T Cells Expressing Activated LFA-1 Are More Susceptible to Infection with Human Immunodeficiency Virus Type 1 Particles Bearing Host-Encoded ICAM-1

The incorporation of host-derived proteins in nascent human immunodeficiency virus type 1 (HIV-1) particles is a well-established phenomenon. We recently demonstrated that the physical presence of host-encoded ICAM-1 glycoproteins on HIV-1 leads to a significant increase in virus infectivity in an ICAM-1/LFA-1-dependent fashion (J.-F. Fortin, R. Cantin, G. Lamontagne, and M. Tremblay, J. Virol. 71:3588–3596, 1997). We show here that conversion of LFA-1 to high affinity for ICAM-1 with the use of anti-LFA-1 antibodies (clones NKI-L16 and MEM83) markedly enhances the susceptibility of different target T-lymphoid cell lines, as well as of primary peripheral blood mononuclear cells, to infection by ICAM-1-bearing HIV-1 particles (6- to 95-fold). It is known that T-cell receptor (TCR) cross-linking induces a transient increase in LFA-1 affinity for ICAM-1. Treatment of peripheral blood mononuclear cells with anti-TCR antibodies (clone OKT3) resulted in a transient increase in susceptibility to infection by ICAM-1-positive virions that parallels the previously reported kinetics of the LFA-1/ICAM-1 adhesion mechanism. Our results led us to postulate that the strong interaction taking place between virally incorporated ICAM-1 and cell surface-activated LFA-1 markedly enhances the efficiency of virus binding and entry, thus favoring greater infection by ICAM-1-bearing HIV-1 particles. In view of the knowledge that primary HIV-1 isolates harbor host-derived ICAM-1 on their surfaces, these results provide new information about the role of host-derived ICAM-1 in the life cycle of HIV-1 and how it could positively modulate the dynamics of the viral infection, mainly in cellular compartments, such as the lymphoid tissues, where the level of cellular activation is high and where the probability of encountering a T cell expressing the activated LFA-1 form is also elevated.In vivo, CD4⁺ T lymphocytes and monocytes-macrophages constitute the main reservoirs for the production and maintenance of human immunodeficiency virus type 1 (HIV-1) (48, 54). Infection of these cells occurs following the high-affinity interaction between the viral surface gp120 and the cell surface CD4 molecule (15). However, in order for the fusion to occur, the sole interaction between gp120 and CD4 is not sufficient (40), and the involvement of other molecules is required. These other cellular components, the so-called coreceptors, have been recently identified and characterized. Formerly called LESTR/HUMSTR/fusin, the chemokine receptor CXCR4 has been shown to act as the coreceptor for T-cell-tropic strains of HIV-1 (22). For macrophage-tropic HIV-1 isolates, the CCR5 molecule has been identified as the major coreceptor (16, 19), even though CCR3 and CCR2b are also used, but to a lesser extent (14, 18). Following ligation of gp120 with CD4, a high-affinity binding site for the chemokine receptor is created, thus leading to membrane fusion and virus entry (36, 58, 59). Besides these essential elements for viral entry, other cellular molecules could play important, although accessory, roles during the process of virus uptake.It has been known for a while that HIV-1 can incorporate, besides its surface glycoproteins, a vast array of cell membrane molecules while budding out from the infected cell. For example, major histocompatibility complex class II (MHC-II) DR molecules were the first host constituents found embedded within HIV-1 particles and these were identified as a potential source of false-positive reactions in enzymatic screening tests (31). Many other cellular structures were found to be acquired by newly formed HIV-1, such as HLA-DP and -DQ, β₂-microglobulin, CD44, CD55, and CD59, as well as LFA-1 and ICAM-1 adhesion molecules (6, 11, 12, 21, 29, 33, 52). It has also been suggested that the profile of virion-bound cellular constituents could be used as a marker to identify the virus-producing cell (1).Recently, several studies investigated the functionality of host-derived molecules when present on the virion surface. The first, although indirect, evidence of the functionality of virally incorporated adhesion molecules came from the demonstration that anti-LFA-1 antibodies can act synergistically with antiserum to neutralize HIV-1 particles (28). More direct proof was provided by the demonstration that an increase in virion-incorporated HLA-DR and ICAM-1 resulted in enhanced infectivity toward CD4-negative cell lines (12). Saiffudin et al. demonstrated that CD55 and CD59, two glycosylphosphatidylinositol-linked complement control proteins, can protect HIV-1 from complement-mediated virolysis when incorporated into budding virions (52), while virion-incorporated host MHC-II molecules were shown to present bacterial superantigens (50). We have been able to demonstrate, by using mutagenized cell lines, that incorporation of MHC-II molecules within the viral envelope enhances the process of viral infection (9). Recently, we developed a transient-transfection-and-expression system that permits the production of virions differing only by the absence or the presence of a specific cell surface molecule on their surfaces. By using this new technical approach, we found that acquisition of cellular HLA-DR1 molecules by budding HIV-1 is associated with a 1.6- to 2.5-fold increase in virus infectivity (10). Moreover, we have shown that incorporation of host-derived ICAM-1 by progeny viruses leads to a 5- to 10-fold increase in HIV-1 infectivity, caused by an interaction between virally incorporated ICAM-1 and cell surface LFA-1 (23), an observation which has been corroborated by another group (49). This finding has great clinical relevance, considering that ICAM-1 is acquired by clinical HIV-1 isolates grown on primary mononuclear cells (4, 11, 24) and the in vivo HIV-1-producing cells are activated CD4⁺ T cells and macrophages, cells which are both known to express high levels of ICAM-1 (55). Therefore, it is likely that HIV-1 isolates found in vivo carry on their surfaces host-derived ICAM-1 glycoproteins.The counterreceptor for ICAM-1 is LFA-1, a member of the integrin family that is expressed mainly on lymphocytes, granulocytes, monocytes, and macrophages, with elevated levels on memory T cells (53). The activation of leukocytes with various agents like phorbol esters and chemoattractant, or cross-linking of specific surface receptors such as the T-cell receptor (TCR)/CD3 complex, CD2, and MHC-II, induces LFA-1-mediated binding to ICAM-1 (17). This transient change in ICAM-1 binding is thought to involve both a variation in the affinity of LFA-1 for ICAM-1 caused by a conformational change and an increase in avidity mediated by clustering of the molecules (20). This dynamic regulation of integrins allows the cells that bear these molecules to convert rapidly from a nonadherent to an adherent phenotype and vice versa. Since LFA-1 can be expressed in two different conformational states, i.e., low versus high affinity for ICAM-1, we therefore examined whether the activation state of LFA-1 on the target cell surface could affect the overall susceptibility to infection by ICAM-1-bearing HIV-1 particles.     

LFA-1 Regulates CD8+ T Cell Activation via T Cell Receptor-mediated and LFA-1-mediated Erk1/2 Signal Pathways

LFA-1 regulates T cell activation and signal transduction through the immunological synapse. T cell receptor (TCR) stimulation rapidly activates LFA-1, which provides unique LFA-1-dependent signals to promote T cell activation. However, the detailed molecular pathways that regulate these processes and the precise mechanism by which LFA-1 contributes to TCR activation remain unclear. We found LFA-1 directly participates in Erk1/2 signaling upon TCR stimulation in CD8⁺ T cells. The presence of LFA-1, not ligand binding, is required for the TCR-mediated Erk1/2 signal pathway. LFA-1-deficient T cells have defects in sustained Erk1/2 signaling and TCR/CD3 clustering, which subsequently prevents MTOC reorientation, cell cycle progression, and mitosis. LFA-1 regulates the TCR-mediated Erk1/2 signal pathway in the context of immunological synapse for recruitment and amplification of the Erk1/2 signal. In addition, LFA-1 ligation with ICAM-1 generates an additional Erk1/2 signal, which synergizes with the existing TCR-mediated Erk1/2 signal to enhance T cell activation. Thus, LFA-1 contributes to CD8⁺ T cell activation through two distinct signal pathways. We demonstrated that the function of LFA-1 is to enhance TCR signaling through the immunological synapse and deliver distinct signals in CD8⁺ T cell activation.Leukocyte function-associated antigen-1 (LFA-1)² plays an important role in regulating leukocyte adhesion and T cell activation (1, 2). LFA-1 consists of the αL (CD11a) and β2 (CD18) subunits. The ligands for LFA-1 include intercellular adhesion molecular-1 (ICAM-1), ICAM-2, and ICAM-3 (3). LFA-1 participates in the formation of the immunological synapse, which regulates T cell activation synergistically with TCR engagement. The immunological synapse is a specialized structure that forms between the T cell and the APC or target cell (1, 2, 4). The function of the immunological synapse is to facilitate T cell activation and signal transduction. Mice deficient in LFA-1 (CD11a KO) have defects in leukocyte adhesion, lymphocyte proliferation, and tumor rejection (5–7).Upon TCR stimulation, the nascent immunological synapse is initiated with surface receptor clustering and cytoskeleton rearrangement, then followed by mature synapse formation after prolonged stimulation (8, 9). In the mature immunological synapse, LFA-1 forms a ring-like pattern at the peripheral supramolecular activation cluster (pSMAC), which surrounds the central supramolecular activation cluster (cSMAC) containing TCR/CD3/lipid rafts (10, 11). The structure of the mature synapse is stable for hours and thought to be important for sustained TCR signaling (12–14). LFA-1 functions via pSMAC to stabilize the cSMAC and is associated with the induction of T cell proliferation, cytokine production, and lytic granule migration toward cSMAC (1, 15). Although LFA-1-containing pSMAC is self-evident in lipid bilayer systems and cell lines, whether it is required for T cell activation under physiological conditions remains controversial (15).TCR stimulation rapidly induces the functional activity of LFA-1, which then provides unique LFA-1-dependent signals to promote T cell activation (16). The process can be divided into two steps. First, the intracellular signaling from TCR regulating LFA-1 activation is known as “inside-out” signaling; second, activated LFA-1, as a signaling receptor, can feedback to transduce the intracellular signal, the “outside-in” signaling (1, 17). It is widely accepted that TCR stimulation activates LFA-1 through affinity and/or avidity regulation, as supported by increased adhesion to ICAM-1 and pSMAC formation (16, 17). The “inside-out” signal process has been investigated extensively (18–21). The TCR proximal signal molecules, Lck, ZAP-70, and PI3K, are known to be important for TCR signaling to LFA-1 activation (22–26). The molecular mechanisms of LFA-1 “outside-in” signaling have been explored only recently. Perez et al. (27) have demonstrated that LFA-1 and ICAM-1 ligation activates the downstream Erk1/2 MAPK signaling pathway upon TCR stimulation, which ultimately leads to the qualitative modulation of CD4⁺ T cell activation through distinct LFA-1-dependent signals. Another recent study provided compelling evidence that LFA-1 reshapes the Ras MAPK pathway downstream of TCR (28). However, the detailed molecular pathways that regulate these processes are poorly defined. Especially, the evidence in support of a distinctive role for LFA-1 in the T cell signaling pathway has lagged behind; whether the function of LFA-1 is to enhance TCR signaling through the immunological synapse and/or deliver distinct signal in T cell activation and whether LFA-1 is indispensable for or merely assists the existing TCR signal pathway. Furthermore, whether and how TCR proximal signal molecules regulate LFA-1 function remains unknown. Further studies are required to understand the LFA-1 and TCR signaling network.In this study, we found that LFA-1 directly participates in CD8⁺ T cell activation. Upon TCR stimulation, LFA-1 regulates both TCR-mediated and LFA-1-mediated Erk1/2 signal pathways. First, the presence of LFA-1, not ligand binding, is required for the sustained Erk1/2 signaling and TCR/CD3 clustering on the surface of CD8⁺ T cells, subsequently leading to MTOC reorientation, cell cycle progression, and mitosis. Second, LFA-1 ligation with ICAM-1 enhances Erk1/2 signaling, which promotes T cell activation with increased IL-2 production and cell proliferation. This LFA-1-mediated Erk1/2 signal pathway integrates with the existing TCR-mediated Erk1/2 signal pathway to enhance T cell activation.     

Syncytium-Inhibiting Monoclonal Antibodies Produced against Human T-Cell Lymphotropic Virus Type 1-Infected Cells Recognize Class II Major Histocompatibility Complex Molecules and Block by Protein Crowding

Four new monoclonal antibodies (MAbs) that inhibit human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation were produced by immunizing BALB/c mice with HTLV-1-infected MT2 cells. Immunoprecipitation studies and binding assays of transfected mouse cells showed that these MAbs recognize class II major histocompatibility complex (MHC) molecules. Previously produced anti-class II MHC antibodies also blocked HTLV-1-induced cell fusion. Coimmunoprecipitation and competitive MAb binding studies indicated that class II MHC molecules and HTLV-1 envelope glycoproteins are not associated in infected cells. Anti-MHC antibodies had no effect on human immunodeficiency virus type 1 (HIV-1) syncytium formation by cells coinfected with HIV-1 and HTLV-1, ruling out a generalized disruption of cell membrane function by the antibodies. High expression of MHC molecules suggested that steric effects of bound anti-MHC antibodies might explain their inhibition of HTLV-1 fusion. An anti-class I MHC antibody and a polyclonal antibody consisting of several nonblocking MAbs against other molecules bound to MT2 cells at levels similar to those of class II MHC antibodies, and they also blocked HTLV-1 syncytium formation. Dose-response experiments showed that inhibition of HTLV-1 syncytium formation correlated with levels of antibody bound to the surface of infected cells. The results show that HTLV-1 syncytium formation can be blocked by protein crowding or steric effects caused by large numbers of immunoglobulin molecules bound to the surface of infected cells and have implications for the structure of the cellular HTLV-1 receptor(s).Human T-cell lymphotropic virus type 1 (HTLV-1) is a type C retrovirus and the etiologic agent of adult T-cell leukemia (43, 56, 59) and HTLV-1-associated myelopathy or tropical spastic paraparesis (15, 17, 49, 61). Although HTLV-1 shows tropism primarily for T cells, it can infect a variety of cell types including cells from some nonhuman species (6, 9, 27, 46, 48, 60, 62). Infection by free HTLV-1 tends to be highly inefficient, and the virus appears to be transmitted primarily by the cell-to-cell route (37). The HTLV-1 envelope glycoprotein is synthesized as a 61-kDa precursor which is cleaved into surface (gp46) and transmembrane (gp21) proteins (40, 57). gp46 is thought to serve as the virus attachment protein, as does gp120 for human immunodeficiency virus (HIV) (40, 57). Although previous reports have identified host cell molecules which might potentially mediate virus binding (9, 14), the cellular receptor for HTLV-1 has not been definitively identified. A recent study in which affinity chromatography was carried out with a gp46 peptide has provided evidence that the heat shock protein HSC70 binds directly to gp46 and may serve as a virus receptor (47).gp21 contains an N-terminal hydrophobic fusion domain and likely serves as a fusion protein similar to HIV gp41 (12, 61). Like many other retroviruses, HTLV-1 can induce syncytium formation between infected cells and certain uninfected cell types (28, 39). However, there are no data to indicate that virus transmission or virus persistence in vivo depends on syncytium formation. It is thought that cell-cell fusion involves the same receptors and occurs in a manner similar to virus-cell fusion. For this reason, HTLV-1 syncytium assays have been used to screen for cell surface molecules that may serve as virus receptors (13, 14, 25, 29). Monoclonal antibodies (MAbs) against a number of membrane proteins including members of the tetraspanner family (30, 31) have been found to block syncytium formation. My colleagues and I recently reported that expression of the cell adhesion molecule vascular cell adhesion molecule 1 (VCAM-1) on uninfected cells can confer sensitivity to HTLV-1-mediated syncytium formation (25). In this previous study, we were not able to block HTLV-1 cell fusion with MAbs against the major VCAM-1 counterreceptor VLA-4 (25). Others have reported that MAbs to other adhesion molecules including intercellular adhesion molecule 3 (ICAM-3) also block HTLV-1 syncytium formation (29). We have demonstrated that adhesion molecules also facilitate HIV type 1 (HIV-1) infection and syncytium formation (16, 24). Thus, adhesion molecules may be important accessory molecules for retroviruses generally.Earlier studies on accessory molecules involved in HTLV-1 biology have been extended by immunizing mice with HTLV-1-infected cells and screening for MAbs that block VCAM-1-supported HTLV-1 syncytium formation. Four new MAbs that completely block HTLV-1-mediated cell fusion have been generated. The MAbs were all determined to be specific for class II major histocompatibility complex (MHC) molecules. These MAbs had no effect on syncytium formation induced by HIV-1. Studies on the mechanism by which the MAbs mediate this effect have revealed a novel mode of antibody blockade of virus-induced cell fusion: protein crowding at the infected cell surface resulting in steric blockade of critical receptor-ligand interactions.     

Identification of a Domain within the Human T-Cell Leukemia Virus Type 2 Envelope Required for Syncytium Induction and Replication

In vitro infection by human T-cell leukemia virus type 1 and 2 (HTLV-1 and HTLV-2) can result in syncytium formation, facilitating viral entry. Using cell lines that were susceptible to HTLV-2-mediated syncytium formation but were nonfusogenic with HTLV-1, we constructed chimeric envelopes between HTLV-1 and -2 and assayed for the ability to induce syncytia in BJAB cells and HeLa cells. We have identified a fusion domain composed of the first 64 amino acids at the amino terminus of the HTLV-2 transmembrane protein, p21, the retention of which was required for syncytium induction. Construction of replication-competent HTLV genomic clones allowed us to correlate the ability of HTLV-2 to induce syncytia with the ability to replicate in BJAB cells. Differences in the ability to induce syncytia were not due to differences in the levels of total or cell membrane-associated envelope or in the formation of multimers. Therefore, we have localized a fusion domain within the amino terminus of the transmembrane protein of HTLV-2 envelope that is necessary for syncytium induction and viral replication.Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are type C retroviruses that have been associated with a variety of human malignancies. HTLV-1 is the etiological agent of adult T-cell leukemia as well as a degenerative neurological disorder, HTLV-1-associated myelopathy/tropical spastic paraparesis (28, 40, 58, 60, 83). Recent reports have also implicated HTLV-1 infection with arthropathy (42, 65), polymyosis (23, 37), and uveitis (48, 49, 51). HTLV-2 has been associated with a rare form of atypical hairy cell leukemia (62, 63, 68) as well as some cases of neuropathy (33, 39). It is estimated that between 10 million and 20 million individuals worldwide are infected with HTLV, with an overall risk of 5% of disease progression in infected individuals (14). HTLV is endemic in southern Japan, the Caribbean Basin, and Central and South America. In the United States, recent reports have identified a high proportion of HTLV, especially HTLV-2, infection in intravenous-drug abusers (44, 61, 64).Cell-to-cell contact is considered critical for the in vivo and in vitro transmission of HTLV-1 and HTLV-2, as infection by cell-free HTLV virus is inefficient in vitro and in vivo. By analogy with other enveloped viruses, HTLV infection of susceptible cells is likely mediated by the envelope glycoprotein. Antibodies against HTLV envelope are protective against infection in vivo (71, 80), and multiple epitopes that elicit neutralizing antibodies have been identified throughout the protein (31, 34, 56). Initially synthesized as a precursor protein, gp61, HTLV envelope is subsequently modified by glycosylation and cleaved into two subunits, gp46 and p21. The external surface glycoprotein, gp46, is anchored to the cell surface by noncovalent association with the transmembrane envelope glycoprotein, p21. Interaction of envelope with the as yet unidentified cellular receptor leads to cell-to-cell fusion and can result in syncytium formation.We were interested in identifying the molecular determinants of HTLV involved in syncytium formation and viral entry. Our laboratory has several cell lines that are permissive to HTLV-2- but not HTLV-1-mediated cell fusion. Therefore, we constructed recombinants between the HTLV-1 and -2 envelope genes and assayed for the loss of syncytium induction in BJAB cells and HeLa cells. Loss of a 64-amino-acid (aa) domain located at the amino terminus of the HTLV-2 transmembrane protein, p21, correlated with a loss in the ability of the envelope chimera to induce cell fusion. When the chimeric envelopes were expressed in the context of replication-competent genomic clones, there was a good correlation between syncytium induction and the ability to replicate in permissive cells. Present within the identified fusion domain is a hydrophobic region and a heptad repeat resembling a leucine zipper. We examined the contribution of the fusion domain to the structural integrity of the HTLV-2 envelope by using a vaccinia virus expression system. None of the recombinants affected the synthesis, transport, or oligomer formation of the HTLV glycoprotein complex.     

Evidence that Antibody-Mediated Neutralization of Human Immunodeficiency Virus Type 1 by Sera from Infected Individuals Is Independent of Coreceptor Usage

Human immunodeficiency virus type 1 (HIV-1) uses a variety of chemokine receptors as coreceptors for virus entry, and the ability of the virus to be neutralized by antibody may depend on which coreceptors are used. In particular, laboratory-adapted variants of the virus that use CXCR4 as a coreceptor are highly sensitive to neutralization by sera from HIV-1-infected individuals, whereas primary isolates that use CCR5 instead of, or in addition to, CXCR4 are neutralized poorly. To determine whether this dichotomy in neutralization sensitivity could be explained by differential coreceptor usage, virus neutralization by serum samples from HIV-1-infected individuals was assessed in MT-2 cells, which express CXCR4 but not CCR5, and in mitogen-stimulated human peripheral blood mononuclear cells (PBMC), where multiple coreceptors including CXCR4 and CCR5 are available for use. Our results showed that three of four primary isolates with a syncytium-inducing (SI) phenotype and that use CXCR4 and CCR5 were neutralized poorly in both MT-2 cells and PBMC. The fourth isolate, designated 89.6, was more sensitive to neutralization in MT-2 cells than in PBMC. We showed that the neutralization of 89.6 in PBMC was not improved when CCR5 was blocked by having RANTES, MIP-1α, and MIP-1β in the culture medium, indicating that CCR5 usage was not responsible for the decreased sensitivity to neutralization in PBMC. Consistent with this finding, a laboratory-adapted strain of virus (IIIB) was significantly more sensitive to neutralization in CCR5-deficient PBMC (homozygous Δ32-CCR5 allele) than were two of two SI primary isolates tested. The results indicate that the ability of HIV-1 to be neutralized by sera from infected individuals depends on factors other than coreceptor usage.Human immunodeficiency virus type 1 (HIV-1), the etiologic agent of AIDS, utilizes the HLA class II receptor, CD4, as its primary receptor to gain entry into cells (17, 30). Entry is initiated by a high-affinity interaction between CD4 and the surface gp120 of the virus (32). Subsequent to this interaction, conformational changes that permit fusion of the viral membrane with cellular membranes occur within the viral transmembrane gp41 (9, 58, 59). In addition to CD4, one or more recently described viral coreceptors are needed for fusion to take place. These coreceptors belong to a family of seven-transmembrane G-protein-coupled proteins and include the CXC chemokine receptor CXCR4 (3, 4, 24, 44), the CC chemokine receptors CCR5 (1, 12, 13, 18, 21, 23, 45) and, less commonly, CCR3 and CCR2b (12, 21), and two related orphan receptors termed BONZO/STRL33 and BOB (19, 34). Coreceptor usage by HIV-1 can be blocked by naturally occurring ligands, including SDF-1 for CXCR4 (4, 44), RANTES, MIP-1α, and MIP-1β in the case of CCR5 (13, 45), and eotaxin for CCR3 (12).The selective cellular tropisms of different strains of HIV-1 may be determined in part by coreceptor usage. For example, all culturable HIV-1 variants replicate initially in mitogen-stimulated human peripheral blood mononuclear cells (PBMC), but only a minor fraction are able to infect established CD4⁺ T-cell lines (43). This differential tropism is explained by the expression of CXCR4 together with CCR5 and other CC chemokine coreceptors on PBMC and the lack of expression of CCR5 on most T-cell lines (5, 10, 19, 35, 39, 50, 53). Indeed, low-passage field strains (i.e., primary isolates) of HIV-1 that fail to replicate in T-cell lines use CCR5 as their major coreceptor and are unable to use CXCR4 (1, 12, 18, 21, 23, 28). Because these isolates rarely produce syncytia in PBMC and fail to infect MT-2 cells, they are often classified as having a non-syncytium-inducing (NSI) phenotype. Primary isolates with a syncytium-inducing (SI) phenotype are able to use CXCR4 alone or, more usually, in addition to CCR5 (16, 20, 51). HIV-1 variants that have been passaged multiple times in CD4⁺ T-cell lines, and therefore considered to be laboratory adapted, exhibit a pattern of coreceptor usage that resembles that of SI primary isolates. Most studies have shown that the laboratory-adapted strain IIIB uses CXCR4 alone (3, 13, 20, 24, 51) and that MN and SF-2 use CXCR4 primarily and CCR5 to a lesser degree (11, 13). Sequences within the V3 loop of gp120 have been shown to be important, either directly or indirectly, for the interaction of HIV-1 with both CXCR4 (52) and CCR5 (12, 14, 54, 60). This region of gp120 contains multiple determinants of cellular tropism (43) and is a major target for neutralizing antibodies to laboratory-adapted HIV-1 but not to primary isolates (29, 46, 57).It has been known for some time that the ability of sera from HIV-1-infected individuals to neutralize laboratory-adapted strains of HIV-1 does not predict their ability to neutralize primary isolates in vitro (7). In general, the former viruses are highly sensitive to neutralization whereas the latter viruses are neutralized poorly by antibodies induced in response to HIV-1 infection (7, 43). Importantly, neutralizing antibodies generated by candidate HIV-1 subunit vaccines have been highly specific for laboratory-adapted viruses (26, 37, 38). In principle, the dichotomy in neutralization sensitivity between these two categories of virus could be related to coreceptor usage. To test this, we investigated whether the use of CXCR4 in the absence of CCR5 would render SI primary isolates highly sensitive to neutralization in vitro by sera from HIV-1-infected individuals. Two similar studies using human monoclonal antibodies and soluble CD4 have been reported (31a, 55).     

A Comprehensive Panel of Near-Full-Length Clones and Reference Sequences for Non-Subtype B Isolates of Human Immunodeficiency Virus Type 1

Non-subtype B viruses cause the vast majority of new human immunodeficiency virus type 1 (HIV-1) infections worldwide and are thus the major focus of international vaccine efforts. Although their geographic dissemination is carefully monitored, their immunogenic and biological properties remain largely unknown, in part because well-characterized virological reference reagents are lacking. In particular, full-length clones and sequences are rare, since subtype classification is frequently based on small PCR-derived viral fragments. There are only five proviral clones available for viruses other than subtype B, and these represent only 3 of the 10 proposed (group M) sequence subtypes. This lack of reference sequences also confounds the identification and analysis of mosaic (recombinant) genomes, which appear to be arising with increasing frequency in areas where multiple sequence subtypes cocirculate. To generate a more representative panel of non-subtype B reference reagents, we have cloned (by long PCR or lambda phage techniques) and sequenced 10 near-full-length HIV-1 genomes (lacking less than 80 bp of long terminal repeat sequences) from primary isolates collected at major epicenters of the global AIDS pandemic. Detailed phylogenetic analyses identified six that represented nonrecombinant members of HIV-1 subtypes A (92UG037.1), C (92BR025.8), D (84ZR085.1 and 94UG114.1), F (93BR020.1), and H (90CF056.1), the last two comprising the first full-length examples of these subtypes. Four others were found to be complex mosaics of subtypes A and C (92RW009.6), A and G (92NG083.2 and 92NG003.1), and B and F (93BR029.4), again emphasizing the impact of intersubtype recombination on global HIV-1 diversification. Although a number of clones had frameshift mutations or translational stop codons in major open reading frames, all the genomes contained a complete set of genes and three had intact genomic organizations without inactivating mutations. Reconstruction of one of these (94UG114.1) yielded replication-competent virus that grew to high titers in normal donor peripheral blood mononuclear cell cultures. This panel of non-subtype B reference genomes should prove valuable for structure-function studies of genetically diverse viral gene products, the generation of subtype-specific immunological reagents, and the production of DNA- and protein-based subunit vaccines directed against a broader spectrum of viruses.One critical question facing current AIDS vaccine development efforts is to what extent human immunodeficiency virus type 1 (HIV-1) genetic variation has to be considered in the design of candidate vaccines (11, 21, 41, 72). Phylogenetic analyses of globally circulating viral strains have identified two distinct groups of HIV-1 (M and O) (33, 45, 61, 62), and 10 sequence subtypes (A to J) have been proposed within the major group (M) (29, 30, 45, 72). Sequence variation among viruses belonging to these different lineages is extensive, with envelope amino acid sequence variation ranging from 24% between different subtypes to 47% between the two different groups. Given this extent of diversity, the question has been raised whether immunogens based on a single virus strain can be expected to elicit immune responses effective against a broad spectrum of viruses or whether vaccine preparations should include mixtures of genetically divergent antigens and/or be tailored toward locally circulating strains (11, 21, 41, 72). This is of particular concern in developing countries, where multiple subtypes of HIV-1 are known to cocirculate and where subtype B viruses (which have been the source of most current candidate vaccine preparations [10, 21]) are rare or nonexistent (5, 24, 40, 72).Although the extent of global HIV-1 variation is well defined, little is known about the biological consequences of this genetic diversity and its impact on cellular and humoral immune responses in the infected host. In particular, it remains unknown whether subtype-specific differences in virus biology exist that have to be considered for vaccine design. Thus far, such differences have not been identified. For example, several studies have shown that there is no correlation between HIV-1 genetic subtypes and neutralization serotypes (38, 42, 46, 68). Some viruses are readily neutralized, while most are relatively neutralization resistant (42). Although the reasons for these different susceptibilities remain unknown, it is clear that neutralization is not a function of the viral genotype (38, 42, 46, 68). Similarly, recent studies have identified vigorous cross-clade cytotoxic T-lymphocyte (CTL) reactivities in individuals infected with viruses from several different clades (3, 6), as well as in recipients of a clade B vaccine (15). These results are very encouraging, since they suggest that CTL cross-recognition among HIV-1 clades is much more prevalent than previously anticipated and that immunogens based on a limited number of variants may be able to elicit a broad CTL response (6). Nevertheless, it would be premature to conclude that HIV-1 variation poses no problem for AIDS vaccine design. Only a comprehensive analysis of genetically defined representatives of the various groups and subtypes will allow us to judge whether certain variants differ in fundamental viral properties and whether such differences will have to be incorporated into vaccine strategies. Obviously, such studies require well-characterized reference reagents, in particular full-length and replication-competent molecular clones that can be used for functional and biological studies.Full-length reference sequences representing the various subtypes are also urgently needed for phylogenetic comparisons. Recent analyses of subgenomic (23, 52, 54, 58) as well as full-length (7, 18, 53, 60) HIV-1 sequences identified a surprising number of HIV-1 strains which clustered in different subtypes in different parts of their genome. All of these originated from geographic regions where multiple subtypes cocirculated and are the results of coinfections with highly divergent viruses (52, 60, 62). Detailed phylogenetic characterization revealed that most of them have a complex genome structure with multiple points of crossover (7, 18, 53, 60). Some recombinants, like the “subtype E” viruses, which are in fact A/E recombinants (7, 18), have a widespread geographic dissemination and are responsible for much of the Asian HIV-1 epidemic (69, 70). In other areas, recombinants appear to be generated with increasing frequencies since many randomly chosen isolates exhibit evidence of mosaicism (4, 8, 31, 66, 71). Since recombination provides the opportunity for evolutionary leaps with genetic consequences that are far greater than those of the steady accumulation of individual mutations, the impact of recombination on viral properties must be monitored. We therefore need full-length nonrecombinant reference sequences for all major HIV-1 groups and subtypes before we can map and characterize the extent of intersubtype recombination.The number of molecular reagents for non-subtype B viruses is very limited. There are currently only five full-length, nonrecombinant molecular clones available for viruses other than subtype B (45), and these represent only three of the proposed (group M) subtypes (A, C, and D). Moreover, only three clones (all derived from subtype D viruses) are replication competent and thus useful for studies requiring functional gene products (45, 48, 65). Given the unknown impact of genetic variation on correlates of immune protection, subtype-specific reagents are critically needed for phylogenetic, immunological, and biological studies. In this paper, we report the cloning (by long PCR and lambda techniques) of 10 near-full-length HIV-1 genomes from isolates previously classified as non-subtype B viruses. Detailed phylogenetic analysis showed that six comprise nonmosaic representatives of five major subtypes, including two for which full-length representatives have not been reported. Four others were identified as complex intersubtype recombinants, again emphasizing the prevalence of hybrid genomes among globally circulating HIV-1 strains. We also describe a strategy for the biological evaluation of long-PCR-derived genomes and report the generation of a replication-competent provirus by this approach. The effect of these reagents on vaccine development is discussed.     

CD4 Glycoprotein Degradation Induced by Human Immunodeficiency Virus Type 1 Vpu Protein Requires the Function of Proteasomes and the Ubiquitin-Conjugating Pathway

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic β subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E₁ or (ii) following expression of a mutant form of ubiquitin (Lys₄₈ mutated to Arg₄₈) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.

The human immunodeficiency virus type 1 (HIV-1)-specific accessory protein Vpu performs two distinct functions in the viral life cycle (11, 12, 29, 34, 46, 47, 50–52; reviewed in references 31 and 55): enhancement of virus particle release from the cell surface, and the selective induction of proteolysis of newly synthesized membrane proteins. Known targets for Vpu include the primary virus receptor CD4 (63, 64) and major histocompatibility complex (MHC) class I molecules (28). Vpu is an oligomeric class I integral membrane phosphoprotein (35, 48, 49) with a structurally and functionally defined domain architecture: an N-terminal transmembrane anchor and C-terminal cytoplasmic tail (20, 34, 45, 47, 50, 65). Vpu-induced degradation of endoplasmic reticulum (ER) membrane proteins involves the phosphorylated cytoplasmic tail of the protein (50), whereas the virion release function is mediated by a cation-selective ion channel activity associated with the membrane anchor (19, 31, 45, 47).CD4 is a 55-kDa class I integral membrane glycoprotein that serves as the primary coreceptor for HIV entry into cells. CD4 consists of a large lumenal domain, a transmembrane peptide, and a 38-residue cytoplasmic tail. It is expressed on the surface of a subset of T lymphocytes that recognize MHC class II-associated peptides, and it plays a pivotal role in the development and maintenance of the immune system (reviewed in reference 30). Down regulation of CD4 in HIV-1-infected cells is mediated through several independent mechanisms (reviewed in references 5 and 55): intracellular complex formation of CD4 with the HIV envelope protein gp160 (8, 14), endocytosis of cell surface CD4 induced by the HIV-1 nef gene product (1, 2), and ER degradation induced by the HIV-1 vpu gene product (63, 64).Vpu-induced degradation of CD4 is an example of ER-associated protein degradation (ERAD). ERAD is a common outcome when proteins in the secretory pathway are unable to acquire their native structure (4). Although it was thought that ERAD occurs exclusively inside membrane vesicles of the ER or other related secretory compartments, this has gained little direct experimental support. Indeed, there are several recent reports that ERAD may actually represent export of the target protein to the cytosol, where it is degraded by cytosolic proteases. It was found that in yeast, a secreted protein, prepro-α-factor (pαF), is exported from microsomes and degraded in the cytosol in a proteasome-dependent manner (36). This process was dependent on the presence of calnexin, an ER-resident molecular chaperone that interacts with N-linked oligosaccharides containing terminal glucose residues (3). In mammalian cells, two human cytomegalovirus (HCMV) proteins, US2 and US11, were found to cause the retranslocation of MHC class I molecules from the ER to the cytosol, where they are destroyed by proteasomes (61, 62). In the case of US2, class I molecules were found to associate with a protein (Sec61) present in the channel normally used to translocate newly synthesized proteins into the ER (termed the translocon), leading to the suggestion that the ERAD substrates are delivered to the cytosol by retrograde transport through the Sec61-containing pore (61). Fujita et al. (24) reported that, similar to these findings, the proteasome-specific inhibitor lactacystin (LC) partially blocked CD4 degradation in transfected HeLa cells coexpressing CD4, Vpu, and HIV-1 Env glycoproteins. In the present study, we show that Vpu-induced CD4 degradation can be completely blocked by proteasome inhibitors, does not require the ER chaperone calnexin, but requires the function of the cytosolic polyubiquitination machinery which apparently targets potential ubiquitination sites within the CD4 cytoplasmic tail. Our findings point to differences between the mechanism of Vpu-mediated CD4 degradation and ERAD processes induced by the HCMV proteins US2 and US11 (61, 62).     

Endosomal Trafficking of HIV-1 Gag and Genomic RNAs Regulates Viral Egress

HIV-1 Gag can assemble and generate virions at the plasma membrane, but it is also present in endosomes where its role remains incompletely characterized. Here, we show that HIV-1 RNAs and Gag are transported on endosomal vesicles positive for TiVamp, a v-SNARE involved in fusion events with the plasma membrane. Inhibition of endosomal traffic did not prevent viral release. However, inhibiting lysosomal degradation induced an accumulation of Gag in endosomes and increased viral production 7-fold, indicating that transport of Gag to lysosomes negatively regulates budding. This also suggested that endosomal Gag-RNA complexes could access retrograde pathways to the cell surface and indeed, depleting cells of TiVamp-reduced viral production. Moreover, inhibition of endosomal transport prevented the accumulation of Gag at sites of cellular contact. HIV-1 Gag could thus generate virions using two pathways, either directly from the plasma membrane or through an endosome-dependent route. Endosomal Gag-RNA complexes may be delivered at specific sites to facilitate cell-to-cell viral transmission.The production of infectious retroviral particles is an ordered process that includes many steps (for review see Refs. 1–3). In particular, three major viral components, Gag, the envelope, and genomic RNAs have to traffic inside the cell to reach their assembly site. Viral biogenesis is driven by the polyprotein Gag, which is able to make viral-like particles when expressed alone (4). Upon release, HIV-1⁴ Gag is processed by the viral protease into matrix (MA(p17)), capsid (CA(p24)), nucleocapsid (NC(p7)), p6, and smaller peptides SP1 and SP2. Gag contains several domains that are essential for viral assembly: a membrane binding domain (M) in MA; a Gag-Gag interaction domain in CA; an assembly domain (I) in NC; and a late domain (L) in p6, which recruits the cellular budding machinery. Genomic RNAs are specifically recognized by NC, and they play fundamental roles in viral biogenesis by acting as a scaffold for Gag multimerization (5).It has been demonstrated that retroviruses bud by hijacking the endosomal machinery that sorts proteins into internal vesicles of multivesicular bodies (for review, see Refs. 6, 7). Indeed, these vesicles bud with the same topology as viral particles. Proteins sorted into this pathway are usually destined for degradation in lysosomes, but some can also recycle to the plasma membrane (for review see Refs. 8, 9). They are also frequently ubiquitinated on their cytoplasmic domain (10, 11), allowing their recognition by ESCRT complexes. ESCRT-0 and ESCRT-I recognize ubiquitinated cargo present at the surface of endosomes and recruit other ESCRT complexes (12–14). ESCRT-III is believed to function directly in the formation of multivesicular body intralumenal vesicles (12), even though its mechanism of action is currently not understood. Remarkably, Gag L domains interact directly with components of the multivesicular body-sorting machinery (for review see Ref. 15). HIV-1 Gag uses a PTAP motif to bind Tsg101, a component of ESCRT-I (16–19), and a YPLTSL motif to interact with Alix, a protein linked to ESCRT-I and -III (20–22). Finally, various ubiquitin ligases are also required directly or indirectly during HIV-1 biogenesis (23, 24; for review see Ref. 25).In many cell lines, Gag is found both at the plasma membrane and in endosomes. This has led to the hypothesis that there are several assembly sites for HIV-1 (1, 3). First, Gag can initiate and complete assembly at the plasma membrane. This is thought to occur predominantly in T lymphocytes, and this process is supported by several lines of evidences: (i) disruption of endosomal trafficking with drugs does not prevent viral production (26, 27); (ii) ESCRT complexes can be recruited at the plasma membrane, at sites where Gag accumulates (28–30); (iii) Gag can be seen multimerizing and budding from the plasma membrane in live cells (31). Second, Gag could initiate assembly in endosomes, and then traffic to the cell surface to be released. This is mainly supported by the presence of Gag in endosomes in several cell lines (32–34), including T cells and more strikingly macrophages (32, 35, 36–39). However, we are currently lacking functional experiments addressing the role of this endosomal pool of Gag, and it is still not clear to what extent it contributes to the production of viral particles. Nevertheless, the presence of Gag in endosomes might facilitate recruitment of ESCRT complexes (34, 40), packaging of viral genomic RNAs (32, 41), and incorporation of the envelope (42). It may also be important for polarized budding (43, 44) and to create a viral reservoir in infected cells (45, 46).Despite great progress, the traffic of HIV-1 components is still not fully elucidated. In particular, the transport of the genomic RNAs is poorly understood. In this study, we have used single molecule techniques to investigate the trafficking of HIV-1 RNAs in fixed and live cells, and we show that they are transported on endosomal vesicles. We also obtained functional evidence that Gag and viral RNAs can use at least two trafficking pathways to produce virions, one going directly from the plasma membrane and another one passing through endosomes.     

Neutralization Sensitivity of Human Immunodeficiency Virus Type 1 Primary Isolates to Antibodies and CD4-Based Reagents Is Independent of Coreceptor Usage

We have investigated whether the identity of the coreceptor (CCR5, CXCR4, or both) used by primary human immunodeficiency virus type 1 (HIV-1) isolates to enter CD4⁺ cells influences the sensitivity of these isolates to neutralization by monoclonal antibodies and CD4-based agents. Coreceptor usage was not an important determinant of neutralization titer for primary isolates in peripheral blood mononuclear cells. We also studied whether dualtropic primary isolates (able to use both CCR5 and CXCR4) were differentially sensitive to neutralization by the same antibodies when entering U87MG-CD4 cells stably expressing either CCR5 or CXCR4. Again, we found that the coreceptor used by a virus did not greatly affect its neutralization sensitivity. Similar results were obtained for CCR5- or CXCR4-expressing HOS cell lines engineered to express green fluorescent protein as a reporter of HIV-1 entry. Neutralizing antibodies are therefore unlikely to be the major selection pressure which drives the phenotypic evolution (change in coreceptor usage) of HIV-1 that can occur in vivo. In addition, the increase in neutralization sensitivity found when primary isolates adapt to growth in transformed cell lines in vitro has little to do with alterations in coreceptor usage.Human immunodeficiency virus type 1 (HIV-1) enters CD4⁺ T cells via an interaction with CD4 and coreceptor molecules, the most important of which yet identified are the chemokine receptors CXCR4 and CCR5 (4, 12, 23, 26, 28, 32). CXCR4 is used by T-cell line-tropic (T-tropic) primary isolates or T-cell line-adapted (TCLA) lab strains, whereas CCR5 is used by primary isolates of the macrophage-tropic (M-tropic) phenotype (4, 12, 23, 26, 28, 32). Most T-tropic isolates and some TCLA strains are actually dualtropic in that they can use both CXCR4 and CCR5 (and often other coreceptors such as CCR3, Bonzo/STRL33, and BOB/gpr15), at least in coreceptor-transfected cells (18, 24, 30, 54, 89). The M-tropic and T-tropic/dualtropic nomenclature has often been used interchangeably with the terms “non-syncytium-inducing” (NSI) and “syncytium-inducing” (SI), although it is semantically imprecise to do so.M-tropic viruses are those most commonly transmitted sexually (3, 33, 87, 106) and from mother to infant (2, 72, 81). If T-tropic strains are transmitted, or when they emerge, this is associated with a more rapid course of disease in both adults (17, 37, 46, 51, 52, 76, 78, 82, 92, 101) and children (6, 45, 84, 90). However, T-tropic viruses emerge in only about 40% of infected people, usually only several years after infection (76, 78). A well-documented, albeit anecdotal, study found that when a T-tropic strain was transmitted by direct transfer of blood, its replication was rapidly suppressed: the T-tropic virus was eliminated from the body, and M-tropic strains predominated (20). These results suggest that there is a counterselection pressure against the emergence of T-tropic strains during the early stages of HIV-1 infection in most people. But what is this pressure?Since the M-tropic and T-tropic phenotypes are properties mediated by the envelope glycoproteins whose function is to associate with CD4 and the coreceptors, a selection pressure differentially exerted on M- and T-tropic viruses could, in principle, act at the level of virus entry. In other words, neutralizing antibodies to the envelope glycoproteins, or the chemokine ligands of the coreceptors, could theoretically interfere more potently with the interactions of T-tropic strains with CXCR4 than with M-tropic viruses and CCR5. A differential effect of this nature could suppress the emergence of T-tropic viruses. Consistent with this possibility, neutralizing antibodies are capable of preventing the CD4-dependent association of gp120 with CCR5 (42, 94, 103), and chemokines can also prevent the coreceptor interactions of HIV-1 (8, 13, 23, 28, 70).Here, we explore whether the efficiency of HIV-1 neutralization is affected by coreceptor usage. Although earlier studies have not found T-tropic strains to be inherently more neutralization sensitive than M-tropic ones (20, 40, 44), previously available reagents and techniques may not have been adequate to fully address this question. One major problem is that even single residue changes can drastically affect both antibody binding to neutralization epitopes and the HIV-1 phenotype (25, 55, 62, 67, 83, 91), and so studies using relatively unrelated viruses and a fixed antibody (polyclonal or monoclonal) preparation have two variables to contend with: the viral phenotype (coreceptor use) and the antigenic structure of the virus and hence the efficiency of the antibody-virion interaction.We have used a new experimental strategy to explore whether coreceptor usage affects neutralization sensitivity in the absence of other confounding variables: the use of dualtropic viruses able to enter CD4⁺ cells via either CCR5 or CXCR4. By using a constant HIV-1 isolate or clone and the same monoclonal antibodies (MAbs) or CD4-based reagents as neutralizing agents, we can ensure that the only variable under study in the neutralization reaction is the nature of the coreceptor used for entry. Our major conclusion is that there is no strong association between coreceptor usage and neutralization sensitivity for primary HIV-1 isolates. Independent studies have reached the same conclusion (53a, 59). The emergence of T-tropic (SI) viruses in vivo may be unlikely to be due to escape from antibody-mediated selection pressure.     

The Cell Tropism of Human Immunodeficiency Virus Type 1 Determines the Kinetics of Plasma Viremia in SCID Mice Reconstituted with Human Peripheral Blood Leukocytes

Most individuals infected with human immunodeficiency virus type 1 (HIV-1) initially harbor macrophage-tropic, non-syncytium-inducing (M-tropic, NSI) viruses that may evolve into T-cell-tropic, syncytium-inducing viruses (T-tropic, SI) after several years. The reasons for the more efficient transmission of M-tropic, NSI viruses and the slow evolution of T-tropic, SI viruses remain unclear, although they may be linked to expression of appropriate chemokine coreceptors for virus entry. We have examined plasma viral RNA levels and the extent of CD4⁺ T-cell depletion in SCID mice reconstituted with human peripheral blood leukocytes following infection with M-tropic, dual-tropic, or T-tropic HIV-1 isolates. The cell tropism was found to determine the course of viremia, with M-tropic viruses producing sustained high viral RNA levels and sparing some CD4⁺ T cells, dual-tropic viruses producing a transient and lower viral RNA spike and extremely rapid depletion of CD4⁺ T cells, and T-tropic viruses causing similarly lower viral RNA levels and rapid-intermediate rates of CD4⁺ T-cell depletion. A single amino acid change in the V3 region of gp120 was sufficient to cause one isolate to switch from M-tropic to dual-tropic and acquire the ability to rapidly deplete all CD4⁺ T cells.The envelope gene of human immunodeficiency virus type 1 (HIV-1) determines the cell tropism of the virus (11, 32, 47, 62), the use of chemokine receptors as cofactors for viral entry (4, 17), and the ability of the virus to induce syncytia in infected cells (55, 60). Cell tropism is closely linked to but probably not exclusively determined by the ability of different HIV-1 envelopes to bind CD4 and the CC or the CXC chemokine receptors and initiate viral fusion with the target cell. Macrophage-tropic (M-tropic) viruses infect primary cultures of macrophages and CD4⁺ T cells and use CCR5 as the preferred coreceptor (2, 5, 15, 23, 26, 31). T-cell-tropic (T-tropic) viruses can infect primary cultures of CD4⁺ T cells and established T-cell lines, but not primary macrophages. T-tropic viruses use CXCR4 as a coreceptor for viral entry (27). Dual-tropic viruses have both of these properties and can use either CCR5 or CXCR4 (and infrequently other chemokine receptors [25]) for viral entry (24, 37, 57). M-tropic viruses are most frequently transmitted during primary infection of humans and persist throughout the duration of the infection (63). Many, but not all, infected individuals show an evolution of virus cell tropism from M-tropic to dual-tropic and finally to T-tropic with increasing time after infection (21, 38, 57). Increases in replicative capacity of viruses from patients with long-term infection have also been noted (22), and the switch to the syncytium-inducing (SI) phenotype in T-tropic or dual-tropic isolates is associated with more rapid disease progression (10, 20, 60). Primary infection with dual-tropic or T-tropic HIV, although infrequent, often leads to rapid disease progression (16, 51). The viral and host factors that determine the higher transmission rate of M-tropic HIV-1 and the slow evolution of dual- or T-tropic variants remain to be elucidated (4).These observations suggest that infection with T-tropic, SI virus isolates in animal model systems with SCID mice grafted with human lymphoid cells or tissue should lead to a rapid course of disease (1, 8, 44–46). While some studies in SCID mice grafted with fetal thymus and liver are in agreement with this concept (33, 34), our previous studies with the human peripheral blood leukocyte-SCID (hu-PBL-SCID) mouse model have shown that infection with M-tropic isolates (e.g., SF162) causes more rapid CD4⁺ T-cell depletion than infection with T-tropic, SI isolates (e.g., SF33), despite similar proviral copy numbers, and that this property mapped to envelope (28, 41, 43). However, the dual-tropic 89.6 isolate (19) caused extremely rapid CD4⁺ T-cell depletion in infected hu-PBL-SCID mice that was associated with an early and transient increase in HIV-1 plasma viral RNA (29). The relationship between cell tropism of the virus isolate and the pattern of disease in hu-PBL-SCID mice is thus uncertain. We have extended these studies by determining the kinetics of HIV-1 RNA levels in serial plasma samples of hu-PBL-SCID mice infected with primary patient isolates or laboratory stocks that differ in cell tropism and SI properties. The results showed significant differences in the kinetics of HIV-1 replication and CD4⁺ T-cell depletion that are determined by the cell tropism of the virus isolate.