Journal of General Virology

Human immunodeficiency virus type 2

Jacqueline D. Reeves and Robert W. Doms

Department of Microbiology, University of Pennsylvania, 301 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104, USA

The AIDS pandemic continues to spread unchecked in many parts of the world, with greater than 34 million individuals currently infected with human immunodeficiency virus (HIV). While most infections are due to HIV type 1 (HIV-1) strains, HIV-2 represents a significant minority of all HIV infections in some countries, such as Guinea-Bissau and Portugal. While similar in many ways, there are important differences between HIV-1 and HIV-2 that provide insights into virus evolution, tropism and pathogenesis. Major differences include reduced pathogenicity of HIV-2 relative to HIV-1, enhanced immune control of HIV-2 infection and often some degree of CD4-independence. This review discusses the origin of HIV-2 and its relationship to simian immunodeficiency virus and HIV-1, its epidemiology, its pathogenic potential and how its Env protein interacts with cell surface receptors to mediate virus infection.

AIDS, resulting from HIV-1 infection, was first recognized in 1981 when a common pattern of symptoms was observed among a small number of homosexual men in the USA (Brennan & Durack, 1981; Gottlieb et al., 1981). AIDS cases were soon reported in other groups, including intravenous drug users and haemophiliacs (CDC, 1982; Davis et al., 1983; Masur et al., 1981). Soon after the identification of AIDS in humans, outbreaks of wasting and severe infections were identified in captive colonies of Asian rhesus macaques in USA primate centres and these symptoms became known as simian AIDS (Henrickson et al., 1983; Letvin et al., 1983). The sera from these animals showed cross-reactivity to HIV-1 antigens by Western blot (Kanki et al., 1985), which led to the identification of a related lentivirus, termed simian immunodeficiency virus (SIV) (Daniel et al., 1985). Surprisingly, sera from Senegalese sex workers were found to cross-react preferentially with SIV antigens compared to HIV-1 by Western blot, indicating exposure to an SIV-like virus (Barin et al., 1985). Subsequently, a virus more closely related to SIV than HIV-1 was isolated from West African AIDS patients from Guinea-Bissau and Cape Verde (Clavel et al., 1986). This virus, referred to as lymphadenopathy-associated virus type 2, is now known as HIV-2.

HIV-2, along with HIV-1 and SIV, comprise the subgenus 'primate lentiviruses'. The genomic organization of these viruses is similar, although HIV-1 and SIV of chimpanzees (SIV_cpz) encode a vpu gene, while HIV-2 and SIV of sooty mangabeys (SIV_sm) have a vpx gene. The function of Vpx is not clear, although it may play a role in nuclear import (Fletcher et al., 1996) and the functions normally associated with HIV-1 Vpr may be provided separately in HIV-2 by Vpr (cell cycle arrest in G₂) and Vpx (nuclear import) in HIV-2. Vpx (but not Vpr) is important for efficient replication of HIV-2 in PBMCs but not T cell lines (Park & Sodroski, 1995). Additionally, Vpx or Vpr SIVs can cause AIDS, while double mutant Vpx/Vpr SIVs are severely attenuated (Gibbs et al., 1995).

Despite similar genomic organizations, there is a high degree of genetic diversity between the primate lentiviruses, especially in their env genes. Genetic variability between HIV-2 strains is comparable to that within HIV-1 groups, with up to 25 % divergence in Gag, Pol and Env (Gao et al., 1994; Schulz et al., 1990; Zagury et al., 1988).

HIV is thought to have originated from zoonotic transmissions from SIV-infected non-human primates (Gao et al., 1992, 1999; Hirsch et al., 1989). SIVs from chimpanzees cluster phylogenetically with HIV-1 (Fig. 1) (Gao et al., 1999); hence, the HIV-1 epidemic is likely to have originated from SIV_cpz. In contrast, all criteria identifying HIV-2 as a zoonosis from the sooty mangabey (Cercocebus atys) are met: i.e. similarity in genomic organization; phylogenetic relatedness (Fig. 1); prevalence in the natural host; geographical overlap; and plausible route of transmission (Sharp et al., 1995). From phylogenetic analysis of divergent HIV-2 strains, it appears that there have been seven independent transmissions from sooty mangabeys to humans, resulting in HIV-2 subtypes A–G (Chen et al., 1997a; Gao et al., 1994; Yamaguchi et al., 2000). Only one member each of subtypes C, E, F and G, and two members of subtype D, have been identified (reviewed by Schim van der Loeff & Aaby, 1999) and it is thought that these rare subtypes may be primary zoonotic infections.

Fig. 1. Phylogenetic relationship of primate lentiviruses. Alignment of pol gene sequences of HIV-1, HIV-2 and SIV strain SIV_mm; SIV of sooty mangabeys or macaques experimentally infected with SIV_mm. Reproduced from Human Retroviruses and AIDS (Los Alamos, New Mexico; Theoretical Biology and Biophysics Group, Los Alamos National Laboratory) (Kuiken et al., 1999), with permission.

Epidemiology

The epicentre of HIV-1 infection is East Africa. In contrast, HIV-2 is restricted primarily to West Africa, although the prevalence of HIV-2 is a growing concern in certain parts of Europe and in the southwestern region of India. In Guinea-Bissau, a former Portuguese colony, there is an HIV-2 prevalence of up to 8–10 % (Poulsen et al., 1989; Wilkins et al., 1993). A lower prevalence of 1–2 % is found in surrounding countries, including The Gambia, Senegal and Guinea, although the prevalence among commercial sex workers in The Gambia is as high as 28 % (Ghys et al., 1997; Langley et al., 1996). In addition to West Africa, countries with past socio-economical links with Portugal, including southwest India (Babu et al., 1993; Kulkarni et al., 1992; Rubsamen-Waigmann et al., 1991), Angola, Mozambique and Brazil, have significant numbers of HIV-2 infections (reviewed by Schim van der Loeff & Aaby, 1999). Portugal has the highest prevalence of HIV-2 infection in Europe, accounting for around 10–13 % of total HIV infections and 4.5 % of AIDS cases (Soriano et al., 2000; UNAIDS/WHO, 1998).

Subtype A accounts for the majority of HIV-2 infections and is the predominant genotype in Guinea-Bissau and Europe (Norrgren et al., 1997; Schim van der Loeff & Aaby, 1999). The prototype HIV-2 strain, ROD, is a subtype A virus that was isolated from a Cape Verdian (Clavel et al., 1986). Subtype B viruses seem to have originated from the eastern parts of West Africa (Ghana and the Ivory Coast) and have occasionally been isolated in Europe (reviewed by Schim van der Loeff & Aaby, 1999). Sierra Leone has a low prevalence of HIV-2, around 0.02 %, but the highest diversity of HIV-2 subtypes (A, B, E and F), probably resulting from zoonoses from local sooty mangabeys infected with diverse strains of SIV_sm (Chen et al., 1996, 1997a; Gao et al., 1994).

Instances of dual infection (HIV-1⁺/HIV-2⁺) are occurring more frequently in HIV-2 endemic regions, such as Guinea-Bissau, due to the rising prevalence of HIV-1 infection in these areas (Andersson et al., 1999; Ishikawa et al., 1998), raising the possibility that recombination events between HIV-1 and HIV-2 will occur. The epidemiology of HIV-2 is detailed further in a review by Schim van der Loeff & Aaby (1999).

Transmission

Due to differences in transmission rates and virulence, HIV-1 is pandemic, with rising prevalence rates in developing countries, while HIV-2 is more endemic, with stable prevalence rates in most countries (Remy, 1998). The transmission of HIV-2 compared to HIV-1 is detailed elsewhere (Schim van der Loeff & Aaby, 1999). Briefly, HIV-2 appears to be transmitted by the same routes as HIV-1; however, the frequency of transmission is reduced, probably due to a very low virus load in many asymptomatic individuals (Adjorlolo-Johnson et al., 1994; Berry et al., 1998; Cavaco-Silva et al., 1998; Kanki et al., 1994; O'Donovan et al., 2000). For instance, sexual and vertical transmissions of HIV-2 are around 5- to 9-fold and 10- to 20-fold reduced relative to HIV-1, respectively.

Treatment of infection

HIV-2-infected individuals in Europe have been treated with anti-retroviral agents (Smith et al., 2001; Soriano et al., 2000; van der Ende, 2000; van der Ende et al., 2000) but there are no reports of large-scale clinical trials involving HIV-2-infected cohorts. As for HIV-1, the use of single anti-retroviral agents, or suboptimal combination therapy, can lead to the development of drug-resistance mutations in vivo (Rodes et al., 2000; Smith et al., 2001; van der Ende et al., 2000). Additionally, many HIV-2 strains naturally have amino acids that confer drug resistance and may thus decrease the therapeutic potential of some anti-retroviral agents (Isaka et al., 2001; van der Ende, 2000). However, others are active against HIV-2 (Clark et al., 1998; Witvrouw et al., 1999) and, with the correct drug combination, the reduced virus load and virulence of HIV-2 may make highly active anti-retroviral therapy extremely effective (Smith et al., 2001; Whittle et al., 1998). The distribution of anti-retroviral drugs within developing countries and the development of an inexpensive vaccine remain priorities.

Naturally infected sooty mangabeys, African green monkeys and chimpanzees do not develop SIV-related disease, even though high virus loads can sometimes be detected in their plasma (Desrosiers, 1990; Rey-Cuille et al., 1998). Disease is only associated with cross-species transmission of the viruses; i.e. from sooty mangabeys into humans (HIV-2) and rhesus macaques (SIV_mac) or from chimpanzees into humans (HIV-1). The clinical features of HIV-2 infection are similar to those of HIV-1 infection (Brun-Vezinet et al., 1987); however, HIV-2 is generally less pathogenic. This may be due to differences in virulence, the lower plasma virus load that is usually associated with HIV-2 infection or better immune control of HIV-2 replication compared to HIV-1 (Andersson et al., 2000; Berry et al., 1998; Simon et al., 1993; Soriano et al., 2000; Whittle et al., 1998). HIV-2-infected individuals usually have a long clinically latent period of 10 years or more, resulting in a mortality rate estimated to be two-thirds lower than that for HIV-1 (Marlink et al., 1994; Pepin et al., 1991; Whittle et al., 1994). Indeed, many HIV-2-infected individuals appear not to progress to AIDS at all (Poulsen et al., 1997). When infection does progress, however, virus load and rate of progression are fairly comparable between HIV-1 and HIV-2 (Berry et al., 1998; Simon et al., 1993; Whittle et al., 1992). Interestingly, the burden of proviral DNA is similar in HIV-1 and HIV-2 infections, hence the lower virus load typically observed in HIV-2-infected individuals may be accounted for by differences in virus production (Popper et al., 2000).

It has been reported that HIV-2 infection can prevent/protect ('immunize') against subsequent HIV-1 infection both in vitro (Arya & Gallo, 1996; Browning et al., 1999; Dern et al., 2001; Kokkotou et al., 2000; Rappaport et al., 1995) and in vivo (Greenberg et al., 1996; Travers et al., 1995, 1998); however, these in vivo findings have proved controversial (Aaby et al., 1997; Ariyoshi et al., 1997; Norrgren et al., 1999; Wiktor et al., 1999). Sera from HIV-2-infected individuals are often better at neutralizing autologous as well as heterologous virus in comparison to HIV-1⁺ sera and some HIV-2 antisera can cross-neutralize HIV-1 strains (Bjorling et al., 1993; Fenyo & Putkonen, 1996; Robert-Guroff et al., 1992; Weiss et al., 1988). Additionally, HIV-2-infected individuals often have a strong cytotoxic T lymphocyte (CTL) response to HIV-2, which can frequently cross-react with HIV-1 strains (Bertoletti et al., 1998; Gotch et al., 1993; Rowland-Jones et al., 1995). CTL responses to HIV-2 infection are reviewed further by Whittle et al. (1998).

The overproduction of chemokines by PBMCs from HIV-2-infected donors can prevent infection of R5 tropic (see below) HIV-1 strains in vitro (Kokkotou et al., 2000). Furthermore, HIV-2 (but not HIV-1) Env protein can interact with CD8 on T cells that are non-permissive to infection, triggering the production of chemokines (Akimoto et al., 1998; Ichiyama et al., 1999). Higher proportions of CD8⁺ T cells from HIV-2-infected individuals retain the ability to simultaneously produce the cytokines IL-2 and IFN-. Likewise, more CD4⁺ T cells are capable of producing IL-2 than those from HIV-1-infected individuals with equivalent CD4⁺ T cell counts (Sousa et al., 2001). These factors may also account in part for the better immune control of HIV-2 infection compared to that of HIV-1 infection. It is also interesting to note that deletions within the nef gene of HIV-2 are quite common (Switzer et al., 1998), while nef deletions are found infrequently in HIV-1, where they significantly reduce replication in vitro and in vivo (Piguet & Trono, 1999). Nef downregulates surface expression of MHC class I molecules (Kerkau et al., 1997; Le Gall et al., 1998), thus a functional Nef protein may aid evasion of host immune responses.

The clinical manifestations of HIV-2 AIDS are similar to those for HIV-1 and only minor differences in pathology resulting from HIV-2, compared to HIV-1, infection have been observed. For example, in the Ivory Coast, encephalitis was shown at autopsy to be almost restricted to individuals with HIV-2-related causes of death (18 %; n=40), compared to HIV-1 (<1 %; n=170) (Lucas et al., 1993). Whether this is because people infected with HIV-2 generally survive longer than those infected with HIV-1 or if HIV-2 is more neurotropic/neuropathogenic than HIV-1 is unknown. Additionally, AIDS-associated Kaposi's sarcoma occurs in around 10 % of HIV-1-infected individuals (Safai, 1997), although it is less frequent in HIV-1-infected Gambians and approximately 12-fold less frequent still in HIV-2-infected Gambians (Ariyoshi et al., 1998).

The env genes of HIV-1 and HIV-2 encode the precursor 160 and 140 kDa glycoproteins (gp160/gp140, respectively). These precursors are cleaved by a host protease into the HIV-1 120 kDa and HIV-2 125 kDa surface (SU) glycoproteins (gp120/gp125) and the 41 and 36 kDa transmembrane (TM) glycoproteins (gp41/gp36) for HIV-1 and HIV-2, respectively (Freed et al., 1989; McCune et al., 1988). SU and TM form glycoprotein 'spikes' in the virus membrane that consist of trimers of non-covalently linked SU and TM proteins (Center et al., 2001; Chan et al., 1997; Lu et al., 1995; Weissenhorn et al., 1997). As for HIV-1, the SU protein of HIV-2 contains five variable (V1–V5) and five conserved domains (C1–C5), numerous N-linked glycosylation sites and conserved disulphide bonds (Modrow et al., 1987; Starcich et al., 1986; Willey et al., 1986). In addition, residues in HIV-1 gp120 implicated in CD4 or coreceptor binding are highly conserved in SIV and HIV-2 (Kwong et al., 1998; Rizzuto et al., 1998), indicating that these proteins likely share similar structures. HIV and SIV TM proteins consist of an N-terminal extracellular domain, containing a fusion peptide and two -helical regions, a membrane-spanning domain and a C-terminal intracellular domain with an endocytosis/basolateral targeting signal and two amphipathic regions. Structural studies indicate that the HIV-1 and SIV TM proteins are highly similar (Blacklow et al., 1995; Chan et al., 1997; Lu et al., 1995; Weissenhorn et al., 1997; Yang et al., 1999), making it likely that the HIV-2 TM subunit closely resembles these proteins.

Interaction with CD4

The Env proteins of HIV are primarily involved in binding and entry. SU proteins interact with cellular receptors to attach virus particles to the cell surface and to induce conformational changes in both the SU and TM proteins, which enable triggering of the fusion process. As for HIV-1, CD4 is the primary receptor for all HIV-2 strains. However, a number of HIV-2 isolates have been described that can infect cells independently of CD4 (see below) (Reeves et al., 1999). For most HIV-1 strains, the Env–CD4 interaction is of high affinity, with dissociation constants occurring in the nM range; for example, around 3–4 nM for the HIV-1 IIIB strain (Lasky et al., 1987; Moore, 1990). The interaction of HIV-2 and SIV Env proteins with CD4 may be of somewhat lower affinity, although relatively few virus strains have been examined carefully (Ivey-Hoyle et al., 1991; Layne et al., 1990; Moore, 1990). For example, the dissociation constant of HIV-2_ROD/A SU protein to CD4 is approximately 45 nM, while that for the SIV_mac SU protein is around 350 nM (Ivey-Hoyle et al., 1991; Moore, 1990).

Interaction with coreceptors

CD4 binding induces conformational changes in the SU subunit of Env that enable it to bind a coreceptor. In the case of HIV-1, CD4 binding induces the formation and/or exposure of a highly conserved domain in the bridging sheet region of the SU protein that has been shown to be important for coreceptor binding (Fig. 2) (Rizzuto et al., 1998). Many residues in this region are conserved between HIV-1, HIV-2 and SIV strains. Thus, it is likely that CD4 binding induces similar conformational changes in HIV-2 Env. As for HIV-1, all HIV-2 strains use either CCR5 and/or CXCR4 as major coreceptors for entry into CD4⁺ cells (Bron et al., 1997; Deng et al., 1997; Hill et al., 1997; Pleskoff et al., 1997a, b; Rucker et al., 1997; Sol et al., 1997). Amino acids in the V3 loop of Env can determine X4 or R5 tropism for both HIV-1 and HIV-2 strains (Choe et al., 1996; Cocchi et al., 1996; Isaka et al., 1999; Speck et al., 1997). Many HIV-2 strains can, however, use a wider range of coreceptors compared to HIV-1 and may use these as efficiently as CCR5 or CXCR4 (Guillon et al., 1998; McKnight et al., 1998; Owen et al., 1998; Reeves et al., 1999; Unutmaz et al., 1998). Additionally, some HIV-2 strains can utilize coreceptors that are not, or are rarely, used by HIV-1 strains, including CCR1 (Guillon et al., 1998; McKnight et al., 1998; Owen et al., 1998), CCR4 (McKnight et al., 1998; Owen et al., 1998) and GPR1 (Liu et al., 2000; Shimizu et al., 1999). Other coreceptors used by HIV-2 strains include CCR2b (Guillon et al., 1998; McKnight et al., 1998; Owen et al., 1998), CCR3 (Bron et al., 1997; Reeves et al., 1997; Sol et al., 1997), CCR8 (Rucker et al., 1997; Simmons et al., 2000), CXCR5 (Kanbe et al., 1999), CX₃CR1 (Reeves et al., 1997; Rucker et al., 1997), GPR15 (Deng et al., 1997; Owen et al., 1998), STRL33 (Deng et al., 1997; Owen et al., 1998), RDC1 (Shimizu et al., 2000), APJ (Liu et al., 2000) and US28 (Pleskoff et al., 1997b). Thus, HIV-2 strains are, in general, more promiscuous that HIV-1 strains in their use of coreceptors. However, it is not clear if use of receptors other than CCR5 or CXCR4 is relevant in vivo. Some alternative coreceptors are not expressed on CD4⁺ cells or are expressed at levels below that needed to support virus infection (Sharron et al., 2000). Nonetheless, in contrast to HIV-1, some HIV-2 and SIV strains are able to infect PBMCs independently of either CCR5 or CXCR4 (Chen et al., 1997b, 1998; Simmons et al., 2000; Sol et al., 1997; Zhang et al., 2000) implicating a potential role of at least some alternative receptor(s) for infection in vivo. Additionally, HIV-2 infection may result in higher levels of chemokine production compared to HIV-1 infection (Akimoto et al., 1998; Kokkotou et al., 2000) and may thus drive evolution of alternative receptor use.

Coreceptor domains that are involved in mediating HIV-2 infection are similar to those required for HIV-1. For example, the N terminus and second extracellular loop of CXCR4 are important in mediating fusion and infection of HIV-2 ROD (Brelot et al., 1997; Potempa et al., 1997; Reeves et al., 1998).

Fig. 2. Predicted coreceptor-binding site on HIV-2 gp120. Space-filling model of HIV-2 gp120 core indicating potential residues involved in coreceptor interactions and the location of the V1/V2 and V3 loop stems. This structure was predicted by SWISSPROT, based upon existing HIV-1 gp120 core crystals (pdb accession codes 1GC1, 1G9N and 1GM9) and rendered with RASMOL, version 2.7.1.

While all HIV-1, HIV-2 and SIV isolates infect primary CD4⁺ T cells, isolates can differ in their relative tropism for macrophages and human T cells lines, as well as in their replication rates, and were thus historically divided into three groups. The first group consists of viruses that infect CD4⁺ T cell lines in vitro, induce syncytia (multinucleated giant cells) and replicate to high titres relatively quickly and are termed syncytium-inducing, T cell tropic or rapid/high viruses. The use of CXCR4 (X4 tropism) is characteristic of these viruses. The second group consists of those that infect macrophage cultures efficiently, do not induce syncytia in infected primary T cells and replicate slowly to relatively low titres and are thus referred to as macrophage tropic, non-syncytium-inducing or slow/low viruses. These viruses typically use CCR5 as their major coreceptor and are referred to as R5 tropic. The third group can infect both T cell lines and macrophages and are thus referred to as dual-tropic viruses. These viruses typically use both CXCR4 and CCR5 and are termed R5/X4. Thus, virus tropism is largely explained by coreceptor usage, although a host of other factors, including receptor density, conformation and post-entry events, can also influence virus infection. Additionally, primary syncytium-inducing isolates can infect macrophages via CXCR4 (Simmons et al., 1998).

For HIV-1, the importance of CCR5 for virus infection was shown by the discovery that individuals who lack CCR5 are highly resistant to virus infection (Liu et al., 1996; Samson et al., 1996). R5 virus strains are predominantly transmitted, are the major virus population in asymptomatic individuals and usually remain present throughout the course of infection (Connor et al., 1997; de Roda Husman et al., 1999; Huang et al., 1996; Li et al., 1999; van't Wout et al., 1998). R5X4 viruses may precede the evolution to X4 tropism, which occurs in less than 50 % of AIDS patients (Connor et al., 1997; Tersmette et al., 1988). An evolution from R5 to X4 is not obvious in HIV-2-infected individuals as many primary isolates use a range of coreceptors including both CCR5 and CXCR4 and only a limited number of X4 viruses have been isolated from symptomatic patients (Guillon et al., 1998; Morner et al., 1999; Reeves et al., 1999). In addition, it is not known if R5 HIV-2 strains are largely responsible for virus transmission, as only a few isolates from asymptomatic individuals predominantly use CCR5 (Guillon et al., 1998; Owen et al., 1998).

One of the most striking differences between HIV-1 and HIV-2 strains is that, while HIV-1 infection usually requires both CD4 and a coreceptor, many primary HIV-2 isolates exhibit some degree of CD4-independence (Reeves et al., 1999). Even some HIV-2 strains that typically require CD4 to infect cells can be induced to infect CD4 cell lines by pretreatment with soluble CD4 (sCD4) (Clapham et al., 1992; Reeves et al., 1999). In addition, CD4-independent HIV-2 variants can arise spontaneously in vitro from chronically infected T cell lines (Clapham et al., 1992; Endres et al., 1996). These viruses primarily use CXCR4 for infection of CD4 cells but can also use other 7TM receptors independently of CD4, including CCR3, CCR5, CCR8, GPR1, GPR15 and STRL33 (Endres et al., 1996; Lin et al., 2001; Reeves et al., 1997, 2000; Simmons et al., 2000). SIV strains that can use CCR5, GPR15 or STRL33 for CD4-independent infection have also been identified (Edinger et al., 1997, 1999; Reeves et al., 1999, 2000; Schenten et al., 1999), while only a few CD4-independent HIV-1 strains that were selected in vitro have been described (Dumonceaux et al., 1998; Kolchinsky et al., 1999; LaBranche et al., 1999).

The Env determinants responsible for the CD4-independence of two HIV-2 strains (ROD/B and vcp) have been analysed in detail (Lin et al., 2001; Reeves & Schulz, 1997). Only two mutations were required to confer a minimal CD4-independent phenotype to ROD/B, a variant of the prototypic CD4-dependent HIV-2 ROD/A strain, and a further two mutations conferred efficient CD4-independent infection (Reeves & Schulz, 1997). These same mutations increase the fusogenicity of the envelope protein as well as increasing the sensitivity of the envelope to undergo conformational changes induced by sCD4. The CD4-independence of the HIV-1 IIIB variant 8x is mediated by mutations in similar regions of envelope as those found in ROD/B (LaBranche et al., 1999), although the CD4-independence of other viruses is conferred by different regions of env (Dumonceaux et al., 1998; Kolchinsky et al., 1999). It is therefore apparent that CD4-independence can be acquired by multiple mechanisms and that only a few amino acid changes are needed to confer CD4-independence.

The fact that HIV-2 and SIV strains more readily infect CD4^– cells compared to HIV-1 strains may indicate that their coreceptor-binding site is at least partially exposed in the absence of CD4, enabling direct contact with a coreceptor (Fig. 3). CD4 binding by these strains is likely to modify the 7TM-binding site to increase the affinity of the Env–7TM interaction or to contribute extra energy to trigger fusion of virus and cell membranes. Either or both of these roles would provide HIV-2 with the capacity to exploit coreceptors that otherwise do not interact with gp120 strongly enough to trigger fusion. Indeed, CD4-independent HIV-2 strains that can use both CCR5 and CXCR4 to infect CD4⁺ cells are only able to use one of these receptors in the absence of CD4, indicating that the use of CD4 enables a wider range of 7TM receptors to be exploited for infection (Reeves et al., 1999).

Fig. 3. CD4-dependent and CD4-independent infection. For CD4-dependent infection, gp120 first interacts with CD4 at the cell surface. This interaction induces conformational changes in Env that allow a secondary interaction with a 7TM coreceptor to occur. Following coreceptor binding, further conformational changes result in fusion of viral and cellular membranes. CD4-independent viruses can bypass CD4 and interact directly with a coreceptor molecule to initiate fusion. The ability to infect cells in the absence of CD4 is associated with HIV-2 and SIV strains and correlates with exposed coreceptor binding sites, broadened cell tropism and enhanced sensitivity to neutralization.

Implications for pathogenesis

While CD4⁺ haematopoietic cells are the major targets of HIV in vivo, infection of CD4^– cells has been reported in a minority of HIV-1-positive individuals. However, the frequencies of infection of many CD4^– cell types in vivo are unknown and there are no reports of HIV-2 tropism for CD4^– cells in vivo. Nonetheless, CD4-independent viruses have the potential to infect a broader range of cell types in vivo. In vitro studies, coupled with the fact that viruses with distinct sequences and phenotypes can become compartmentalized in different tissues within an individual, such as the brain, lymphoid tissue and lungs (Epstein et al., 1991; Itescu et al., 1994; Sankale et al., 1996), indicate that selection can occur at different sites in vivo and suggest some possible sites where CD4-independent viruses may be important. In the brain, for example, CD4^– astrocytes are susceptible to HIV-1 infection in vivo (Bagasra et al., 1996; Stoler et al., 1986; Ward et al., 1987; Wiley et al., 1986), particularly in paediatric AIDS cases (Saito et al., 1994; Tornatore et al., 1994). Such infection is usually restrictive, however, with structural gag and env genes poorly expressed. HIV-2- and SIV-infected astrocytes have been cultured ex vivo from infected macaques, indicating that both HIV-2 and SIV can also infect astrocytes in vivo (Guillemin et al., 1998). In addition, CD4-independent HIV-2 ROD/B can productively infect astrocytes in vitro via CXCR4 (Reeves et al., 1999). A neurovirulent SIV strain can infect CD4^– brain capillary endothelial cells (BCECs) both in vitro and in vivo (Edinger et al., 1997; Mankowski et al., 1994) and HIV-1 infection of BCECs has also been observed in vitro via CCR5 and in vivo (Bagasra et al., 1996; Moses et al., 1993; Ward et al., 1987; Wiley et al., 1986). Therefore, both HIV-2 and SIV strains have the potential to infect CD4-independently in vivo via an interaction with CCR5 or CXCR4.

Implications for vaccine design and therapy

HIV Env contains highly variable regions that may present antigenic decoys to the immune system. In addition, it is heavily glycosylated and attempts to generate broadly cross-reactive neutralizing antibodies have been largely unsuccessful. With the identification of the receptors used by HIV to infect cells coupled with structural studies, conserved, functionally important regions in Env have been identified that could be targets for neutralizing antibodies. Env proteins that elicit antibodies to conserved epitopes, such as the coreceptor-binding domain, may prove to be better vaccine candidates. CD4-independent virus strains can differ from CD4-dependent isolates by only a few amino acids, yet are invariably far more sensitive to neutralizing antibodies and HIV-positive human serum (Edwards et al., 2001; Hoffman et al., 1999). Therefore, the conformation of Env glycoproteins that allows a direct interaction with coreceptors may reveal epitopes to neutralizing antibodies that are usually exposed only after CD4 binding and thus favourably influence the capacity of the host to control virus replication. Monoclonal antibodies to such epitopes do interact preferentially with CD4-independent HIV-1 strains (Edwards et al., 2001; Hoffman et al., 1999). The very low virus load observed during asymptomatic HIV-2 infection may result partly from better immune control of viruses with 'open' Env proteins, which, in turn, could explain why many HIV-2-infected individuals often survive significantly longer than those infected with HIV-1. Indeed, sera from HIV-2-infected individuals are often broadly cross neutralizing (Bjorling et al., 1993; Robert-Guroff et al., 1992; Whittle et al., 1998). It will be important to determine if genetically triggered, CD4-independent Env proteins can preferentially elicit neutralizing antibodies in addition to being more susceptible to them.

Implications for virus evolution

The ability of many HIV-2 and SIV strains to infect cells independently of CD4 suggests that the virus ancestors of HIV and SIV may have originally used a 7TM receptor alone. Acquisition of a second receptor, i.e. CD4, may have provided selective advantages to a virus that persistently replicates in the face of a vigorous host immune response. On HIV-1 envelopes, the gp120 site for binding a 7TM receptor is only fully exposed following contact with CD4 (Figs 2 and 3). This mechanism may enable potential neutralizing epitopes on or around the 7TM-binding site to be hidden until the fusion reaction is triggered. Binding to CD4 also enables a wider range of 7TM receptors to be exploited for infection (Reeves et al., 1999) and may assist adaptation or switching to new coreceptors in vivo. Indeed for HIV-1 strains, CD4 use may aid the evolution from R5 to R5X4 tropism, which seems to compromise or weaken the interaction of Env with CCR5, as infection via CCR5 for R5X4 strains is especially sensitive both to CCR5 amino acid substitutions (Bieniasz et al., 1997; Picard et al., 1997) and to inhibition by RANTES (Kledal et al., 1997).

HIV-2 provides an interesting contrast to HIV-1. While HIV-1 has spread rapidly throughout the world, HIV-2 has remained more restricted in its distribution, being relatively rare outside of West Africa. For the most part, HIV-2 behaves similarly to HIV-1 in vitro, with its ability to frequently infect cells CD4-independently being a notable exception. Yet, in vivo, HIV-2 is clearly less pathogenic than HIV-1; it is transmitted less efficiently, establishes lower virus loads in asymptomatic infection and results in slower disease progression. HIV-2 therefore provides an important opportunity to study immune control and pathogenesis of lentivirus infections in humans. In effect, HIV-2 appears to be a mildly attenuated form of HIV-1, yet is clearly more pathogenic than SIV in its natural host, the sooty mangabey. Identifying the virus and host factors that account for this are likely to clarify the mechanisms that account for the pathogenic nature of HIV-1 and may help to identify immune factors responsible for the control of HIV-2 infection. This, in turn, could assist in HIV vaccine development. Increased study of primary HIV-2 strains as well as HIV-2-infected individuals will be needed to accomplish these goals.

We would like to thank Paul Clapham and James Hoxie for helpful comments on this manuscript and Mark Biscone for modelling the CCR5-binding site on gp120. Our work is supported by NIH grants AI40880 and AI35383, by a Burroughs Wellcome Fund Translational Research Award and by an Elizabeth Glaser Scientist Award from the Pediatric AIDS Foundation.

Aaby, P., Poulsen, A. G., Larsen, O., Christiansen, C. B., Jensen, H., Melbye, M., Naucler, A. & Dias, F. (1997). Does HIV-2 protect against HIV-1 infection? AIDS 11, 939–940.

Adjorlolo-Johnson, G., De Cock, K. M., Ekpini, E., Vetter, K. M., Sibailly, T., Brattegaard, K., Yavo, D., Doorly, R., Whitaker, J. P., Kestens, L., Ou, C.-Y., George, J. R. & Gayle, H. (1994). Prospective comparison of mother-to-child transmission of HIV-1 and HIV-2 in Abidjan, Ivory Coast. The Journal of the American Medical Association 272, 462–466.

Akimoto, H., Kaneko, H., Sekigawa, I., Hashimoto, H., Kaneko, Y. & Yamamoto, N. (1998). Binding of HIV-2 envelope glycoprotein to CD8 molecules and related chemokine production. Immunology 95, 214–218.

Andersson, S., Norrgren, H., Dias, F., Biberfeld, G. & Albert, J. (1999). Molecular characterization of human immunodeficiency virus (HIV)-1 and -2 in individuals from Guinea-Bissau with single or dual infections: predominance of a distinct HIV-1 subtype A/G recombinant in West Africa. Virology 262, 312–320.

Andersson, S., Norrgren, H., da Silva, Z., Biague, A., Bamba, S., Kwok, S., Christopherson, C., Biberfeld, G. & Albert, J. (2000). Plasma viral load in HIV-1 and HIV-2 singly and dually infected individuals in Guinea-Bissau, West Africa: significantly lower plasma virus set point in HIV-2 infection than in HIV-1 infection. Archives of Internal Medicine 160, 3286–3293.

Ariyoshi, K., Schim van der Loeff, M., Sabally, S., Cham, F., Corrah, T. & Whittle, H. (1997). Does HIV-2 infection provide cross-protection against HIV-1 infection? AIDS 11, 1053–1054.

Ariyoshi, K., Cham, F., Berry, N., Harding, E., Sabally, S., N'Gom, P. T., Ishikawa, K., Corrah, T., Tedder, R. & Whittle, H. (1998). Diagnosis of HIV-1/2 dual infection using dilution analysis of type-specific antibody. AIDS 12, 2504–2505.

Arya, S. K. & Gallo, R. C. (1996). Human immunodeficiency virus (HIV) type 2-mediated inhibition of HIV type 1: a new approach to gene therapy of HIV-infection. Proceedings of the National Academy of Sciences, USA 93, 4486–4491.

Babu, P. G., Saraswathi, N. K., Devapriya, F. & John, T. J. (1993). The detection of HIV-2 infection in southern India. Indian Journal of Medical Research 97, 49–52.

Bagasra, O., Lavi, E., Bobroski, L., Khalili, K., Pestaner, J. P., Tawadros, R. & Pomerantz, R. J. (1996). Cellular reservoirs of HIV-1 in the central nervous system of infected individuals: identification by the combination of in situ polymerase chain reaction and immunohistochemistry. AIDS 10, 573–585.

Barin, F., M'Boup, S., Denis, F., Kanki, P., Allan, J. S., Lee, T. H. & Essex, M. (1985). Serological evidence for virus related to simian T-lymphotropic retrovirus III in residents of west Africa. Lancet 2, 1387–1389.

Berry, N., Ariyoshi, K., Jaffar, S., Sabally, S., Corrah, T., Tedder, R. & Whittle, H. (1998). Low peripheral blood viral HIV-2 RNA in individuals with high CD4 percentage differentiates HIV-2 from HIV-1 infection. Journal of Human Virology 1, 457–468.

Bertoletti, A., Cham, F., McAdam, S., Rostron, T., Rowland-Jones, S., Sabally, S., Corrah, T., Ariyoshi, K. & Whittle, H. (1998). Cytotoxic T cells from human immunodeficiency virus type 2-infected patients frequently cross-react with different human immunodeficiency virus type 1 clades. Journal of Virology 72, 2439–2448.

Bieniasz, P. D., Fridell, R. A., Aramori, I., Ferguson, S. S., Caron, M. G. & Cullen, B. R. (1997). HIV-1-induced cell fusion is mediated by multiple regions within both the viral envelope and the CCR-5 co-receptor. EMBO Journal 16, 2599–2609.

Bjorling, E., Scarlatti, G., von Gegerfelt, A., Albert, J., Biberfeld, G., Chiodi, F., Norrby, E. & Fenyo, E. M. (1993). Autologous neutralizing antibodies prevail in HIV-2 but not in HIV-1 infection. Virology 193, 528–530.

Blacklow, S. C., Lu, M. & Kim, P. S. (1995). A trimeric subdomain of the simian immunodeficiency virus envelope glycoprotein. Biochemistry 34, 14955–14962.

Brelot, A., Heveker, N., Pleskoff, O., Sol., N. & Alizon, M. (1997). Role of the first and third extracellular domains of CXCR-4 in human immunodeficiency virus coreceptor activity. Journal of Virology 71, 4744–4751.

Brennan, R. O. & Durack, D. T. (1981). Gay compromise syndrome. Lancet 2, 1338–1339.

Bron, R., Klasse, P. J., Wilkinson, D., Clapham, P. R., Pelchen-Matthews, A., Power, C., Wells, T. N., Kim, J., Peiper, S. C., Hoxie, J. A. & Marsh, M. (1997). Promiscuous use of CC and CXC chemokine receptors in cell-to-cell fusion mediated by a human immunodeficiency virus type 2 envelope protein. Journal of Virology 71, 8405–8415.

Browning, C. M., Cagnon, L., Good, P. D., Rossi, J., Engelke, D. R. & Markovitz, D. M. (1999). Potent inhibition of human immunodeficiency virus type 1 (HIV-1) gene expression and virus production by an HIV-2 tat activation-response RNA decoy. Journal of Virology 73, 5191–5195.

Brun-Vezinet, F., Rey, M. A., Katlama, C., Girard, P. M., Roulot, D., Yeni, P., Lenoble, L., Clavel, F., Alizon, M., Gadelle, S. & Harzic, M. (1987). Lymphadenopathy-associated virus type 2 in AIDS and AIDS-related complex. Clinical and virological features in four patients. Lancet 1, 128–132.

Cavaco-Silva, P., Taveira, N. C., Rosado, L., Lourenco, M. H., Moniz-Pereira, J., Douglas, N. W., Daniels, R. S. & Santos-Ferreira, M. O. (1998). Virological and molecular demonstration of human immunodeficiency virus type 2 vertical transmission. Journal of Virology 72, 3418–3422.

CDC (1982). Epidemiologic aspects of the current outbreak of Kaposi's sarcoma and opportunistic infections. New England Journal of Medicine 306, 248–252.

Center, R. J., Schuck, P., Leapman, R. D., Arthur, L. O., Earl, P. L., Moss, B. & Lebowitz, J. (2001). Oligomeric structure of virion-associated and soluble forms of the simian immunodeficiency virus envelope protein in the prefusion activated conformation. Proceedings of the National Academy of Sciences, USA 98, 14877–14882.

Chan, D. C., Fass, D., Berger, J. M. & Kim, P. S. (1997). Core structure of gp41 from the HIV envelope glycoprotein. Cell 89, 263–273.

Chen, Z., Telfier, P., Gettie, A., Reed, P., Zhang, L., Ho, D. D. & Marx, P. A. (1996). Genetic characterization of new West African simian immunodeficiency virus SIV_sm: geographic clustering of household-derived SIV strains with human immunodeficiency virus type 2 subtypes and genetically diverse viruses from a single feral sooty mangabey troop. Journal of Virology 70, 3617–3627.

Chen, Z., Luckay, A., Sodora, D. L., Telfer, P., Reed, P., Gettie, A., Kanu, J. M., Sadek, R. F., Yee, J., Ho, D. D., Zhang, L. & Marx, P. A. (1997a). Human immunodeficiency virus type 2 (HIV-2) seroprevalence and characterization of a distinct HIV-2 genetic subtype from the natural range of simian immunodeficiency virus-infected sooty mangabeys. Journal of Virology 71, 3953–3960.

Chen, Z., Zhou, P., Ho, D. D., Landau, N. R. & Marx, P. A. (1997b). Genetically divergent strains of simian immunodeficiency virus use CCR5 as a coreceptor for entry. Journal of Virology 71, 2705–2714.

Chen, Z., Gettie, A., Ho, D. D. & Marx, P. A. (1998). Primary SIV_sm isolates use the CCR5 coreceptor from sooty mangabeys naturally infected in West Africa: a comparison of coreceptor usage of primary SIV_sm, HIV-2, and SIV_mac. Virology 246, 113–124.

Choe, H., Farzan, M., Sun, Y., Sullivan, N., Rollins, B., Ponath, P. D., Wu, L., Mackay, C. R., LaRosa, G., Newman, W., Gerard, N., Gerard, C. & Sodroski, J. (1996). The -chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85, 1135–1148.

Clapham, P. R., McKnight, A. & Weiss, R. A. (1992). Human immunodeficiency virus type 2 infection and fusion of CD4-negative human cell lines: induction and enhancement by soluble CD4. Journal of Virology 66, 3531–3537.

Clark, N. M., Dieng Sarr, A., Sankale, J. L., Kanki, P. J., Kazanjian, P., Winfield, R. & Markovitz, D. M. (1998). Immunologic and virologic response of HIV-2 infection to antiretroviral therapy. AIDS 12, 2506–2507.

Clavel, F., Guetard, D., Brun-Vezinet, F., Chamaret, S., Rey, M. A., Santos-Ferreira, M. O., Laurent, A. G., Dauguet, C., Katlama, C., Rouzioux, C., Klatzmann, D., Champalimaud, J. L. & Montagnier, L. (1986). Isolation of a new human retrovirus from West African patients with AIDS. Science 233, 343–346.

Cocchi, F., DeVico, A. L., Garzino-Demo, A., Cara, A., Gallo, R. C. & Lusso, P. (1996). The V3 domain of the HIV-1 gp120 envelope glycoprotein is critical for chemokine-mediated blockade of infection. Nature Medicine 2, 1244–1247.

Connor, R. I., Sheridan, K. E., Ceradini, D., Choe, S. & Landau, N. R. (1997). Change in coreceptor use correlates with disease progression in HIV-1 infected individuals. Journal of Experimental Medicine 185, 621–628.

Daniel, M. D., Letvin, N. L., King, N. W., Kannagi, M., Sehgal, P. K., Hunt, R. D., Kanki, P. J., Essex, M. & Desrosiers, R. C. (1985). Isolation of T-cell tropic HTLV-III-like retrovirus from macaques. Science 228, 1201–1204.

Davis, K. C., Horsburgh, C. R., Jr, Hasiba, U., Schocket, A. L. & Kirkpatrick, C. H. (1983). Acquired immunodeficiency syndrome in a patient with hemophilia. Annals of Internal Medicine 98, 284–286.

Deng, H. K., Unutmaz, D., KewalRamani, V. N. & Littman, D. R. (1997). Expression cloning of new receptors used by simian and human immunodeficiency viruses. Nature 388, 296–300.

Dern, K., Rubsamen-Waigmann, H. & Unger, R. E. (2001). Inhibition of HIV type 1 replication by simultaneous infection of peripheral blood lymphocytes with human immunodeficiency virus types 1 and 2. AIDS Research and Human Retroviruses 17, 295–309.

de Roda Husman, A. M., van Rij, R. P., Blaak, H., Broersen, S. & Schuitemaker, H. (1999). Adaptation to promiscuous usage of chemokine receptors is not a prerequisite for human immunodeficiency virus type 1 disease progression. Journal of Infectious Diseases 180, 1106–1115.

Desrosiers, R. C. (1990). The simian immunodeficiency viruses. Annual Review of Immunology 8, 557–578.

Dumonceaux, J., Nisole, S., Chanel, C., Quivet, L., Amara, A., Baleux, F., Briand, P. & Hazan, U. (1998). Spontaneous mutations in the env gene of the human immunodeficiency virus type 1 NDK isolate are associated with a CD4-independent entry phenotype. Journal of Virology 72, 512–519.

Edinger, A. L., Mankowski, J. L., Doranz, B. J., Margulies, B. J., Lee, B., Rucker, J., Sharron, M., Hoffman, T. L., Berson, J. F., Zink, M. C., Hirsch, V. M., Clements, J. E. & Doms, R. W. (1997). CD4-independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain. Proceedings of the National Academy of Sciences, USA 94, 14742–14747.

Edinger, A. L., Blanpain, C., Kunstman, K. J., Wolinsky, S. M., Parmentier, M. & Doms, R. W. (1999). Functional dissection of CCR5 coreceptor function through the use of CD4-independent simian immunodeficiency virus strains. Journal of Virology 73, 4062–4073.

Edwards, T. G., Hoffman, T. L., Baribaud, F., Wyss, S., LaBranche, C. C., Romano, J., Adkinson, J., Sharron, M., Hoxie, J. A. & Doms, R. W. (2001). Relationships between CD4 independence, neutralization sensitivity, and exposure of a CD4-induced epitope in a human immunodeficiency virus type 1 envelope protein. Journal of Virology 75, 5230–5239.

Endres, M. J., Clapham, P. R., Marsh, M., Ahuja, M., Turner, J. D., McKnight, A., Thomas, J. F., Stoebenau-Haggarty, B., Choe, S., Vance, P. J., Wells, T. N., Power, C. A., Sutterwala, S. S., Doms, R. W., Landau, N. R. & Hoxie, J. A. (1996). CD4-independent infection by HIV-2 is mediated by fusin/CXCR4. Cell 87, 745–756.

Epstein, L. G., Kuiken, C., Blumberg, B. M., Hartman, S., Sharer, L. R., Clement, M. & Goudsmit, J. (1991). HIV-1 V3 domain variation in brain and spleen of children with AIDS: tissue-specific evolution within host-determined quasispecies. Virology 180, 583–590.

Fenyo, E. M. & Putkonen, P. (1996). Broad cross-neutralizing activity in serum is associated with slow progression and low risk of transmission in primate lentivirus infections. Immunology Letters 51, 95–99.

Fletcher, T. M., III, Brichacek, B., Sharova, N., Newman, M. A., Stivahtis, G., Sharp, P. M., Emerman, M., Hahn, B. H. & Stevenson, M. (1996). Nuclear import and cell cycle arrest functions of the HIV-1 Vpr protein are encoded by two separate genes in HIV-2/SIV_sm. EMBO Journal 15, 6155–6165.

Freed, E. O., Myers, D. J. & Risser, R. (1989). Mutational analysis of the cleavage sequence of the human immunodeficiency virus type 1 envelope glycoprotein precursor gp160. Journal of Virology 63, 4670–4675.

Gao, F., Yue, L., White, A. T., Pappas, P. G., Barchue, J., Hanson, A. P., Greene, B. M., Sharp, P. M., Shaw, G. M. & Hahn, B. H. (1992). Human infection by genetically diverse SIV_SM-related HIV-2 in West Africa. Nature 358, 495–499.

Gao, F., Yue, L., Robertson, D. L., Hill, S. C., Hui, H., Biggar, R. J., Neequaye, A. E., Whelan, T. M., Ho, D. D., Shaw, G. M., Sharp, P. M. & Hahn, B. H. (1994). Genetic diversity of human immunodeficiency virus type 2: evidence for distinct sequence subtypes with differences in virus biology. Journal of Virology 68, 7433–7447.

Gao, F., Bailes, E., Robertson, D. L., Chen, Y., Rodenburg, C. M., Michael, S. F., Cummins, L. B., Arthur, L. O., Peeters, M., Shaw, G. M., Sharp, P. M. & Hahn, B. H. (1999). Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes. Nature 397, 436–441.

Ghys, P. D., Fransen, K., Diallo, M. O., Ettiegne-Traore, V., Coulibaly, I. M., Yeboue, K. M., Kalish, M. L., Maurice, C., Whitaker, J. P., Greenberg, A. E. & Laga, M. (1997). The associations between cervicovaginal HIV shedding, sexually transmitted diseases and immunosuppression in female sex workers in Abidjan, Cote d'Ivoire. AIDS 11, F85–93.

Gibbs, J. S., Lackner, A. A., Lang, S. M., Simon, M. A., Sehgal, P. K., Daniel, M. D. & Desrosiers, R. C. (1995). Progression to AIDS in the absence of a gene for Vpr or Vpx. Journal of Virology 69, 2378–2383.

Gotch, F., McAdam, S. N., Allsopp, C. E., Gallimore, A., Elvin, J., Kieny, M. P., Hill, A. V., McMichael, A. J. & Whittle, H. C. (1993). Cytotoxic T cells in HIV2 seropositive Gambians. Identification of a virus-specific MHC-restricted peptide epitope. Journal of Immunology 151, 3361–3369.

Gottlieb, M. S., Schroff, R., Schanker, H. M., Weisman, J. D., Fan, P. T., Wolf, R. A. & Saxon, A. (1981). Pneumocystis carinii pneumonia and mucosal candidiasis in previously healthy homosexual men: evidence of a new acquired cellular immunodeficiency. New England Journal of Medicine 305, 1425–1431.

Greenberg, A. E., Wiktor, S. Z., DeCock, K. M., Smith, P., Jaffe, H. W. & Dondero, T. J., Jr (1996). HIV-2 and natural protection against HIV-1 infection. Science 272, 1959–1960.

Guillemin, G., Croitoru, J., Boussin, F. D., Le Grand, R., Franck-Duchenne, M. & Dormont, D. (1998). Astrocytes and lentivirus infection in an experimental model of macaque infected with SIV_mac251. Comptes Rendus des Séances de la Société de Biologie et de Ses Filiales 192, 179–186.

Guillon, C., van der Ende, M. E., Boers, P. H., Gruters, R. A., Schutten, M. & Osterhaus, A. D. (1998). Coreceptor usage of human immunodeficiency virus type 2 primary isolates and biological clones is broad and does not correlate with their syncytium-inducing capacities. Journal of Virology 72, 6260–6263.

Henrickson, R. V., Maul, D. H., Osborn, K. G., Sever, J. L., Madden, D. L., Ellingsworth, L. R., Anderson, J. H., Lowenstine, L. J. & Gardner, M. B. (1983). Epidemic of acquired immunodeficiency in rhesus monkeys. Lancet 1, 388–390.

Hill, C. M., Deng, H., Unutmaz, D., Kewalramani, V. N., Bastiani, L., Gorny, M. K., Zolla-Pazner, S. & Littman, D. R. (1997). Envelope glycoproteins from human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus can use human CCR5 as a coreceptor for viral entry and make direct CD4-dependent interactions with this chemokine receptor. Journal of Virology 71, 6296–6304.

Hirsch, V. M., Olmsted, R. A., Murphey-Corb, M., Purcell, R. H. & Johnson, P. R. (1989). An African primate lentivirus (SIV_sm) closely related to HIV-2. Nature 339, 389–392.

Hoffman, T. L., LaBranche, C. C., Zhang, W., Canziani, G., Robinson, J., Chaiken, I., Hoxie, J. A. & Doms, R. W. (1999). Stable exposure of the coreceptor-binding site in a CD4-independent HIV-1 envelope protein. Proceedings of the National Academy of Sciences, USA 96, 6359–6364.

Huang, Y., Paxton, W. A., Wolinsky, S. M., Neumann, A. U., Zhang, L., He, T., Kang, S., Ceradini, D., Jin, Z., Yazdanbakhsh, K., Kunstman, K., Erickson, D., Dragon, E., Landau, N. R., Phair, J., Ho, D. D. & Koup, R. A. (1996). The role of a mutant CCR5 allele in HIV-1 transmission and disease progression. Nature Medicine 2, 1240–1243.

Ichiyama, K., Akimoto, H., Neoh, L. P., Takeda-Hirokawa, N., Kaneko, H., Hishikawa, T., Hashimoto, H., Hirose, S., Kaneko, Y., Yamamoto, N. & Sekigawa, I. (1999). HIV type 2 can bind to CD8 molecules but does not infect CD8⁺ T cells. AIDS Research and Human Retroviruses 15, 687–688.

Isaka, Y., Sato, A., Miki, S., Kawauchi, S., Sakaida, H., Hori, T., Uchiyama, T., Adachi, A., Hayami, M., Fujiwara, T. & Yoshie, O. (1999). Small amino acid changes in the V3 loop of human immunodeficiency virus type 2 determine the coreceptor usage for CXCR4 and CCR5. Virology 264, 237–243.

Isaka, Y., Miki, S., Kawauchi, S., Suyama, A., Sugimoto, H., Adachi, A., Miura, T., Hayami, M., Yoshie, O., Fujiwara, T. & Sato, A. (2001). A single amino acid change at Leu-188 in the reverse transcriptase of HIV-2 and SIV renders them sensitive to non-nucleoside reverse transcriptase inhibitors. Archives of Virology 146, 743–755.

Ishikawa, K., Fransen, K., Ariyoshi, K., Nkengasong, J. N., Janssens, W., Heyndrickx, L., Whittle, H., Diallo, M. O., Ghys, P. D., Coulibaly, I. M., Greenberg, A. E., Piedade, J., Canas-Ferreira, W. & van der Groen, G. (1998). Improved detection of HIV-2 proviral DNA in dually seroreactive individuals by PCR. AIDS 12, 1419–1425.

Itescu, S., Simonelli, P. F., Winchester, R. J. & Ginsberg, H. S. (1994). Human immunodeficiency virus type 1 strains in the lungs of infected individuals evolve independently from those in peripheral blood and are highly conserved in the C-terminal region of the envelope V3 loop. Proceedings of the National Academy of Sciences, USA 91, 11378–11382.

Ivey-Hoyle, M., Culp, J. S., Chaikin, M. A., Hellmig, B. D., Matthews, T. J., Sweet, R. W. & Rosenberg, M. (1991). Envelope glycoproteins from biologically diverse isolates of immunodeficiency viruses have widely different affinities for CD4. Proceedings of the National Academy of Sciences, USA 88, 512–516.

Kanbe, K., Shimizu, N., Soda, Y., Takagishi, K. & Hoshino, H. (1999). A CXC chemokine receptor, CXCR5/BLR1, is a novel and specific coreceptor for human immunodeficiency virus type 2. Virology 265, 264–273.

Kanki, P. J., McLane, M. F., King, N. W., Jr, Letvin, N. L., Hunt, R. D., Sehgal, P., Daniel, M. D., Desrosiers, R. C. & Essex, M. (1985). Serologic identification and characterization of a macaque T-lymphotropic retrovirus closely related to HTLV-III. Science 228, 1199–1201.

Kanki, P. J., Travers, K. U., MBoup, S., Hsieh, C. C., Marlink, R. G., Gueye, N. A., Siby, T., Thior, I., Hernandez-Avila, M., Sankale, J. L., Ndoye, I. & Essex, M. E. (1994). Slower heterosexual spread of HIV-2 than HIV-1. Lancet 343, 943–946.

Kerkau, T., Bacik, I., Bennink, J. R., Yewdell, J. W., Hunig, T., Schimpl, A. & Schubert, U. (1997). The human immunodeficiency virus type 1 (HIV-1) Vpu protein interferes with an early step in the biosynthesis of major histocompatibility complex (MHC) class I molecules. Journal of Experimental Medicine 185, 1295–1305.

Kledal, T. N., Rosenkilde, M. M., Coulin, F., Simmons, G., Johnsen, A. H., Alouani, S., Power, C. A., Luttichau, H. R., Gerstoft, J., Clapham, P. R., Clark-Lewis, I., Wells, T. N. C. & Schwartz, T. W. (1997). A broad-spectrum chemokine antagonist encoded by Kaposi's sarcoma-associated herpesvirus. Science 277, 1656–1659.

Kokkotou, E. G., Sankale, J. L., Mani, I., Gueye-Ndiaye, A., Schwartz, D., Essex, M. E., Mboup, S. & Kanki, P. J. (2000). In vitro correlates of HIV-2-mediated HIV-1 protection. Proceedings of the National Academy of Sciences, USA 97, 6797–6802.

Kolchinsky, P., Mirzabekov, T., Farzan, M., Kiprilov, E., Cayabyab, M., Mooney, L. J., Choe, H. & Sodroski, J. (1999). Adaptation of a CCR5-using, primary human immunodeficiency virus type 1 isolate for CD4-independent replication. Journal of Virology 73, 8120–8126.

Kuiken, C., Foley, B., Hahn, B., Marx, P., McCutchan, F., Mellors, J. W., Mullins, J., Wolinsky, S. & Korber, B. (1999). A compilation and analysis of nucleic acid and amino acid sequences. In Human Retroviruses and AIDS. Los Alamos, New Mexico: Theoretical Biology and Biophysics Group, Los Alamos National Laboratory.

Kulkarni, S., Thakar, M., Rodrigues, J. & Banerjee, K. (1992). HIV-2 antibodies in serum samples from Maharashtra state. Indian Journal of Medical Research 95, 213–215.

Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J. & Hendrickson, W. A. (1998). Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393, 648–659.

LaBranche, C. C., Hoffman, T. L., Romano, J., Haggarty, B. S., Edwards, T. G., Matthews, T. J., Doms, R. W. & Hoxie, J. A. (1999). Determinants of CD4-independence for a human immunodeficiency virus type 1 variant map outside regions required for coreceptor specificity. Journal of Virology 73, 10310–10319.

Langley, C. L., Benga-De, E., Critchlow, C. W., Ndoye, I., Mbengue-Ly, M. D., Kuypers, J., Woto-Gaye, G., Mboup, S., Bergeron, C., Holmes, K. K. & Kiviat, N. B. (1996). HIV-1, HIV-2, human papillomavirus infection and cervical neoplasia in high-risk African women. AIDS 10, 413–417.

Lasky, L. A., Nakamura, G., Smith, D. H., Fennie, C., Shimasaki, C., Patzer, E., Berman, P., Gregory, T. & Capon, D. J. (1987). Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor. Cell 50, 975–985.

Layne, S. P., Merges, M. J., Dembo, M., Spouge, J. L. & Nara, P. L. (1990). HIV requires multiple gp120 molecules for CD4-mediated infection. Nature 346, 277–279.

Le Gall, S., Erdtmann, L., Benichou, S., Berlioz-Torrent, C., Liu, L., Benarous, R., Heard, J. M. & Schwartz, O. (1998). Nef interacts with the mu subunit of clathrin adaptor complexes and reveals a cryptic sorting signal in MHC I molecules. Immunity 8, 483–495.

Letvin, N. L., Eaton, K. A., Aldrich, W. R., Sehgal, P. K., Blake, B. J., Schlossman, S. F., King, N. W. & Hunt, R. D. (1983). Acquired immunodeficiency syndrome in a colony of macaque monkeys. Proceedings of the National Academy of Sciences, USA 80, 2718–2722.

Li, S., Juarez, J., Alali, M., Dwyer, D., Collman, R., Cunningham, A. & Naif, H. M. (1999). Persistent CCR5 utilization and enhanced macrophage tropism by primary blood human immunodeficiency virus type 1 isolates from advanced stages of disease and comparison to tissue-derived isolates. Journal of Virology 73, 9741–9755.

Lin, G., Lee, B., Haggarty, B. S., Doms, R. W. & Hoxie, J. A. (2001). CD4-independent use of rhesus CCR5 by human immunodeficiency virus type 2 implicates an electrostatic interaction between the CCR5 N terminus and the gp120 C4 domain. Journal of Virology 75, 10766–10778.

Liu, R., Paxton, W. A., Choe, S., Ceradini, D., Martin, S. R., Horuk, R., MacDonald, M. E., Stuhlmann, H., Koup, R. A. & Landau, N. R. (1996). Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86, 367–377.

Liu, H. Y., Soda, Y., Shimizu, N., Haraguchi, Y., Jinno, A., Takeuchi, Y. & Hoshino, H. (2000). CD4-Dependent and CD4-independent utilization of coreceptors by human immunodeficiency viruses type 2 and simian immunodeficiency viruses. Virology 278, 276–288.

Lu, M., Blacklow, S. C. & Kim, P. S. (1995). A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nature Structural Biology 2, 1075–1082.

Lucas, S. B., Hounnou, A., Peacock, C., Beaumel, A., Djomand, G., N'Gbichi, J. M., Yeboue, K., Honde, M., Diomande, M., Giordano, C., Doorly, R., Brattefaard, D., Kestens, L., Smithwick, R., Kadio, A., Ezani, N., Yapi, A. & De Cock, K. M. (1993). The mortality and pathology of HIV infection in a West African city. AIDS 7, 1569–1579.

McCune, J. M., Rabin, L. B., Feinberg, M. B., Lieberman, M., Kosek, J. C., Reyes, G. R. & Weissman, I. L. (1988). Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus. Cell 53, 55–67.

McKnight, A., Dittmar, M. T., Moniz-Periera, J., Ariyoshi, K., Reeves, J. D., Hibbitts, S., Whitby, D., Aarons, E., Proudfoot, A. E., Whittle, H. & Clapham, P. R. (1998). A broad range of chemokine receptors are used by primary isolates of human immunodeficiency virus type 2 as coreceptors with CD4. Journal of Virology 72, 4065–4071.

Mankowski, J. L., Spelman, J. P., Ressetar, H. G., Strandberg, J. D., Laterra, J., Carter, D. L., Clements, J. E. & Zink, M. C. (1994). Neurovirulent simian immunodeficiency virus replicates productively in endothelial cells of the central nervous system in vivo and in vitro. Journal of Virology 68, 8202–8208.

Marlink, R., Kanki, P., Thior, I., Travers, K., Eisen, G., Siby, T., Traore, I., Hsieh, C. C., Dia, M. C., Gueye, E. H., Hellinger, J., Gueye-Ndiaye, A., Sankale, J.-L., Ndoye, I., Mboup, S. & Essex, M. (1994). Reduced rate of disease development after HIV-2 infection as compared to HIV-1. Science 265, 1587–1590.

Masur, H., Michelis, M. A., Greene, J. B., Onorato, I., Stouwe, R. A., Holzman, R. S., Wormser, G., Brettman, L., Lange, M., Murray, H. W. & Cunningham-Rundles, S. (1981). An outbreak of community-acquired Pneumocystis carinii pneumonia: initial manifestation of cellular immune dysfunction. New England Journal of Medicine 305, 1431–1438.

Modrow, S., Hahn, B. H., Shaw, G. M., Gallo, R. C., Wong-Staal, F. & Wolf, H. (1987). Computer-assisted analysis of envelope protein sequences of seven human immunodeficiency virus isolates: prediction of antigenic epitopes in conserved and variable regions. Journal of Virology 61, 570–578.

Moore, J. P. (1990). Simple methods for monitoring HIV-1 and HIV-2 gp120 binding to soluble CD4 by enzyme-linked immunosorbent assay: HIV-2 has a 25-fold lower affinity than HIV-1 for soluble CD4. AIDS 4, 297–305.

Morner, A., Bjorndal, A., Albert, J., Kewalramani, V. N., Littman, D. R., Inoue, R., Thorstensson, R., Fenyo, E. M. & Bjorling, E. (1999). Primary human immunodeficiency virus type 2 (HIV-2) isolates, like HIV-1 isolates, frequently use CCR5 but show promiscuity in coreceptor usage. Journal of Virology 73, 2343–2349.

Moses, A. V., Bloom, F. E., Pauza, C. D. & Nelson, J. A. (1993). Human immunodeficiency virus infection of human brain capillary endothelial cells occurs via a CD4/galactosylceramide-independent mechanism. Proceedings of the National Academy of Sciences, USA 90, 10474–10478.

Norrgren, H., Marquina, S., Leitner, T., Aaby, P., Melbye, M., Poulsen, A. G., Larsen, O., Dias, F., Escanilla, D., Andersson, S., Albert, J. & Naucler, A. (1997). HIV-2 genetic variation and DNA load in asymptomatic carriers and AIDS cases in Guinea-Bissau. Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology 16, 31–38.

Norrgren, H., Andersson, S., Biague, A. J., da Silva, Z. J., Dias, F., Naucler, A. & Biberfeld, G. (1999). Trends and interaction of HIV-1 and HIV-2 in Guinea-Bissau, west Africa: no protection of HIV-2 against HIV-1 infection. AIDS 13, 701–707.

O'Donovan, D., Ariyoshi, K., Milligan, P., Ota, M., Yamuah, L., Sarge-Njie, R. & Whittle, H. (2000). Maternal plasma viral RNA levels determine marked differences in mother-to-child transmission rates of HIV-1 and HIV-2 in The Gambia. AIDS 14, 441–448.

Owen, S. M., Ellenberger, D., Rayfield, M., Wiktor, S., Michel, P., Grieco, M. H., Gao, F., Hahn, B. H. & Lal, R. B. (1998). Genetically diverg