Daan Mulder studied the rampage of HIV through East Africa. In particular his work in south-west Uganda gave us a picture of the burgeoning AIDS epidemic that regrettably was to become all too familiar in neighbouring regions and nations. I vividly recall the exciting days not long after the discovery of HIV ( Barré-Sinoussi et al. 1983 ), then known as LAV or HTLV-III, when we first coaxed the virus to grow to high titre in cell culture. This provided sufficient viral antigen to develop the first reliable serological assay for epidemiological studies ( Cheingsong-Popov et al. 1984 ). This ‘HIV test’ proved to be robust in the African setting ( Bayley et al. 1985 ) and enabled our Ugandan colleagues to show that ‘slim’ disease was indeed AIDS caused by HIV ( Serwadda et al. 1985 ).
Fifteen years later we know a great deal about the replication, variability and cell tropism of HIV. However, we still do not really understand what eventually tips the balance of infection away from host immunity and towards the viral pathogenesis to cause AIDS ( Coutinho 2000), or how to design a thoroughly safe yet efficacious vaccine ( Gotch 2000). From the early days we found that in comparison to oncogenic human retroviruses, HIV elicited only weak protective humoral immunity and that neutralization antigens in the HIV envelope were highly variable ( Weiss et al. 1986 ). Here, a brief sketch is provided of HIV's replication, origins and evolution, cell biology and pathogenesis. For a more detailed account the reader is referred to major texts ( Coffin et al. 1997 ; Levy 1998).
Human immunodeficiency viruses comprise two distinct viruses, HIV-1 and HIV-2, which differ in origin and gene sequence. However, both viruses cause AIDS with a similar spectrum of symptoms, though CNS disease may be more frequent in HIV-2 disease ( Lucas et al. 1993 ). It appears that HIV-2 is less virulent than HIV-1 in that HIV-2 infection takes longer to progress to AIDS ( Whittle et al. 1994 ). This is true as a generalization, but it may obscure a bimodal pattern of disease, in which some HIV-2 infected people progress to AIDS at a similar rate as those with HIV-1 infection, while a higher proportion than is the case for HIV-1 remain long-term nonprogressors. More detailed longitudinal studies in West Africa are required to determine if this is so.
The genomes of HIV-1 and HIV-2 are shown in Figure 1. Both of them belong to the lentivirus subfamily of retroviruses and have a similar structure and order of genes ( Coffin et al. 1997 ). However, HIV-1 carries a vpu gene where HIV-2 and most simian immunodeficiency viruses (SIVs) carry vpx. In common with all retroviruses the gag gene encodes the structural proteins of the core (p24, p7, p6) and matrix (p17) proteins of the virus particle, and the env gene encodes the glycoproteins (gp120, gp41) that comprise the viral envelope antigens, which interact with cell surface receptors. The p24m gp120 proteins are the ones most commonly incorporated into diagnostic tests for HIV antibodies.
The pol gene encodes the enzymes crucial for viral replication: reverse transcriptase to convert viral RNA into DNA, integrase to incorporate the viral DNA into host chromosomal DNA (the provirus) and protease to cleave large precursor Gag and Pol proteins into their component parts. Because these enzymes are unique to retroviruses, molecules that inhibit them or act as ‘junk’ substrates competing for their activity serve as promising drugs to block HIV replication without causing too severe toxicity to the host. Reverse transcriptase and protease inhibitors represent the current generation of antiretroviral drugs ( Darbyshire 2000).
The tat gene encodes a protein that promotes transcription or production of HIV RNA from the DNA provirus. Rev ensures that the correctly processed messenger RNA and genomic RNA is exported from nucleus to cytoplasm. The function of the other, accessory HIV genes is less well understood. Vpr helps to arrest the cell cycle. In HIV-1 vpr also enables the reverse transcribed DNA to gain access to the nucleus in nondividing cells such as macrophages, a function performed by vpx in HIV-2. Vpu is important for virus particle release. Vif encodes a small protein that enhances the infectiveness of progeny virus particles. Nef has multiple functions including signal transduction and removing the CD4 receptor from the cell surface to allow the virus to bud from the surface late in the cellular infection cycle.
Figure 2 depicts the major steps in viral replication. Reverse transcription and integration of the proviral genome are the hallmarks of retroviruses which thus establish persistent infection in the host. The integrated provirus can remain latent, and be passed to daughter cells during chromosomal replication and cell division. In activated cells, however, RNA molecules for messenger RNA and for new genomes are forward transcribed from the DNA provirus to produce the next generation of virus particles. Full replication of HIV in T-lymphocytes usually results in cell death, whereas macrophages may sustain lower levels of viable virus production for long periods. In addition to the well-known drugs that inhibit reverse transcription (stage 4) and protease cleavage in maturation (stage 13), other molecules could conceivably block each stage of replication which represents a potential drug target. For example, there is considerable current interest in experimental drugs that prevent interaction with the cell surface early in infection (stages 1 and 2), and that block insertion into chromosomal DNA (integrase inhibitors, stage 6).
Origins and diversity
It is generally agreed that both HIV-1 and HIV-2 represent novel, zoonotic introductions into the human population within the past 100 years (Hahn et al. 2000). The animal lentivirus most related to HIV-1 is SIVcpz of chimpanzees. Several SIVcpz genomes have been sequenced and those resident in the subspecies Pan troglodytes troglodytes are closest to HIV-1 ( Gao et al. 1999 ). In human populations, there are three major groups of HIV-1, named M, N and O. Group M represents all the subtypes or ‘clades’ A-H that have spread to cause the worldwide pandemic ( De Cock & Weiss 2000). HIV-1 groups O and N, in contrast, are largely confined to Gabon, Cameroon and neighbouring countries ( Peeters et al. 1997 ), close to the natural habitat of P.t. troglodytes. The gene sequences of groups M, N and O are as distinct from each other as they are from SIVcpz ( Gao et al. 1999 ). This implies that they derive from three separate introductions from chimpanzees to humans, yet only one of them has become pandemic ( Weiss & Wrangham 1999; Hahn et al. 2000 ).
HIV-2 is endemic in West Africa, but has spread to Europe (especially Portugal) and to India. Like HIV-1, HIV-2 can be subdivided into a number of major groups which appear to represent separate zoonoses from a primate host. In this case the primate reservoir is not a great ape but a West African monkey, the sooty mangabey, which is infected with SIVsmm. Many species of African monkey harbour other types of SIV, but there is no evidence that any of these SIV strains have infected humans. African green monkeys are frequently infected by SIV. Despite 35 years' use of unscreened African green monkey kidneys to propagate poliovirus for the live attenuated vaccine, SIVagm has not crossed to humans. Thus the hypothesis that HIV emerged from contaminated polio vaccines appears tenuous ( Weiss 1999).
Even though HIV-1 Group M has probably not infected humans for more than about 70 years, and did not emerge epidemically until 20 years ago, the virus has generated enormous variation ( Leigh Brown 1999). The reasons for this great diversity are several. First, the process of reverse transcription, when DNA is replicated from the viral RNA template, does not include an editing device to correct mutations. Second, the RNA genomes of retrovirus particles are diploid and genetic recombination occurs during reverse transcription. In dually infected persons therefore, recombinant forms can emerge and some of the HIV-1 clades show evidence of past recombinational origin, e.g. clade E in Thailand. However, recombinants between HIV-1 and HIV-2 have not yet been reported although dual infections occur in West Africa. Third, during the long asymptomatic incubation period before AIDS develops, the virus is not latent, but is actively replicating, producing as many as 109 infectious viral progeny and over 107 newly infected cells each day ( Phillips 1999). This high rate of virus replication allows numerous immune escape and drug-resistant mutants to be generated, and permits substantial genetic drift within each infected individual. As discussed below, the evolution and selection of variants within one infected host may therefore be substantial, allowing colonization of new cells and tissues, although there are certain constraints on passing infection from one human to another which help to reset the evolutionary clock each time transmission occurs. In other words, viral fitness for infection to spread within an individual host does not necessarily equate with fitness for transmission.
The natural history of infection and disease is described by Coutinho (2000). In addition, the cell biology of HIV infection has illuminated our understanding of HIV infection, transmission and disease. From the beginning AIDS was recognized to result from selective depletion of CD4-positive helper T-lymphocytes ( Gottlieb et al. 1981 ). The function of CD4 cells is to help CD8-positive cytotoxic T-lymphocytes (CTL or killer cells) to destroy other cells expressing foreign antigens, and also to enhance antibody production by B-lymphocytes. Thus CD4 cells represent a key component of the immune system. In the healthy individual about 1200 CD4 cells circulate per μL blood; when CD4 counts drop below 400/μL, opportunistic infections can occur. Not long after HIV-1 was first isolated, Klatzman et al. (1984) showed that the virus selectively infected and destroyed CD4 cells in culture, and we then demonstrated that it does so by binding to the CD4 antigen itself ( Dalgleish et al. 1984 ). Thus the cellular tropism of the virus already explained much about the pattern of disease.
Another cell type that expresses low levels of CD4 antigen is the tissue macrophage, derived from monocytes circulating in the blood. These cells, which function as scavengers and as antigen-presenting cells, become infected by HIV, and act as an important reservoir of the virus in the body ( Levy 1998). It is macrophage infection that probably accounts for the wasting syndrome in AIDS, due to aberrant signalling of cytokines and other short-range molecules (chemokines) that traffic between different types of blood and tissue cells. Microglia in the brain are a type of macrophage. Their infection by HIV leads to aberrant signalling via astrocytes to cause loss of neurones and the dementia that sometimes occur in AIDS. Dendritic cells, another important type of antigen-presenting cell derived from monocytes, are also affected by HIV. Dendritic cells include the Langerhans cells of the mucous membranes and these may be involved as an early target of infection during sexual transmission. Dendritic cells carry HIV to the lymph nodes where CD4-positive lymphocytes become infected ( Levy 1998).
Whereas the CD4 antigen is important for HIV attachment to the cell surface ( Dalgleish et al. 1984 ), it soon became apparent that while CD4 is necessary it is not sufficient for HIV entry as an intracellular parasite ( Maddon et al. 1986 ). After much research by many groups, two chemokine receptors, known as CCR5 and CXCR4, were identified as coreceptors to CD4 that permitted virus entry ( Weiss 1996; Norcross 1999). Most HIV strains of each subtype use CCR5 as a coreceptor, which is expressed both on lymphocytes and on macrophages. However, in about 50% of HIV-infected persons progressing to AIDS, CXCR4-using viruses emerge late in the course of disease. These ‘X4’ strains ( Berger et al. 1998 ) are more virulent than the initial ‘R5’ strains and probably hasten the depletion of CD4 cells and the onset of disease. It is remarkable that while X4 viruses are much less efficient for person-to-person transmission than R5 viruses, they emerge as new variants in so many infected individuals as disease progresses. However, X4 viruses seldom arise in infection by HIV-1 subtype C ( Abebe et al. 1999 ; Ping et al. 1999 ).
Certain individuals carry genetically defective CCR5 receptors ( Carrington et al. 1999 ; Michael 1999). A 32 base-pair deletion in the CCR5 gene occurs as a frequent polymorphism in the Caucasian population and approximately 1 in 400 people are homozygous for the deleted gene. Such homozygotes are highly resistant to HIV infection and they are over-represented among the uninfected partners of discordant couples who are frequently exposed to HIV. Moreover, the few exceptions among the homozygotes who have become infected by HIV exclusively harbour X4 strains of virus which do not require the CCR5 coreceptor. Since CCR5 does not seem to be essential for leucocyte function (because homozygotes lacking functional CCR5 are healthy) it is an attractive target for new drug development ( Norcross 1999). CCR5 also is a possible target for immunization, if an invariant host antigen of this type could be used to block HIV infection prophylactically. Deletions in the CCR5 gene are rare among Africans. Mutations in the promoter region of this gene, however, occur frequently and can affect the incubation period from infection to AIDS ( Carrington et al. 1999 ; Michael 1999).
While the foregoing story of the cellular tropism of HIV explains why the virus causes the syndrome we call AIDS, it does not explain why the virus eventually wins the balance of power over the immune system. Other viruses that infect CD4 lymphocytes (e.g. human herpes virus 7) and macrophages (e.g. cytomegalovirus) typically do not cause disease unless the immune system is already impaired. Furthermore, the nonhuman primates whence HIV-1 and HIV-2 came, chimpanzees and sooty mangabey monkeys, carry SIV without becoming sick. Indeed, the sooty mangabeys carry viral loads of SIV as high as many humans do with HIV, yet only the humans (and also SIV-infected Asian macaques) succumb to AIDS. Part of the answer to this conundrum may be that HIV infection in humans elicits a general activation and proliferation of lymphocytes, which in turn assists virus replication, whereas in infected chimpanzees and sooty mangabeys, perturbation of lymphocyte regulation is less evident. If a means could be found of converting humans to a simian-type, commensal coexistence with the virus, people might not develop AIDS, although it would do nothing to prevent HIV transmission.
Conclusions and prospects
HIV continues to spread. A safe, efficacious vaccine is not yet in sight, although a number of interesting leads may soon be tested ( Gotch 2000). Nonetheless, research into HIV has been useful not only in understanding the nature of the disease, but also in prevention, diagnosis and treatment. With regard to progress in medical research, the study of HIV/AIDS has been remarkably rapid. AIDS was first recognized as a new disease in 1981, and to be transmissible in 1982. The first HIV isolate was made in 1983. By the end of 1985, within a year or so of learning to propagate the virus to high levels, screening of blood donations had been introduced throughout developed countries. A year later, zidovudine therapy was introduced to clinical trial, though it took a further 10 years to achieve the efficacy of combination antiretroviral therapy that has so dramatically reduced the mortality from AIDS in developed countries. Delivering such therapy where it is most needed is prohibitively expensive and adherence for the patient also has its difficulties ( Darbyshire 2000). We do not yet know whether the therapeutic success in the West is a temporary pause before multiresistant strains of HIV appear and spread, or whether virus load can be permanently controlled, thus abrogating progression to AIDS. Behavioural control of HIV transmission is notoriously difficult to introduce and maintain ( De Cock & Weiss 2000; Hart 2000). Thus the most important, yet daunting task facing virologists and immunologists is to provide an efficacious vaccine for mass immunization.