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Biomedical Science The AIDS Virus What We Know and What We Can Do About It FLOSSIE WONG-STAAL, PhD, La Jolla, California
Presented at the annual meeting of the Western Association of Physicians, Cannel, California, February 6-7, 1991.
nfectious diseases have long been the scourge ofhumanity. In the past few decades, tremendous medical advances have been made, leading to a complacency that mortality from infectious diseases is no longer a major concern. That optimism was shown to be premature, however, with the appearance of the acquired immunodeficiency syndrome (AIDS) as the epidemic of the century. In certain areas of the world, AIDS is literally wiping out entire populations. The causative agent of AIDS, the human immunodeficiency virus (HIV), is a retrovirus. It is not the first retrovirus shown to cause disease in humans. Several years before, the first human retrovirus to be isolated was identified as the causative agent of an aggressive leukemia called adult T-cell leukemia. To date, the four human retroviruses that have been isolated and characterized all share one major feature, the propensity to infect T cells. For this reason, they have sometimes been referred to as human T-lymphotropic viruses (HTLVs). These viruses also fall neatly into two subgroups: the leukemia viruses, HTLV-I and HTLV-II, and the immunodeficiency viruses, HIV type 1 and HIV type 2. The prototype AIDS virus, HIV- 1, probably originated in Central and East Africa, but it is now found in most of the world. Also associated with AIDS, HIV-2 is still largely confined to countries of western Africa. This presentation will pertain predominantly to HIV-1, although much of it should also be relevant to HIV-2. There may be interesting parallels to be drawn from and to the leukemia viruses as well. I
Pathophysiology of the Acquired Immunodeficiency Syndrome The clinical spectrum of HIV infection is broad, affecting as it does almost all parts of the human body. The resulting symptoms reflect three major manifestations: * The immune system is profoundly impaired. As a result, a variety of agents are able to produce opportunistic infections. * The human immunodeficiency virus also infects the brain, resulting in central nervous system disorders, includ-
ing dementia. *
sia)
Hyperproliferative disorders (hyperplasia and neoplaincreased in incidence, the most notable example
are
being Kaposi's sarcoma, the incidence of which is increased about 1,000-fold in certain HIV-infected populations. The role of HIV in associated neoplasias is probably indirect as HIV does not infect and transform these tumor cells. Indirect mechanisms, such as the action of diffusible viral or viral-induced protein, may initiate the uncontrolled hyperproliferation and eventual malignant transformation. On the other hand, the immune and neurologic manifestations of AIDS probably relate to the nature of the two types of target cells of the virus. The early target is a cell of the monocytemacrophage lineage. This includes monocytes in the peripheral blood, Langerhans' cells, follicular dendritic cells, and microglial cells. These cells are refractory to the cytotoxic effect of the virus and therefore may serve as reservoirs for it. Furthermore, a cell of this lineage infected in the peripheral blood could migrate to different tissue sites, including the brain, where it finds sanctuary and becomes refractory to the immune surveillance of the host. The mechanisms by which primary central nervous system disorders are produced so far remain unknown. The other major target for HIV is the T-helper cell. In fact, the antigen that defines this cell, the CD4 antigen, is the specific receptor for HIV. The receptor for HIV in monocytes is also CD4, of which a low level is sufficient for viral infection. The CD4+ lymphocyte has been compared to the maestro of an orchestra because it is the critical cell for all immune regulation. Infection with HIV severely depletes the number ofthese cells. When it was first observed that HIV can kill the CD4 or T4 cells in vitro, the mechanism of this depletion seemed clear. My colleagues and I observed, however, that in an infected person, only a small percentage of the cells is actually infected and expressing virus. Therefore, a direct cytopathic effect of the virus on cells may contribute to, but does not fully account for, the global and quantitative T4-cell depletion in patients with AIDS. Other alternative mechanisms have since been invoked. A possible mechanism is that the infected cells, by virtue of the expression of the viral envelope, can adhere to uninfected cells that express the CD4 receptor. Such an interaction may result in the formation of large syncytia involving both infected and uninfected cells. The cells in such syncytia
(Wong-Staal F: The AIDS virus-What we know and what we can do about it. West J Med 1991 Nov; 155:481-487) From the Departments of Medicine and Biology, University of Califomia, San Diego, School of Medicine, La Jolla, Califomia. Reprint requests to Flossie Wong-Staal, PhD, Department of Medicine, 0613-M, University of Califomia, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0613.
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THE AIDS VIRUS
ABBREVIATIONS USED IN TEXT AIDS = acquired immunodeficiency syndrome AZT = zidovudine HIV = human immunodeficiency virus HTLV = human T-lymphotropic virus LTR = long terminal repeat mRNA = messenger RNA NFRRE = nuclear factor that binds to RRE RRE = rev response element SIV = simian immunodeficiency virus SIVmac = macaque strain of SIV SIVsm = sooty mangabey strain of SIV
are not able to divide or to proliferate. By this means, the replication of both infected and uninfected CD4 cells would be halted. Circulating envelope glycoprotein gpl20 can also adsorb to CD4 cells and can be taken up and processed in such a way that converts the cells into targets for cellular immune destruction. Further, there is evidence that the binding of gp 120 to the CD4 molecule can activate a signal transduction pathway that decreases the expression of some cytokine products, most notably interleukin 2. This would decrease or inhibit the proliferation of T cells. Finally, a viral protein product can directly inhibit the proliferation of T cells. Whatever the mechanism, CD4-cell depletion, the resulting impaired immune function of the host, and the progression in clinical AIDS require viral expression, replication, and the continuous recruitment of new infected cells. Soon after infection, the virus typically replicates actively and induces an antiviral response in the host. With the mounting of that immune response, viremia is suppressed, bringing on a period of clinical latency. With time, however, viremia almost invariably returns because of increased viral replication. Immune function progressively decreases, and an infected person progresses from prodromal conditions to overt clinical AIDS. These events are presented schematically in Figure 1.
Exposure to Enhancing Factors
Asymptomatic ARC AIDS Figure 1.-The schematic shows the correlation of host immunity, virus load, and viral variation with clinical progression (see text for details). AIDS=acquired immunodeficiency syndrome, ARC=AIDS-related complex, HIV=human immunodeficiency virus
What is the cause and what is the effect? Is there more viral replication because the immune system is suppressed, or is the immune system suppressed because there is more viral replication? The answer is unclear, but both factors may pertain. The continued low replication of virus in the socalled latent period is presumably enough to erode the immune system, as a consequence of which more replicative forms of the virus can emerge. These viruses, replicating at greater efficiency, cause greater damage to the immune system, which in turn allows for the emergence of even more rapidly replicating virus. The resulting vicious spiral can account for the rapid disease progression in the later course of infection. Other determinants can also facilitate disease progression, namely the exogenous and environmental factors that can enhance viral expression. These factors include stress, exposure to UV light (which is known to activate the virus), other DNA damaging agents, and opportunistic infections of other viral agents-such as the leukemia viruses, HTLV-I and HTLV-II, the herpesviruses, and hepatitis virus-that are able to activate the expression of HIV. Furthermore, immunosuppressive agents can also contribute to AIDS progression. These can be considered as cofactors for AIDS induction. It is unlikely, however, that other than HIV, any other cofactors are essential; rather, a multitude of factors can facilitate disease progression. Of central importance is the conclusion that viral expression and host immunity are opposing forces that determine AIDS progression. This provides a rational basis for both antiviral therapy and immunotherapy. Inducing a high level of immunity against the virus before the establishment of infection to prevent infection is the rationale for developing a vaccine.
The Replication Cycle of Retroviruses as Target for Therapy Because HIV is a retrovirus, any inhibitors that would suppress retroviral replication would be suitable or possible anti-HIV agents. The typical retrovirus is a rather simple genetic entity, consisting of only three genes, the gag, pol, and env genes. The gag gene encodes for proteins that form the capsid of the particle, thepol gene encodes three separate proteins with four enzyme activities that are necessary for the replication of the virus, and the env gene encodes the envelope proteins that form both the surface glycoprotein and the transmembrane protein. Expression of all of these viral gene products is regulated by the interaction of cellular factors with the virus long terminal repeat (LTR). Infection begins with the binding of the viral envelope to the cellular receptor, which then allows entry of the viral core containing the RNA genetic information. A viral enzyme, reverse transcriptase, converts the RNA to DNA provirus, which can then move to the nucleus, where it is integrated into the host chromosomal DNA, catalyzed by another viral enzyme, integrase. When the virus thus becomes a permanent endowment of the host chromosome, the infection is established and cannot be cured; only viral expression can be suppressed. The integrated provirus serves as a template for messenger RNA (mRNA) that can then be processed and translated into precursor proteins. The proteins then assemble and incorporate the genomic RNA to form virion particles. The maturation of these particles requires processing of the precursor proteins, using yet another viral enzyme, protease. Every step in this replication cycle is a possible target for
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antiviral therapy. Logically, the easiest targets to focus on are the specific viral enzymes that are essential for replication. The most effective treatment so far of HIV infection is zidovudine (AZT), which is a nucleoside analogue directed at the reverse transcriptase. There are limitations to the success of AZT because of its high toxicity and the possibility of the emergence of AZT-resistant mutants in the course of treatment. This underscores the importance of seeking additional means of therapy. This would allow combined regimens that might lower the toxicity, lessen the chance of the emergence of resistant mutants, and increase the antiviral potency. The steps described above are general strategies for inhibiting retroviral infections, but there have been few successes in inhibiting retroviral diseases, even though retroviruses have been known since the beginning of this century to be associated with a variety of diseases in animals. Recent work on AIDS has actually provided guidance for other systems. For example, AZT now can be used in feline leukemia, which is another retrovirus-induced disease.
CD4 and Infection With the Human Immunodeficiency Virus There are also unique strategies for therapy against HIV. For example, the receptor of HIV infection is the CD4 molecule. It could be possible to exploit this information to develop antiviral drugs. Indeed, such possibilities have stirred some early excitement concerning the potential benefits of such drugs. The CD4 molecule, a protein of the immunoglobulin family, contains variable transmembrane and cytoplasmic domains. The binding site for the virus has been mapped to the first variable domain of this molecule. Soluble fragments of CD4 that retain the binding site for the virus but lack the transmembrane and cytoplasm domains have been used as competitive inhibitors for virus infection; Specifically, soluble CD4 binds to the envelope protein (gpl20) of the virus and prevents it from binding to the intact cellular receptor. There are variations of this scheme. For example, antiidiotypic antibodies that would mimic the soluble CD4 would also compete for the binding. Binding a toxin molecule to the CD4 molecule can specifically target the toxin to destroy virus-expressing cells. Soluble CD4 is now in clinical trial. Although it does not seem to have significant toxicity even at high doses, it also does not have demonstrable efficacy. Among a number of inherent problems for CD4 therapy, the most formidable is that of pharmacokinetics. Soluble CD4 is rapidly cleared from the circulation by the kidney, such that its levels are likely to be insufficient. Furthermore, most clinical isolates of HIV seem to be much more refractory to inhibition by soluble CD4 than are laboratory passaged isolates. Therefore, the effective level of CD4 is even more difficult to attain. There are methods to refine this approach and to increase the stability of the CD4 molecule. For example, chimeric immunoadhesion molecules with one chain of the antibody coupled to a fragment of CD4 have been constructed to produce greater stability. Whether the refinement is sufficient to do the job remains to be seen. If CD4 is not likely to be the magic bullet, where else can we look? The genome of HIV is much more complex than that of the usual retrovirus. Not only is the virus itself more complex, but the mechanisms that the virus uses to regulate
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its expression are also completely novel. Research in this area will provide new insights into eukaryotic gene regulation that will have broad implications for all biology. Moreover, the means to control infection may ultimately come from understanding the complexity and the regulatory mechanisms of the virus. HIV Regulatory Genes The first indication that not all retroviruses are as simple as gag, pol, and env came from the studies of the human leukemia viruses where additional coding information for at least two regulatory genes, tax and rex, is found. For the HIVs, the complexity is greater, with at least six extra genes (Figure 2). Several ofthese genes are completely dispensable for viral replication and may exert a modest effect on viral production, transmission, and perhaps tropism. Their mechanisms of action or their in vivo role in pathogenesis are not well understood at present. This review will focus on two genes, tat and rev, because they are essential for viral replication. There is a division of labor for tat and rev. The tat gene regulates the expression of all viral genes, and rev is a differential activator. Its expression is required only for the production of viral structural products, such as the gag, pol, and env proteins, but not for the regulatory genes themselves. Interestingly, human leukemia viruses have analogous genes to carry out the same functions. The tax gene for HTLV is like tat in its overall control of virus gene expression, and the rex gene, amazingly, has exactly the same function as rev even though the two genes have no sequence homology. Indeed, rex can even substitute for rev in activity. tat
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THE AIDS VIRUS
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Through the interplay of these two genes, the human retroviruses have developed a strategy to regulate their expression either coordinately or in a temporal and differential manner. In the early phase of infection, only the regulatory genes are expressed, but in the late phase the full complement of viral proteins is made, including those that are essential for the production of viral particles. This switch from early phase to late phase is not seen in most retroviruses, and because it is brought about by the differential processing of a single primary transcript, is distinct from the early to late switch found in DNA viruses. The regulatory proteins of HIV are encoded by small, multiply spliced RNAs, whereas the structural proteins are expressed from larger RNAs, which are either unspliced or singly spliced. The control of gene expression is divided in that tat is needed to turn on the gene expression of both classes of gene products, while the transition from early to late is mediated by the rev gene product. In a single round of viral expression, the first mRNA species that appear are the multiply spliced low-molecular-weight RNAs, with a transition to express the higher molecular weight forms in 17 to 24 hours. A mutation in tat results in no viral expression at all. In contrast, with a mutation in rev, viral mRNA is expressed, but only the lower molecular weight, multiply spliced RNAs are detected in the cytoplasm. Although the mechanism of Rev action has not been defined, it clearly represents a novel pathway of posttranscriptional regulation. Most precursor mRNAs expressed from cellular genes follow one of two fates: they either form a spliceosome complex and get spliced and exported into cytoplasm, or they turn over in the nucleus. The Rev protein, by interacting with a specific target sequence contained in the intron of the viral precursor mRNA, is able to disengage the RNA from the spliceosome and facilitate export into the cytoplasm (Figure 3). By doing so, Rev actually diverts the distribution of multiply spliced RNA to unspliced or less spliced forms, so that Rev functions at the expense of its own production and the production of the other regulatory proteins, such as Tat. This is a way for the virus to allow a quick
burst of replication and then to revert back to a more controlled steady state. The mechanism of tat function is equally complex. A lot of activities have been attributed to this protein, all of them resulting in the activation of gene expression from the viral LTR. The target sequence that tat recognizes is called TAR, located immediately downstream of the site of transcription initiation. The tat gene has been known to increase the frequency of transcriptional initiation and to act as a processivity factor for transcriptional elongation (Figure 4). There is some evidence that Tat may also work as a posttranscriptional activator. The best evidence for this comes from the Xenopus laevis oocyte system in which it was shown that preformed mRNA injected into the nucleus can respond to tat transactivation. Despite these differences in functional mechanisms, tat and rev also have several parallel features. They are both small nucleoproteins that are found predominantly in the nucleolus. Both of them recognize RNA rather than DNA targets, a feature that distinguishes them from most other viral transactivators. The target for rev is called the rev response element, or RRE, and the target for tat is TAR. In both cases, the RNA targets are highly structured molecules, and the recognition of them by the viral transactivators is largely structural. The rev gene product binds to a specific "hammerhead" (stem-loop II) region of the RRE, and tat binds to three unpaired bases in the so-called bulge of TAR. That binding of these viral transactivators to their target RNAs is not sufficient for function and that cellular factors are also involved are evident because mutants of both proteins that are fully capable of binding to their targets may not be functional. For example, the domain for binding to RRE has been mapped on the Rev protein. This region also doubles as the nucleolar targeting domain. In addition, there is another critical domain in which mutations do not affect the capability of the protein to bind to RRE, and yet the resultant proteins are completely inactive. Moreover, they display a negative dominant phenotype; that is, they can inhibit the function of wild-type Rev protein. It is proposed that this region is an activator domain that mediates interaction of the Rev protein with cellular factor(s). Essential functional domains including the regions for RNA binding or nuclear localization have also been mapped on the Tat protein. An important dividend of these observations is that these viral proteins and mRNA molecules allow us to fish out cellular factors involved in their respective activities, factors that are likely to be involved in novel mechanisms of eukaryotic gene regulation and that are otherwise inaccessible. One ofthe major focuses in my laboratory at present is to examine several of the factors that may be involved in the tat and rev transactivation pathways. We have identified a nuclear factor, NF,,,, that specifically binds to the same hammerhead structure of the RRE molecule that Rev binds. Furthermore, NF,,, can bind simultaneously with Rev in close proximity, with possible further protein-protein interaction in the infected cell. This nuclear factor is highly conserved and is widely distributed in mammalian cells but absent in the X laevis oocytes. Interestingly, Rev is active in most cell types but not in Xlaevis oocytes. Preliminary studies showed that if partially purified NFRIl protein is injected into the oocytes, Rev activity is indeed restored. Thus, NFRRE is likely to have a role in rev-mediated transactivation. We have just cloned the NF.,, gene, and it would be interesting to determine not only
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what its exact role is in rev function but also what its role is in eukaryotic gene regulation. Similarly, not one but several cellular factors have been shown to bind to either Tat or TAR. For example, a 68kilodalton (kd) protein that recognizes the loop sequence of the TAR region has been described. A 45-kd protein with the same TAR-binding specificity is prevalent in T cells. A similar-sized protein binds specifically to the stem between the loop and the bulge (Figure 4). Again, it would be interesting to determine the functions of these proteins in both the infected and uninfected cells. As noted, tat and rev are both required for viral replication and therefore play important roles in the pathogenesis of HIV. In addition, the potential effects of these molecules, particularly for Tat, may go beyond their role as viral transactivators. There is now evidence that Tat may act as an exogenous factor. It can be released from infected cells or Tat-producing cells and be found in the extracellular medium. Exogenous Tat can also be taken up by uninfected cells in a functional form-that is, the internalized Tat retains its ability to transactivate its target, the LTR. Therefore, its sphere of action is not restricted to the infected cell. Also, its target is not restricted to the viral LTR. A first demonstrated example of Tat activating a heterologous promoter is that of the JC virus late promoter, suggesting that along with immune suppression as a consequence of HIV infection, Tat may contribute to the reactivation of latent JC virus infection. This may lead to the induction of the neurologic disorder, progressive multifocal leukoencephalopathy, associated with active JC virus infection. Furthermore, Tat may have a greater effect on cellular function than we had previously thought, as it was also found recently to activate some cytokine genes, specifically the transforming growth factor-$ gene. Processivity
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Figure 4.-The mechanisms of Tat activation are shown. Tat has been proposed to increase the processivity of transcription or the frequency of initiation. The role of various cell factors that bind to the target sequence, TAR, in either mechanism has not been defined. HIV LTR=human immunodeficiency virus long terminal repeat
Exogenous Tat protein can apparently modulate cell growth. It specifically suppresses antigen-activated T-cell proliferation and increases the proliferation of cells derived from Kaposi's sarcoma-derived cells. These activities can contribute to immunosuppression and to the progression of the Kaposi's sarcoma tumors in HIV-infected persons. The remarkable parallels between HIV and HTLV have often prompted workers in both areas to take cues from each other. An illustration of this point comes from recent investigations of the Tax protein of HTLV. Analogous to the observations on Tat, Tax is found extracellularly, can be internalized in a functional form, and can activate T cells through the NFxB pathway and stimulate their proliferation. These features of Tax are in line with the pathogenic potential of the leukemia viruses, which is T-cell activation and transformation.
Regulatory Genes as Possible Targets Because the tat and rev genes are important for viral replication and pathogenesis, a useful antiviral strategy obviously would be to interfere with their functions. A straightforward way has been to inhibit the expression of both genes using antisense oligonucleotides directed at the tat/rev coding region. Indeed, viral expression can be specifically inhibited by such compounds, as measured by expression of the core protein p24 or viral reverse transcriptase. The sensestrand of the same sequence had no effect. When the expression of RNA is examined in the antisense-treated cells, neither tat nor rev is completely inhibited-that is, some RNA, including singly spliced viral RNA, is expressed. The unspliced genomic RNA, however (the gag-pol message), appears to be highly sensitive to rev depletion and is completely repressed. As a result, viral production is also repressed. Therefore, even though the antisense approach is not an efficient means of gene inhibition, viral production can be completely repressed in the absence of total gene inhibition, making the rev gene an attractive target for virus inhibition. As discussed earlier, both tat and rev transactivations involve a number of players: the viral protein, a viral RNA element, and cellular factors, each interacting with the others. It is possible to devise means of interrupting those interactions that are crucial to their functions. For example, some mutant proteins are negative dominant. These proteins can retain binding activity to their RNA targets but are not able to interact with the proper cellular factors and therefore would not function. They can thus act as competitive inhibitors of the wild-type protein. Alternatively, oligonucleotides that can hybridize to the stem-loop sequences of the RNA target can presumably disrupt the structure or the folding of these molecules, thereby rendering them incapable of interacting with both viral and cellular factors. Synthetic oligonucleotides directed at regions of RRE that have previously been shown to be important for rev function have been used. As a control, oligonucleotides complementary to a dispensable loop in RRE were used. All the antisense oligonucleotides directed at the critical regions were capable of inhibiting rev activity and viral production, but the control oligonucleotides directed at the dispensable loop, as well as random-sequence oligonucleotides, were not inhibitory (M. Klotman, S. Daefler, F. Wong-Staal, unpublished data,
1991).
There are many variations on this theme. One example is
486 THE AIDS VIRUS 48lH
the expression of multiple copies of TAR RNA as a decoy to compete with normal TAR in the virus genome for expression. A potential problem of expressing a TAR or RRE decoy, however, is that it may also sequester cellular factors that may be important for cell function. To overcome this problem, a vector has been devised in which the expression of TAR is conditional on tat acting on its promoter, which is the HIV LTR. Therefore, these decoy molecules would only be expressed in infected cells, where the first sign of the expression of tat can then unleash a whole army of these TAR decoys to limit the extent of viral replication (J. Lisziewicz, MD, written communication, April 1991). The foregoing discussion only relates to laboratory successes on inhibiting viral functions. This is a far cry from turning them into pharmaceutic products. Many considerations are important in drug development: toxicity of the compounds, pharmacokinetics, and cost effectiveness, to mention only a few. Moreover, the major recurrent difficulty is to have continuous delivery of these agents to maintain a persistent high level in the bloodstream or appropriate target tissues. In this sense, these novel antiviral approaches should dovetail ultimately with the development of gene therapy, which is itself a rapidly evolving field and which may have finally come of age. The Potential for Vaccine Development Optimism for the prospects of an AIDS vaccine has waxed and waned since the discovery of the virus. There are two major hurdles for the development of an HIV vaccine: the lack of an appropriate animal model; and the diversity of the virus, which makes it questionable whether it is feasible to develop a broadly cross-reactive vaccine. Lately there is renewed optimism because substantial progress has been made relating to both issues. A candidate vaccine would have to be evaluated in an animal model where protection from infection can be verified. For HIV-1, the only animals other than humans that can be infected are chimpanzees and apes. Because these primates are on the endangered species list, it is hard to get sufficiently large numbers for vaccine evaluation. Furthermore, these animals do not get AIDS with HIV-l infection. It is not known if they are appropriate models because their immune responses may be different from those in humans. The simian model may be better. A number of Old World monkeys are naturally infected with a virus similar to HIV- 1, designated the simian immunodeficiency virus (SIV). Four groups of primate immunodeficiency viruses have now been identified: HIV- 1 and rare viruses identified from chimpanzees (Pan troglodytes); viruses isolated from African green monkeys (Cercopithecus sabaeus); viruses from mandrills (Mandrillus mormon); and those isolated from sooty mangabey monkeys (Ceroccebusfuliginosus) (SIVsm), the West African virus HIV-2, and SIV isolated from captive macaque monkeys (Macaca species) (SIVmac). Because the sooty mangabey monkeys, but not macaques, are prevalently infected in the wild, it is thought that HIV-2 and SIVmac might have actually been derived from a cross-species infection of (SIVsm) or a virus closely related to it. All four groups are at about equal distance from each other. The monkeys that have been infected in the wild have a high prevalence of infection (30% to 50% of African green monkeys are seropositive for SIV) but do not become sick. This suggests a coadaptation of the virus and host in the
course of evolution. The same virus when inoculated into macaques, which are naive animals, induces a disease much
like AIDS. Type 2 HIV must have entered the human population recently, and it causes disease. Macaques are also susceptible to AIDS induction by some HIV-2 strains. The macaques then provide a relevant model that is a bit more accessible. Progress has been made in the simian model. Most important, immunization with killed virus preparations protects against a challenge infection by SIV. Even in some cases where vaccination had not protected against infection, it seemed to protect against disease. This may be the best that we can hope for from an AIDS vaccine. Previous infections with nonpathogenic variants can also protect against disease induction by the more virulent forms of virus. Thus, we are encouraged that in principle a vaccine against this group of viruses can work. Because of its potential hazards, a vaccine made from intact inactivated virus is not one of choice, however. Most investigators are focusing on using viral subunits for an HIV vaccine. The added advantage of this approach is that correct epitopes can be chosen and the wrong ones excluded. For example, the envelope for HIV contains a number of "good epitopes": neutralizing epitopes and epitopes that would elicit cellular immunity. There are also "bad epitopes," namely, those that elicit enhancing antibodies, immunosuppressive epitopes, and epitopes that may induce autoimmunity by molecular mimicry. The region on the HIV envelope that is the most potent for eliciting neutralizing antibodies has been sublocalized to a peptide of about 20 amino acids. This region contains the principal neutralizing epitope(s). Not only can peptides derived thereof induce high-titer neutralizing antibodies, but they can also adsorb most of the neutralizing activities elicited by the whole envelope. That recombinant proteins or synthetic peptides were effective in eliciting a potent neutralization response created much excitement and suggested the feasibility of a synthetic peptide vaccine approach. The same envelope region also contains a cytotoxic T-cell epitope, suggesting that this region may turn out to be the Achilles' heel of the virus. Enthusiasm was greatly dimmed, however, when this region of the viral envelope was found to map into a highly variable region, now referred to as V3 or the third hypervariable stretch on gpl20. The V3 sequence potentially forms a loop by a cysteine-cysteiqW disulfide bridge. The antigenic region of this loop has been mapped to close to the crown of the sequence. In fact, a synthetic peptide that comprises the sequence only at the crown has also been shown to elicit high titers of neutralizing antibodies. Antibodies directed at this peptide were initially thought to be strictly type-specific; that is, they would not crossneutralize any heterologous strains. This assessment has turned out to be unduly pessimistic. An alignment of the V3 loop among the dozen or so published sequences of HIV revealed that the early prototypes that were used (HXB-2, LAV, and RF) are rare representatives of the universe of HIV. The sequences of hundreds of isolated HIV organisms have now been determined, of which about 30% have a common consensus sequence (with the MN virus) at the tip of the V3 loop. Another significant fraction shares seven out of nine of these residues. The early prototypes (III-B, LAV, and RF) were rarely represented. More important, antibodies to the MN peptide now can block the infectivity of many of these organisms. These results raise the possibility that a cocktail
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of the right representation of V3-derived sequences may be incorporated into a broadly cross-reactive vaccine. In conclusion, the principles of a vaccine have been shown to work. Now we need to confirm and extend these observations and put them into practice. GENERAL REFERENCES Bolognesi D: HIV antibodies and vaccine design. AIDS 1989; 3:S1I1-118 Gallo RC: HIV-The cause of AIDS: An overview on its biology, mechanisms of disease induction and our attempts to control it. J AIDS 1988; 1:521-535 Gallo RC, Montagnier L: AIDS In 1988. Sci Am 1988; 259:4148
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487 Greene W: Mechanisms of disease-The molecular biology of human immunodeficiency virus-type 1 infection. N Engl J Med 1991; 324:308-317 Mitsuya H, Yarchoan R, Broder S: Molecular targets for AIDS therapy. Science 1990; 249:1533-1544 Putney SD, Javaherian K, Rusche J, Matthews T, Bolognesi D: Features of the HIV envelope and development of a subunit vaccine, In Putney SD, Bolognesi DP (Eds): AIDS Vaccine Research and Clinical Trials. New York, NY, Marcel Dekker, 1990, pp 3-62 Vaishnav Y, Wong-Staal F: The biochemistry of AIDS. Annu Rev Biochem 1991; 60:577-630 Wong-Staal F: Human immunodeficiency viruses and their replication, In Fields BN, Knipe DN, Channock RM, et al (Eds): Virology 2nd Edition. New York, NY, Raven Press, 1990, pp 1529-1543
Biomedical Science The AIDS Virus What We Know and What We Can Do About It FLOSSIE WONG-STAAL, PhD, La Jolla, California
Presented at the annual meeting of the Western Association of Physicians, Cannel, California, February 6-7, 1991.
nfectious diseases have long been the scourge ofhumanity. In the past few decades, tremendous medical advances have been made, leading to a complacency that mortality from infectious diseases is no longer a major concern. That optimism was shown to be premature, however, with the appearance of the acquired immunodeficiency syndrome (AIDS) as the epidemic of the century. In certain areas of the world, AIDS is literally wiping out entire populations. The causative agent of AIDS, the human immunodeficiency virus (HIV), is a retrovirus. It is not the first retrovirus shown to cause disease in humans. Several years before, the first human retrovirus to be isolated was identified as the causative agent of an aggressive leukemia called adult T-cell leukemia. To date, the four human retroviruses that have been isolated and characterized all share one major feature, the propensity to infect T cells. For this reason, they have sometimes been referred to as human T-lymphotropic viruses (HTLVs). These viruses also fall neatly into two subgroups: the leukemia viruses, HTLV-I and HTLV-II, and the immunodeficiency viruses, HIV type 1 and HIV type 2. The prototype AIDS virus, HIV- 1, probably originated in Central and East Africa, but it is now found in most of the world. Also associated with AIDS, HIV-2 is still largely confined to countries of western Africa. This presentation will pertain predominantly to HIV-1, although much of it should also be relevant to HIV-2. There may be interesting parallels to be drawn from and to the leukemia viruses as well. I
Pathophysiology of the Acquired Immunodeficiency Syndrome The clinical spectrum of HIV infection is broad, affecting as it does almost all parts of the human body. The resulting symptoms reflect three major manifestations: * The immune system is profoundly impaired. As a result, a variety of agents are able to produce opportunistic infections. * The human immunodeficiency virus also infects the brain, resulting in central nervous system disorders, includ-
ing dementia. *
sia)
Hyperproliferative disorders (hyperplasia and neoplaincreased in incidence, the most notable example
are
being Kaposi's sarcoma, the incidence of which is increased about 1,000-fold in certain HIV-infected populations. The role of HIV in associated neoplasias is probably indirect as HIV does not infect and transform these tumor cells. Indirect mechanisms, such as the action of diffusible viral or viral-induced protein, may initiate the uncontrolled hyperproliferation and eventual malignant transformation. On the other hand, the immune and neurologic manifestations of AIDS probably relate to the nature of the two types of target cells of the virus. The early target is a cell of the monocytemacrophage lineage. This includes monocytes in the peripheral blood, Langerhans' cells, follicular dendritic cells, and microglial cells. These cells are refractory to the cytotoxic effect of the virus and therefore may serve as reservoirs for it. Furthermore, a cell of this lineage infected in the peripheral blood could migrate to different tissue sites, including the brain, where it finds sanctuary and becomes refractory to the immune surveillance of the host. The mechanisms by which primary central nervous system disorders are produced so far remain unknown. The other major target for HIV is the T-helper cell. In fact, the antigen that defines this cell, the CD4 antigen, is the specific receptor for HIV. The receptor for HIV in monocytes is also CD4, of which a low level is sufficient for viral infection. The CD4+ lymphocyte has been compared to the maestro of an orchestra because it is the critical cell for all immune regulation. Infection with HIV severely depletes the number ofthese cells. When it was first observed that HIV can kill the CD4 or T4 cells in vitro, the mechanism of this depletion seemed clear. My colleagues and I observed, however, that in an infected person, only a small percentage of the cells is actually infected and expressing virus. Therefore, a direct cytopathic effect of the virus on cells may contribute to, but does not fully account for, the global and quantitative T4-cell depletion in patients with AIDS. Other alternative mechanisms have since been invoked. A possible mechanism is that the infected cells, by virtue of the expression of the viral envelope, can adhere to uninfected cells that express the CD4 receptor. Such an interaction may result in the formation of large syncytia involving both infected and uninfected cells. The cells in such syncytia
(Wong-Staal F: The AIDS virus-What we know and what we can do about it. West J Med 1991 Nov; 155:481-487) From the Departments of Medicine and Biology, University of Califomia, San Diego, School of Medicine, La Jolla, Califomia. Reprint requests to Flossie Wong-Staal, PhD, Department of Medicine, 0613-M, University of Califomia, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0613.
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THE AIDS VIRUS
ABBREVIATIONS USED IN TEXT AIDS = acquired immunodeficiency syndrome AZT = zidovudine HIV = human immunodeficiency virus HTLV = human T-lymphotropic virus LTR = long terminal repeat mRNA = messenger RNA NFRRE = nuclear factor that binds to RRE RRE = rev response element SIV = simian immunodeficiency virus SIVmac = macaque strain of SIV SIVsm = sooty mangabey strain of SIV
are not able to divide or to proliferate. By this means, the replication of both infected and uninfected CD4 cells would be halted. Circulating envelope glycoprotein gpl20 can also adsorb to CD4 cells and can be taken up and processed in such a way that converts the cells into targets for cellular immune destruction. Further, there is evidence that the binding of gp 120 to the CD4 molecule can activate a signal transduction pathway that decreases the expression of some cytokine products, most notably interleukin 2. This would decrease or inhibit the proliferation of T cells. Finally, a viral protein product can directly inhibit the proliferation of T cells. Whatever the mechanism, CD4-cell depletion, the resulting impaired immune function of the host, and the progression in clinical AIDS require viral expression, replication, and the continuous recruitment of new infected cells. Soon after infection, the virus typically replicates actively and induces an antiviral response in the host. With the mounting of that immune response, viremia is suppressed, bringing on a period of clinical latency. With time, however, viremia almost invariably returns because of increased viral replication. Immune function progressively decreases, and an infected person progresses from prodromal conditions to overt clinical AIDS. These events are presented schematically in Figure 1.
Exposure to Enhancing Factors
Asymptomatic ARC AIDS Figure 1.-The schematic shows the correlation of host immunity, virus load, and viral variation with clinical progression (see text for details). AIDS=acquired immunodeficiency syndrome, ARC=AIDS-related complex, HIV=human immunodeficiency virus
What is the cause and what is the effect? Is there more viral replication because the immune system is suppressed, or is the immune system suppressed because there is more viral replication? The answer is unclear, but both factors may pertain. The continued low replication of virus in the socalled latent period is presumably enough to erode the immune system, as a consequence of which more replicative forms of the virus can emerge. These viruses, replicating at greater efficiency, cause greater damage to the immune system, which in turn allows for the emergence of even more rapidly replicating virus. The resulting vicious spiral can account for the rapid disease progression in the later course of infection. Other determinants can also facilitate disease progression, namely the exogenous and environmental factors that can enhance viral expression. These factors include stress, exposure to UV light (which is known to activate the virus), other DNA damaging agents, and opportunistic infections of other viral agents-such as the leukemia viruses, HTLV-I and HTLV-II, the herpesviruses, and hepatitis virus-that are able to activate the expression of HIV. Furthermore, immunosuppressive agents can also contribute to AIDS progression. These can be considered as cofactors for AIDS induction. It is unlikely, however, that other than HIV, any other cofactors are essential; rather, a multitude of factors can facilitate disease progression. Of central importance is the conclusion that viral expression and host immunity are opposing forces that determine AIDS progression. This provides a rational basis for both antiviral therapy and immunotherapy. Inducing a high level of immunity against the virus before the establishment of infection to prevent infection is the rationale for developing a vaccine.
The Replication Cycle of Retroviruses as Target for Therapy Because HIV is a retrovirus, any inhibitors that would suppress retroviral replication would be suitable or possible anti-HIV agents. The typical retrovirus is a rather simple genetic entity, consisting of only three genes, the gag, pol, and env genes. The gag gene encodes for proteins that form the capsid of the particle, thepol gene encodes three separate proteins with four enzyme activities that are necessary for the replication of the virus, and the env gene encodes the envelope proteins that form both the surface glycoprotein and the transmembrane protein. Expression of all of these viral gene products is regulated by the interaction of cellular factors with the virus long terminal repeat (LTR). Infection begins with the binding of the viral envelope to the cellular receptor, which then allows entry of the viral core containing the RNA genetic information. A viral enzyme, reverse transcriptase, converts the RNA to DNA provirus, which can then move to the nucleus, where it is integrated into the host chromosomal DNA, catalyzed by another viral enzyme, integrase. When the virus thus becomes a permanent endowment of the host chromosome, the infection is established and cannot be cured; only viral expression can be suppressed. The integrated provirus serves as a template for messenger RNA (mRNA) that can then be processed and translated into precursor proteins. The proteins then assemble and incorporate the genomic RNA to form virion particles. The maturation of these particles requires processing of the precursor proteins, using yet another viral enzyme, protease. Every step in this replication cycle is a possible target for
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antiviral therapy. Logically, the easiest targets to focus on are the specific viral enzymes that are essential for replication. The most effective treatment so far of HIV infection is zidovudine (AZT), which is a nucleoside analogue directed at the reverse transcriptase. There are limitations to the success of AZT because of its high toxicity and the possibility of the emergence of AZT-resistant mutants in the course of treatment. This underscores the importance of seeking additional means of therapy. This would allow combined regimens that might lower the toxicity, lessen the chance of the emergence of resistant mutants, and increase the antiviral potency. The steps described above are general strategies for inhibiting retroviral infections, but there have been few successes in inhibiting retroviral diseases, even though retroviruses have been known since the beginning of this century to be associated with a variety of diseases in animals. Recent work on AIDS has actually provided guidance for other systems. For example, AZT now can be used in feline leukemia, which is another retrovirus-induced disease.
CD4 and Infection With the Human Immunodeficiency Virus There are also unique strategies for therapy against HIV. For example, the receptor of HIV infection is the CD4 molecule. It could be possible to exploit this information to develop antiviral drugs. Indeed, such possibilities have stirred some early excitement concerning the potential benefits of such drugs. The CD4 molecule, a protein of the immunoglobulin family, contains variable transmembrane and cytoplasmic domains. The binding site for the virus has been mapped to the first variable domain of this molecule. Soluble fragments of CD4 that retain the binding site for the virus but lack the transmembrane and cytoplasm domains have been used as competitive inhibitors for virus infection; Specifically, soluble CD4 binds to the envelope protein (gpl20) of the virus and prevents it from binding to the intact cellular receptor. There are variations of this scheme. For example, antiidiotypic antibodies that would mimic the soluble CD4 would also compete for the binding. Binding a toxin molecule to the CD4 molecule can specifically target the toxin to destroy virus-expressing cells. Soluble CD4 is now in clinical trial. Although it does not seem to have significant toxicity even at high doses, it also does not have demonstrable efficacy. Among a number of inherent problems for CD4 therapy, the most formidable is that of pharmacokinetics. Soluble CD4 is rapidly cleared from the circulation by the kidney, such that its levels are likely to be insufficient. Furthermore, most clinical isolates of HIV seem to be much more refractory to inhibition by soluble CD4 than are laboratory passaged isolates. Therefore, the effective level of CD4 is even more difficult to attain. There are methods to refine this approach and to increase the stability of the CD4 molecule. For example, chimeric immunoadhesion molecules with one chain of the antibody coupled to a fragment of CD4 have been constructed to produce greater stability. Whether the refinement is sufficient to do the job remains to be seen. If CD4 is not likely to be the magic bullet, where else can we look? The genome of HIV is much more complex than that of the usual retrovirus. Not only is the virus itself more complex, but the mechanisms that the virus uses to regulate
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its expression are also completely novel. Research in this area will provide new insights into eukaryotic gene regulation that will have broad implications for all biology. Moreover, the means to control infection may ultimately come from understanding the complexity and the regulatory mechanisms of the virus. HIV Regulatory Genes The first indication that not all retroviruses are as simple as gag, pol, and env came from the studies of the human leukemia viruses where additional coding information for at least two regulatory genes, tax and rex, is found. For the HIVs, the complexity is greater, with at least six extra genes (Figure 2). Several ofthese genes are completely dispensable for viral replication and may exert a modest effect on viral production, transmission, and perhaps tropism. Their mechanisms of action or their in vivo role in pathogenesis are not well understood at present. This review will focus on two genes, tat and rev, because they are essential for viral replication. There is a division of labor for tat and rev. The tat gene regulates the expression of all viral genes, and rev is a differential activator. Its expression is required only for the production of viral structural products, such as the gag, pol, and env proteins, but not for the regulatory genes themselves. Interestingly, human leukemia viruses have analogous genes to carry out the same functions. The tax gene for HTLV is like tat in its overall control of virus gene expression, and the rex gene, amazingly, has exactly the same function as rev even though the two genes have no sequence homology. Indeed, rex can even substitute for rev in activity. tat
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THE AIDS VIRUS
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Through the interplay of these two genes, the human retroviruses have developed a strategy to regulate their expression either coordinately or in a temporal and differential manner. In the early phase of infection, only the regulatory genes are expressed, but in the late phase the full complement of viral proteins is made, including those that are essential for the production of viral particles. This switch from early phase to late phase is not seen in most retroviruses, and because it is brought about by the differential processing of a single primary transcript, is distinct from the early to late switch found in DNA viruses. The regulatory proteins of HIV are encoded by small, multiply spliced RNAs, whereas the structural proteins are expressed from larger RNAs, which are either unspliced or singly spliced. The control of gene expression is divided in that tat is needed to turn on the gene expression of both classes of gene products, while the transition from early to late is mediated by the rev gene product. In a single round of viral expression, the first mRNA species that appear are the multiply spliced low-molecular-weight RNAs, with a transition to express the higher molecular weight forms in 17 to 24 hours. A mutation in tat results in no viral expression at all. In contrast, with a mutation in rev, viral mRNA is expressed, but only the lower molecular weight, multiply spliced RNAs are detected in the cytoplasm. Although the mechanism of Rev action has not been defined, it clearly represents a novel pathway of posttranscriptional regulation. Most precursor mRNAs expressed from cellular genes follow one of two fates: they either form a spliceosome complex and get spliced and exported into cytoplasm, or they turn over in the nucleus. The Rev protein, by interacting with a specific target sequence contained in the intron of the viral precursor mRNA, is able to disengage the RNA from the spliceosome and facilitate export into the cytoplasm (Figure 3). By doing so, Rev actually diverts the distribution of multiply spliced RNA to unspliced or less spliced forms, so that Rev functions at the expense of its own production and the production of the other regulatory proteins, such as Tat. This is a way for the virus to allow a quick
burst of replication and then to revert back to a more controlled steady state. The mechanism of tat function is equally complex. A lot of activities have been attributed to this protein, all of them resulting in the activation of gene expression from the viral LTR. The target sequence that tat recognizes is called TAR, located immediately downstream of the site of transcription initiation. The tat gene has been known to increase the frequency of transcriptional initiation and to act as a processivity factor for transcriptional elongation (Figure 4). There is some evidence that Tat may also work as a posttranscriptional activator. The best evidence for this comes from the Xenopus laevis oocyte system in which it was shown that preformed mRNA injected into the nucleus can respond to tat transactivation. Despite these differences in functional mechanisms, tat and rev also have several parallel features. They are both small nucleoproteins that are found predominantly in the nucleolus. Both of them recognize RNA rather than DNA targets, a feature that distinguishes them from most other viral transactivators. The target for rev is called the rev response element, or RRE, and the target for tat is TAR. In both cases, the RNA targets are highly structured molecules, and the recognition of them by the viral transactivators is largely structural. The rev gene product binds to a specific "hammerhead" (stem-loop II) region of the RRE, and tat binds to three unpaired bases in the so-called bulge of TAR. That binding of these viral transactivators to their target RNAs is not sufficient for function and that cellular factors are also involved are evident because mutants of both proteins that are fully capable of binding to their targets may not be functional. For example, the domain for binding to RRE has been mapped on the Rev protein. This region also doubles as the nucleolar targeting domain. In addition, there is another critical domain in which mutations do not affect the capability of the protein to bind to RRE, and yet the resultant proteins are completely inactive. Moreover, they display a negative dominant phenotype; that is, they can inhibit the function of wild-type Rev protein. It is proposed that this region is an activator domain that mediates interaction of the Rev protein with cellular factor(s). Essential functional domains including the regions for RNA binding or nuclear localization have also been mapped on the Tat protein. An important dividend of these observations is that these viral proteins and mRNA molecules allow us to fish out cellular factors involved in their respective activities, factors that are likely to be involved in novel mechanisms of eukaryotic gene regulation and that are otherwise inaccessible. One ofthe major focuses in my laboratory at present is to examine several of the factors that may be involved in the tat and rev transactivation pathways. We have identified a nuclear factor, NF,,,, that specifically binds to the same hammerhead structure of the RRE molecule that Rev binds. Furthermore, NF,,, can bind simultaneously with Rev in close proximity, with possible further protein-protein interaction in the infected cell. This nuclear factor is highly conserved and is widely distributed in mammalian cells but absent in the X laevis oocytes. Interestingly, Rev is active in most cell types but not in Xlaevis oocytes. Preliminary studies showed that if partially purified NFRIl protein is injected into the oocytes, Rev activity is indeed restored. Thus, NFRRE is likely to have a role in rev-mediated transactivation. We have just cloned the NF.,, gene, and it would be interesting to determine not only
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what its exact role is in rev function but also what its role is in eukaryotic gene regulation. Similarly, not one but several cellular factors have been shown to bind to either Tat or TAR. For example, a 68kilodalton (kd) protein that recognizes the loop sequence of the TAR region has been described. A 45-kd protein with the same TAR-binding specificity is prevalent in T cells. A similar-sized protein binds specifically to the stem between the loop and the bulge (Figure 4). Again, it would be interesting to determine the functions of these proteins in both the infected and uninfected cells. As noted, tat and rev are both required for viral replication and therefore play important roles in the pathogenesis of HIV. In addition, the potential effects of these molecules, particularly for Tat, may go beyond their role as viral transactivators. There is now evidence that Tat may act as an exogenous factor. It can be released from infected cells or Tat-producing cells and be found in the extracellular medium. Exogenous Tat can also be taken up by uninfected cells in a functional form-that is, the internalized Tat retains its ability to transactivate its target, the LTR. Therefore, its sphere of action is not restricted to the infected cell. Also, its target is not restricted to the viral LTR. A first demonstrated example of Tat activating a heterologous promoter is that of the JC virus late promoter, suggesting that along with immune suppression as a consequence of HIV infection, Tat may contribute to the reactivation of latent JC virus infection. This may lead to the induction of the neurologic disorder, progressive multifocal leukoencephalopathy, associated with active JC virus infection. Furthermore, Tat may have a greater effect on cellular function than we had previously thought, as it was also found recently to activate some cytokine genes, specifically the transforming growth factor-$ gene. Processivity
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Figure 4.-The mechanisms of Tat activation are shown. Tat has been proposed to increase the processivity of transcription or the frequency of initiation. The role of various cell factors that bind to the target sequence, TAR, in either mechanism has not been defined. HIV LTR=human immunodeficiency virus long terminal repeat
Exogenous Tat protein can apparently modulate cell growth. It specifically suppresses antigen-activated T-cell proliferation and increases the proliferation of cells derived from Kaposi's sarcoma-derived cells. These activities can contribute to immunosuppression and to the progression of the Kaposi's sarcoma tumors in HIV-infected persons. The remarkable parallels between HIV and HTLV have often prompted workers in both areas to take cues from each other. An illustration of this point comes from recent investigations of the Tax protein of HTLV. Analogous to the observations on Tat, Tax is found extracellularly, can be internalized in a functional form, and can activate T cells through the NFxB pathway and stimulate their proliferation. These features of Tax are in line with the pathogenic potential of the leukemia viruses, which is T-cell activation and transformation.
Regulatory Genes as Possible Targets Because the tat and rev genes are important for viral replication and pathogenesis, a useful antiviral strategy obviously would be to interfere with their functions. A straightforward way has been to inhibit the expression of both genes using antisense oligonucleotides directed at the tat/rev coding region. Indeed, viral expression can be specifically inhibited by such compounds, as measured by expression of the core protein p24 or viral reverse transcriptase. The sensestrand of the same sequence had no effect. When the expression of RNA is examined in the antisense-treated cells, neither tat nor rev is completely inhibited-that is, some RNA, including singly spliced viral RNA, is expressed. The unspliced genomic RNA, however (the gag-pol message), appears to be highly sensitive to rev depletion and is completely repressed. As a result, viral production is also repressed. Therefore, even though the antisense approach is not an efficient means of gene inhibition, viral production can be completely repressed in the absence of total gene inhibition, making the rev gene an attractive target for virus inhibition. As discussed earlier, both tat and rev transactivations involve a number of players: the viral protein, a viral RNA element, and cellular factors, each interacting with the others. It is possible to devise means of interrupting those interactions that are crucial to their functions. For example, some mutant proteins are negative dominant. These proteins can retain binding activity to their RNA targets but are not able to interact with the proper cellular factors and therefore would not function. They can thus act as competitive inhibitors of the wild-type protein. Alternatively, oligonucleotides that can hybridize to the stem-loop sequences of the RNA target can presumably disrupt the structure or the folding of these molecules, thereby rendering them incapable of interacting with both viral and cellular factors. Synthetic oligonucleotides directed at regions of RRE that have previously been shown to be important for rev function have been used. As a control, oligonucleotides complementary to a dispensable loop in RRE were used. All the antisense oligonucleotides directed at the critical regions were capable of inhibiting rev activity and viral production, but the control oligonucleotides directed at the dispensable loop, as well as random-sequence oligonucleotides, were not inhibitory (M. Klotman, S. Daefler, F. Wong-Staal, unpublished data,
1991).
There are many variations on this theme. One example is
486 THE AIDS VIRUS 48lH
the expression of multiple copies of TAR RNA as a decoy to compete with normal TAR in the virus genome for expression. A potential problem of expressing a TAR or RRE decoy, however, is that it may also sequester cellular factors that may be important for cell function. To overcome this problem, a vector has been devised in which the expression of TAR is conditional on tat acting on its promoter, which is the HIV LTR. Therefore, these decoy molecules would only be expressed in infected cells, where the first sign of the expression of tat can then unleash a whole army of these TAR decoys to limit the extent of viral replication (J. Lisziewicz, MD, written communication, April 1991). The foregoing discussion only relates to laboratory successes on inhibiting viral functions. This is a far cry from turning them into pharmaceutic products. Many considerations are important in drug development: toxicity of the compounds, pharmacokinetics, and cost effectiveness, to mention only a few. Moreover, the major recurrent difficulty is to have continuous delivery of these agents to maintain a persistent high level in the bloodstream or appropriate target tissues. In this sense, these novel antiviral approaches should dovetail ultimately with the development of gene therapy, which is itself a rapidly evolving field and which may have finally come of age. The Potential for Vaccine Development Optimism for the prospects of an AIDS vaccine has waxed and waned since the discovery of the virus. There are two major hurdles for the development of an HIV vaccine: the lack of an appropriate animal model; and the diversity of the virus, which makes it questionable whether it is feasible to develop a broadly cross-reactive vaccine. Lately there is renewed optimism because substantial progress has been made relating to both issues. A candidate vaccine would have to be evaluated in an animal model where protection from infection can be verified. For HIV-1, the only animals other than humans that can be infected are chimpanzees and apes. Because these primates are on the endangered species list, it is hard to get sufficiently large numbers for vaccine evaluation. Furthermore, these animals do not get AIDS with HIV-l infection. It is not known if they are appropriate models because their immune responses may be different from those in humans. The simian model may be better. A number of Old World monkeys are naturally infected with a virus similar to HIV- 1, designated the simian immunodeficiency virus (SIV). Four groups of primate immunodeficiency viruses have now been identified: HIV- 1 and rare viruses identified from chimpanzees (Pan troglodytes); viruses isolated from African green monkeys (Cercopithecus sabaeus); viruses from mandrills (Mandrillus mormon); and those isolated from sooty mangabey monkeys (Ceroccebusfuliginosus) (SIVsm), the West African virus HIV-2, and SIV isolated from captive macaque monkeys (Macaca species) (SIVmac). Because the sooty mangabey monkeys, but not macaques, are prevalently infected in the wild, it is thought that HIV-2 and SIVmac might have actually been derived from a cross-species infection of (SIVsm) or a virus closely related to it. All four groups are at about equal distance from each other. The monkeys that have been infected in the wild have a high prevalence of infection (30% to 50% of African green monkeys are seropositive for SIV) but do not become sick. This suggests a coadaptation of the virus and host in the
course of evolution. The same virus when inoculated into macaques, which are naive animals, induces a disease much
like AIDS. Type 2 HIV must have entered the human population recently, and it causes disease. Macaques are also susceptible to AIDS induction by some HIV-2 strains. The macaques then provide a relevant model that is a bit more accessible. Progress has been made in the simian model. Most important, immunization with killed virus preparations protects against a challenge infection by SIV. Even in some cases where vaccination had not protected against infection, it seemed to protect against disease. This may be the best that we can hope for from an AIDS vaccine. Previous infections with nonpathogenic variants can also protect against disease induction by the more virulent forms of virus. Thus, we are encouraged that in principle a vaccine against this group of viruses can work. Because of its potential hazards, a vaccine made from intact inactivated virus is not one of choice, however. Most investigators are focusing on using viral subunits for an HIV vaccine. The added advantage of this approach is that correct epitopes can be chosen and the wrong ones excluded. For example, the envelope for HIV contains a number of "good epitopes": neutralizing epitopes and epitopes that would elicit cellular immunity. There are also "bad epitopes," namely, those that elicit enhancing antibodies, immunosuppressive epitopes, and epitopes that may induce autoimmunity by molecular mimicry. The region on the HIV envelope that is the most potent for eliciting neutralizing antibodies has been sublocalized to a peptide of about 20 amino acids. This region contains the principal neutralizing epitope(s). Not only can peptides derived thereof induce high-titer neutralizing antibodies, but they can also adsorb most of the neutralizing activities elicited by the whole envelope. That recombinant proteins or synthetic peptides were effective in eliciting a potent neutralization response created much excitement and suggested the feasibility of a synthetic peptide vaccine approach. The same envelope region also contains a cytotoxic T-cell epitope, suggesting that this region may turn out to be the Achilles' heel of the virus. Enthusiasm was greatly dimmed, however, when this region of the viral envelope was found to map into a highly variable region, now referred to as V3 or the third hypervariable stretch on gpl20. The V3 sequence potentially forms a loop by a cysteine-cysteiqW disulfide bridge. The antigenic region of this loop has been mapped to close to the crown of the sequence. In fact, a synthetic peptide that comprises the sequence only at the crown has also been shown to elicit high titers of neutralizing antibodies. Antibodies directed at this peptide were initially thought to be strictly type-specific; that is, they would not crossneutralize any heterologous strains. This assessment has turned out to be unduly pessimistic. An alignment of the V3 loop among the dozen or so published sequences of HIV revealed that the early prototypes that were used (HXB-2, LAV, and RF) are rare representatives of the universe of HIV. The sequences of hundreds of isolated HIV organisms have now been determined, of which about 30% have a common consensus sequence (with the MN virus) at the tip of the V3 loop. Another significant fraction shares seven out of nine of these residues. The early prototypes (III-B, LAV, and RF) were rarely represented. More important, antibodies to the MN peptide now can block the infectivity of many of these organisms. These results raise the possibility that a cocktail
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of the right representation of V3-derived sequences may be incorporated into a broadly cross-reactive vaccine. In conclusion, the principles of a vaccine have been shown to work. Now we need to confirm and extend these observations and put them into practice. GENERAL REFERENCES Bolognesi D: HIV antibodies and vaccine design. AIDS 1989; 3:S1I1-118 Gallo RC: HIV-The cause of AIDS: An overview on its biology, mechanisms of disease induction and our attempts to control it. J AIDS 1988; 1:521-535 Gallo RC, Montagnier L: AIDS In 1988. Sci Am 1988; 259:4148
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487 Greene W: Mechanisms of disease-The molecular biology of human immunodeficiency virus-type 1 infection. N Engl J Med 1991; 324:308-317 Mitsuya H, Yarchoan R, Broder S: Molecular targets for AIDS therapy. Science 1990; 249:1533-1544 Putney SD, Javaherian K, Rusche J, Matthews T, Bolognesi D: Features of the HIV envelope and development of a subunit vaccine, In Putney SD, Bolognesi DP (Eds): AIDS Vaccine Research and Clinical Trials. New York, NY, Marcel Dekker, 1990, pp 3-62 Vaishnav Y, Wong-Staal F: The biochemistry of AIDS. Annu Rev Biochem 1991; 60:577-630 Wong-Staal F: Human immunodeficiency viruses and their replication, In Fields BN, Knipe DN, Channock RM, et al (Eds): Virology 2nd Edition. New York, NY, Raven Press, 1990, pp 1529-1543
