still a lethal disease. A detailed knowledge of protein structure and their interaction has brought great insight to the field of HIV assembly and entry. As a result of this research, new drugs able to block either early events of virus attachment or the later events leading to membrane fusion are in development.

Research on HIV accessory gene and identification of novel cellular factors has broadened our understanding of the regulation of HIV replication in its host cells. Since these genes are playing an important role in HIV pathogenesis, they might be potential targets for new antiviral therapies.

The following is a short overview, representing a selection of knowledge that has been accumulated within the field of HIV-1 including virology, immunology and HAART. (In this review, we have not covered vaccine development, since prevention and vaccine topics were extensively covered in the last issue of SEARCHLIGHT.)

HIV-1 entry and membrane fusion

HIV, like other retroviruses, enters cells by fusing its envelope with a cellular membrane. This fusion allow the nucleocapsid (inner contents of the virus) to enter the cell cytoplasm. The process of HIV entry is completed through several steps including adhesion, receptor binding and membrane fusion. The envelope of HIV is a lipid bilayer formed by viral and cellular components. Some cellular proteins such as MHC class I, II, cholesterol and intracellular adhesion molecule-1 are very abundant in viral envelope, while other cellular proteins like (CD45) are absent.

Thus budding of virus may occur from a specific area of cellular membrane. (1) The envelope of HIV-1 is composed of two types of glycoproteins (named after their molecular weight), the gp120 (surface subunit, SU) and gp41 (transmembrane subunit, TM) (fig.1). Both proteins are the results of the cleavage of a viral precursor, gp160. The gp41 becomes anchored to cellular or viral membranes and interacts non-covalently with extracellularly expressed gp120 to form the native oligomer. The gp120 surface protein is responsible for a high degree of genetic variability between HIV-1 isolates and also between HIV-1 and HIV-2.

Like other retroviruses, the role of the gp120 is to establish nonspecific contact with target cells and then to engage interaction with receptors (e.g. CD4, CCR5, CXCR4) (fig.1). This interaction leads to a conformational change of the gp120/gp41 complex, unfolding domains of gp41 responsible for fusion of viral and cell membrane and formation of pores between cell membrane and nucleocapsid. The following steps of the viral entry consist on expansion of these pores and translocation of nucleocapsid. Each step in viral entry process represents a potential target for development of antiviral drugs.

Adhesion

The first contact between HIV-1 and target cells is established through non-specific interaction between gp120 and charged groups at the cell surface, specifically sugars of glycolipids and glycoproteins. These interactions are not sufficient for virus entry but are important for the subsequent recognition of the receptors by gp120, either by modification of gp120 conformation or by concentrating virus at the surface of target cells. Indeed some anionic polymers and heparin can inhibit virus entry by blocking this non-specific interaction with sugars. Most probably HIV-1 entry does not occur through endocytosis in vivo, since HIV-1 fusion with cell membrane occurs at neutral pH, in contrast to influenza virus for which hemaglutinin is activated by low pH (requirement for transit to late endosome)(2).

Receptor Binding

In 1983 the CD4 molecule was identified as a receptor for HIV-16. However, the involvement of the CD4 receptor in HIV infection could not explain the fact that the presence of CD4 was not always necessary for HIV-1 infection and that CD4 alone is not sufficient for infection of macrophages or mouse cells. Feng and colleagues in 1996 discovered that chemokine receptors are also involved in HIV entry process. Over the past years several studies have established that the presence of chemokine receptors on the cell surface are necessary for HIV infection (and are also necessary for infection with related retroviruses), while the presence of CD4 is not always required (7).

The only chemokine receptors playing a role in HIV-1 infection in vivo are CXCR4 and CCR5; even though other receptors can permit laboratory infection. Interaction between gp120 and chemokine receptor was subject to extensive studies. In brief, residues belonging to conserved region of gp120 as well as a cavity situated close to the base of V3 and V1/V2 loop of gp120 constitute a chemokine binding site. The region of chemokine receptors apparently involve in interaction with gp120 are the amino-terminal domain and the second extracellular loop (ECL2)9 (fig.1). Binding of gp120 to CD4 receptor induces a conformational change in gp120, unmasking the binding site for chemokine receptors at the base of V3 loop and the conserved region. Interaction with the chemokine receptor allows close proximity between virus and cell membrane, and further conformational change in the gp120/gp41 complex. The latter allows exposure of fusion peptide and its insertion into the cell membrane (fig.1).

Footnote: Recently, interest in understanding the step of cell adhesion was increased by the finding that dendritic cells (DC, antigen-presenting cells in skin and mucosa) can enhance HIV infection by capturing and retaining infectious HIV-1 for up to 4 days. They are capable of presenting these virions to appropriate T cells and subsequently enhance infection. Apparently, the interaction between DCs and HIV-1 occurs through specific protein present at the surface of some DC cells called DC-SIGN (mannose-binding C-type lectin domain of a type II membrane protein). Several questions such as reasons for virion stability at the surface or in intracellular compartment have to be elucidated. If scientists prove the importance of dendritic cells in the establishment of HIV infection, DC-SIGN may be a potential target for antiviral development (3-5).

Viral tropism is defined by the ability of a virus to interact with CXCR4 (X4 virus) or CCR5 (R5 virus) or both (R5X4 virus) [10]. In vivo, X4 and R5X4 virus emerge at later stages of disease, while R5 virus can be isolated at any stage. This may be because at later stages a weak immune system allows for the selection of viruses that can infect a larger spectrum of cells, such as resting T cells and CD4 negative cell11,12. However it is still unclear why HIV-1 has selected CXCR4 and CCR5 as cellular receptors or why the X4 virus is selected only at the later stage of disease.

The natural and synthetic ligands for CCR5 (MIP-1, MIP-1, RANTES) and CXCR4 (SDF-1) receptors can be envisioned as a potential antiviral drugs, since they are able to block HIV infection through steric hindrance and stimulation of receptor endocytosis. However, the costs of production of these ligands are very high and their bioviability very limited. Several synthetic inhibitors are under investigation including TAK-779 (Takeda) antagonist and Sch-C and Sch-D compounds (Schering-Plough). One potential problem with targeting only CCR5 receptors in an antiviral approach is that we may select for more pathogenic X4 variants, so a strategy targeting both receptors simultaneously might be safer (13).

Fusion

Important progress has been made in the understanding of gp41 structure, but we still have limited knowledge of the fusion process. The extracellular part of gp41 is formed from the association of two helix-forming domains called proximal (P) and distal (D) (fig. 2.), relative to amino terminal of the protein. The P helixes of three gp41 monomers are associated through hydrophobic interaction and form the "leucine zipper". The leucine zipper is a stable coiled-coil structure around, which is packed three D helixes in an anti-parallel orientation. The hydrophobic amino-terminal parts of each gp41 monomer are called fusion peptides and must point toward the viral membrane. The region between P and D helixes forms a loop that is in contact with gp120. This loop is characterized by a di-cysteine motif with a disulfide bridge, which seems to be involved in interaction with gp120 and is an immunodominant epitope of gp41 (fig.2).

Two models have been proposed for unmasking of fusion peptide and their insertion into cell membrane. The first one suggests the formation of a transient, pre-hairpin structure, in which the six helixes are dissociated letting the fusion peptide to come in contact with target cells membrane. The major argument in favor of this model was the unmasking of epitopes in the P and D helixes and the antiviral activity of the peptide derived from these domains such as T20. However, evidence demonstrated that a peptide from D helixes was active on a step posterior to lipid mixing, suggesting that the peptide acts on native gp41 and not on pre-hairpin structure.(14,15)

Footnote: Chemokines are member of the family of receptors with seven membrane-spanning domains, and are involved in signal transduction through coupling to heterodimeric G proteins. They are involved in the activation of leukocytes chemotaxis and are classified as CC or CXC based on the relative position of conserved cysteines in their amino-terminal region (8).

Therefore a second model was proposed in which a simple tilting of the gp41 axis is required so that the fusion peptide can access cell membrane. As mentioned before, a major argument in favor of this model is the mode of action of peptides such as T20.16 Another more efficient peptide in investigation, T1249 (Trimeris) acts on HIV-1, HIV-2 and SIV. By binding to gp41, these peptides could prevent the local recruitment of gp41, probably necessary to complete membrane fusion. It is clear that a better understanding of the role of gp41 in the membrane fusion process will allow us to define new targets for development of antiviral compounds.

HIV Accessory Proteins

Like all lentiviruses, HIV contains three structural genes (gag, env, pol) essential for viral expression. In addition to these structural genes, HIV contains six accessory genes not required for viral replication but which play an important role in viral pathogenesis. These genes encode for accessory proteins such as Nef, Vpr, Vpu and Vif. Because of their importance in promoting the clinical manifestations of HIV disease, they might be potential targets for new antiviral therapies. To this end, extensive basic research investigation has been focused to gain a better understanding of their mode of action. In this review we highlight important new information that have been emerged into how this class of HIV proteins function.

Vpr

The Vpr protein is present in viral particles and is implicated in multiple functions. The Vpr protein causes induction of cell-cycle arrest, transactivation of viral and cellular gene expression, participation in nuclear import of the HIV genome, and induction of apoptosis. Recent studies demonstrated that some of these activities can be separated through mutagenesis and are not the consequence of single activity of the protein.(17,18) It has been shown that Vpr can infect non-dividing cells by facilitating the nuclear localization of the pre-integration complex. Subsequent studies demonstrated that Vpr contains 2 sequences each mediating nuclear localization. The two nuclear localization signal (NLS) functions through distinct pathways. One is thought to act as a nucleocytoplasmic transport factor, since it is found that Vpr is associated with the nuclear pore when expressed in cells and can bind to nuclear pore complex. Furthermore, the presence of nuclear export signal, in addition to NLS suggests that Vpr can shuttle between the nuclear and cytoplasmic compartment. However, the exact reason for this is yet to be identified.

Vpu

Vpu is an integral membrane phosphoprotein, localized in the internal membrane of the cells. Vpu is implicated in down-regulation of CD4 expression and the enhancement of virion release. Both functions can be separated genetically. Simultaneous expression of env and CD4 in infected cells leads to formation of a complex in reticulum endoplasmic that traps both proteins in this compartment, decreasing the amount of envelope protein available for viral assembly. The role of Vpu is to liberate env protein by facilitating degradation of CD4 molecule that is complexed with env. A recent study also shows that Vpu facilitates the virion release from the surface of infected cells by stimulating the formation of ion like channel in lipid bilayer.(19)

Vif

The Vif protein is essential for HIV replication in some cells, including peripheral blood lymphocytes and macrophages. In most cell lines, the presence of Vif is not required. These cell lines are called permissive for Vif mutants. HIV virions produced in permissive cells can infect non-permissive cells but the virus produced subsequently is not infectious. There are two models for the permissive phenotype of Vif; i. non-permissive cell line lack a factor that has a Vif-like function; ii. non-permissive cells contain an anti-viral factor that is blocked by Vif. Several studies revealed that non-permissive cells contain an anti-viral factor that is overcome by Vif.20,21 Furthermore, Vif defective virions can enter cells but cannot synthesize proviral DNA. The infectivity of Vif defective virions can be restored by exposing them to high concentrations of nucleotides that stimulate reverse transcription within virion.(22) Therefore Vif may be involved in a post-entry step essential for the completion of reverse transcription.

Nef

Nef protein is a 27 KDa myristolated protein necessary for the maintenance of high viral load and viral pathogenesis in HIV-infected individuals. HIV Nef protein has multiple activities including activation of T-cells, alteration of intracellular trafficking of cell surface proteins, and enhancement of infectivity. These activities can be separated by their sensitivity to certain mutations.

Through enhancement of T cell activation, Nef provides an optimal environment for viral replication. Interaction with lipid rafts is required for Nef to promote T-cell activation.23 Nef-mediated T cell activation is thought to occur by mimicking the signal via T cell receptor. Nef protein also contributes to HIV pathogenesis by altering intracellular trafficking of cell surface proteins. Indeed Nef is involved in down regulation of CD4, CD28, and MHC class I (for the purpose of immune evasion) through different mechanisms.23-25 Additionally Nef protein is able to stimulate viral infectivity, through uncoating of viral core after fusion.26 An alternative model proposed that Nef stimulate infectivity by increasing the efficiency of viral entry (for review.27 However, more studies are necessary to determine the exact role of this protein in stimulation of HIV infectivity.

Footnote: MHC class I protein is required for the immune system to recognize infected cells. Down-regulation of this protein allows infected cells to be "invisible" to the immune system.

New anti-viral agents and alternative treatment strategies

The continued long-term success of the available HAART is impended by the emergence of drug resistance, the persistence of virus replication in successfully suppressed patients, and toxicities associated with long term use of HAART. Alternative treatment strategies that efficiently suppress and/or eradicate viral replication with minimal side effects and decrease viral reservoirs or enhance immune response to control infection are needed. Here we briefly review some new antiviral agents in development and summarize the latest outcome of alternative strategies under investigation for review. (28,29)

New Reverse Transcriptase Inhibitors

Nucleoside Reverse Transcriptase Inhibitors (NRTI) are nucleoside analogues that act by direct competitive inhibition of HIV reverse transcriptase and also behave as chain terminators by blocking the elongation of nascent DNA. They represent the first class of antiviral drugs tested in human against HIV-1 (e.g. AZT, 3TC). The toxic effects associated with NRTI include bone marrow suppression, anemia, neuropathy and pancreatitis. Furthermore, emergence of drug resistance and transmission of multi-drug resistant virus are barrier to successful prolonged treatment. Table 1. summarizes new NRTIs in various stages of development. (28)

New Protease Inhibitors

HIV-1 produces a small, dimeric protease that specifically cleaves the polyprotein precursors encoding the structure proteins and enzymes of the virus that is necessary for the production of mature, infectious virions. The protease inhibitors (PI) effectively block the protease within HIV-1 infected cells and therefore block virus propagation. The attractive feature of this class of anti-HIV is that they block infection in both acutely and chronically infected cells. The long term use of this class of anti-viral is also associated with emergence of resistance and toxicities such as high cholesterol and tryglicerides. Table 1. summarizes new PIs in various stage of development. (28)

Fusion Inhibitors

As mentioned earlier, fusion of HIV with the cell membrane requires the formation of a hairpin structure between the proximal and distal helixes. T20 peptide, derived from gp41, binds to this region and prevents HIV fusion. T20 displays a short-term antiviral activity when administered intravenous and subcutaneous in infected individuals. T1249 (Trimeris), another peptide under investigation, is a 39 amino acid peptide with anti-viral activity against HIV-1, HIV-2, SIV and T-20 resistant isolates.

More interestingly, it has been shown that the fusion inhibitors may act synergistically with chemokine receptors in vitro. The rational for this activity is that any factor that may increase the recruitment time of co-receptors increases the time that gp41 molecule remain exposed to binding of fusion inhibitors.

Alternative Strategies

Viral eradication is not possible with current HAART therapy, but several alternative strategies are presently under investigation. These strategies are designed to restore or enhance the HIV immune response in both primary infected individuals and in chronically infected people. The concept behind this approach is the observation that long term non-progressors have a high number of HIV specific T-helper cells leading to continuous suppression of HIV replication. In primary infected individuals, early treatment with HAART may preserve the T-helper cells while in chronically infected individuals it may boost the immune response which in turn can control HIV replication in the absence of treatment.

Studies investigating the effects of early treatment in primary HIV infection (before or early after seroconversion), demonstrated that in patients with a preserved HIV specific immune response, viral replication can be suppressed for a prolonged period of time after treatment interruption. In one patient, they also demonstrated an increase in HIV specific T-lymphocytes and an increase in HIV neutralizing antibodies. (28,30-32) However, these are preliminary results and are subject to more investigation.

Footnote: Conflicting results appear in literature concerning the correlation between the frequency of CD8 T cells and plasma viral load in these patients. Studies on 21 long-term non-progressors have not shown any correlation between these two parameters. (33) However, evidence from other studies demonstrated that CD8 T cell response is important in controlling viral replication and that efficient activity of these cells requires the presence of strong CD4-cells.

Several studies demonstrated that in chronically infected individuals receiving HAART, restoration of immune response is slow but existent. The major concern in using structured treatment interruption in these patients is the emergence of resistance virus. Data obtained from clinical trials in chronically infected patients subjected to structured intermittent therapy suggest that restoration of HIV specific immunity in these patients is difficult and supports the concept that early intervention will help to preserve CD4 cells.

Another strategy under investigation is boosting the immune response with HIV immunogens in suppressed patients. Several studies are underway to investigate the effects of various immunogens alone or in combination with IL-2 and/or with treatment interruption. The success of this strategy will depend on finding immunogens, which stimulate a broad range of immune responses against various strain of HIV-1. In one study recombinant canarypox vaccine combined with rpgp160, increased HIV specific neutralizing antibody in all individuals suppressed with HAART during primary infection. HIV specific CD8 responses were increased transiently in 6 out of 14 individuals. (28)

In cytokine-based strategies, investigators are exploring the possibility of boosting HIV specific immune response with IL-2, IL-12, and or interferon.(29) Two large phase III clinical endpoint studies of IL-2 in HIV infected individuals are in progress. The results from these studies may answer the question of the therapeutic role of IL-2 administration in HIV infected patients.

Social and Economic Issues

The success of antiviral treatment in reducing death among people living with HIV should not be minimized. However, governments, scientist and public opinion agree that is no longer acceptable that HIV positive people in developing countries do not have access to drugs. Tremendous amounts of effort have been made in the past 2 years to reduce the cost of drugs. As a consequence of these efforts treatment is within reach of more people.

However, cost is not the only barrier to AIDS treatment in developing countries. A common experience with HAART in these countries shows that patients usually begin HAART when already severely immune-compromised. They are at high risk of opportunistic infections and the diagnosis and the treatments of such infections are difficult, due to the lack of medical infrastructure. Therefore, specific efforts should be made to simplify and adapt the antiviral therapies for developing countries.

Additionally, an estimated additional 5.3 million new infections were reported in the year 2001 alone, demonstrating that the epidemic is not under control and that new HIV prevention strategies are desperately needed. Therefore, we need to continue our efforts to develop effective vaccines and microbicides to prevent the spread of infection. For those 30 million people currently living with HIV, it is important to continue our efforts in price reduction and in helping these countries set up the infrastructure necessary to deliver and monitor the use of antiviral drugs.

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