Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLa^K44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.     

cDNA transfection of amino-terminal fragment of urokinase efficiently inhibits cancer cell invasion and metastasis.

Focusing of urokinase-type plasminogen activator (uPA) to the cell surface via binding to its specific receptor (uPAR, CD87) is critical for tumor invasion and metastasis. Consequently, the inhibition of uPA-uPAR interaction on the cell surface might be a promising anti-invasion and anti-metastasis strategy. We examined the effects of cDNA transfection of the human uPA amino-terminal fragment (ATF) on invasion and metastasis of cancer cells. First, a highly metastatic human lung giant-cell carcinoma cell line (PG), used as the target cell for evaluation of this effect, was demonstrated to express both uPA and uPAR. Then, ATF, which contains an intact uPAR binding site but is catalytically inactive, was designed as an antagonist of uPA-uPAR interaction and was transfected into PG cells. [(3)H]-Thymidine incorporation and cell growth curves indicated that expressed ATF did not affect the proliferation of transfected cells. However, analysis by scanning electron microscopy revealed that ATF changed the host cells from the typical invasive phenotype to a noninvasive one. Correspondingly, the modified Boyden chamber test in vitro showed that ATF expression significantly decreased the invasive capacity of transfected cells. Furthermore, in the spontaneous metastasis model, it was confirmed in vivo that expressed ATF remarkably inhibited lung metastasis of implanted ATF-transfected PG cells. In summary, autocrine ATF could act as an antagonist of uPA-uPAR interaction, and ATF cDNA transfection could efficiently inhibit the invasion and metastasis of the cancer cells. Inhibition of uPA-uPAR interaction on the cell surface might be a promising anti-invasion and anti-metastasis strategy.     

Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect.

Physiological concentrations of urokinase plasminogen activator (uPA) stimulated a chemotactic response in human monocytic THP-1 through binding to the urokinase receptor (uPAR). The effect did not require the protease moiety of uPA, as stimulation was achieved also with the N-terminal fragment (ATF), while the 33 kDa low molecular weight uPA was ineffective. Co-immunoprecipitation experiments showed association of uPAR with intracellular kinase(s), as demonstrated by in vitro kinase assays. Use of specific antibodies identified p56/p59hck as a kinase associated with uPAR in THP-1 cell extracts. Upon addition of ATF, p56/p59hck activity was stimulated within 2 min and returned to normal after 30 min. Since uPAR lacks an intracellular domain capable of interacting with intracellular kinase, activation of p56/p59hck must require a transmembrane adaptor. Evidence for this was strongly supported by the finding that a soluble form of uPAR (suPAR) was capable of inducing chemotaxis not only in THP-1 cells but also in cells lacking endogenous uPAR (IC50, 5 pM). However, activity of suPAR require chymotrypsin cleavage between the N-terminal domain D1 and D2 + D3. Chymotrypsin-cleaved suPAR also induced activation of p56/p59hck in THP-1 cells, with a time course comparable with ATF. Our data show that uPA-induced signal transduction takes place via uPAR, involves activation of intracellular tyrosine kinase(s) and requires an as yet undefined adaptor capable of connecting the extracellular ligand binding uPAR to intracellular transducer(s).     

Efficient Infection Mediated by Viral Receptors Incorporated into Retroviral Particles

Many host cell surface proteins, including viral receptors, are incorporated into enveloped viruses. To address the functional significance of these host proteins, murine leukemia viruses containing the cellular receptors for Rous sarcoma virus (Tva) or ecotropic murine leukemia virus (MCAT-1) were produced. These receptor-pseudotyped viruses efficiently infect cells expressing the cognate viral envelope glycoproteins, with titers of up to 10⁵ infectious units per milliliter for the Tva pseudotypes. Receptor and viral glycoprotein specificity and functional requirements are maintained, suggesting that receptor pseudotype infection recapitulates events of normal viral entry. The ability of the Tva and MCAT-1 pseudotypes to infect cells efficiently suggests that, in contrast to human immunodeficiency virus type 1 entry, neither of these retroviral receptors requires a coreceptor for membrane fusion. In addition, the ability of receptor pseudotypes to target infected cells suggests that they may be useful therapeutic reagents for directing infection of viral vectors. Receptor-pseudotyped viruses may be useful for identifying new viral receptors or for defining functional requirements of known receptors. Moreover, this work suggests that the production of receptor pseudotypes in vivo could provide a mechanism for expanded viral tropism with potential effects on the pathogenesis and evolution of the virus.     

Structural basis of interaction between urokinase-type plasminogen activator and its receptor

Recent studies indicate that binding of the urokinase-type plasminogen activator (uPA) to its high-affinity receptor (uPAR) orchestrates uPAR interactions with other cellular components that play a pivotal role in diverse (patho-)physiological processes, including wound healing, angiogenesis, inflammation, and cancer metastasis. However, notwithstanding the wealth of biochemical data available describing the activities of uPAR, little is known about the exact mode of uPAR/uPA interactions or the presumed conformational changes that accompany uPA/uPAR engagement. Here, we report the crystal structure of soluble urokinase plasminogen activator receptor (suPAR), which contains the three domains of the wild-type receptor but lacks the cell-surface anchoring sequence, in complex with the amino-terminal fragment of urokinase-type plasminogen activator (ATF), at the resolution of 2.8 A. We report the 1.9 A crystal structure of free ATF. Our results provide a structural basis, represented by conformational changes induced in uPAR, for several published biochemical observations describing the nature of uPAR/uPA interactions and provide insight into mechanisms that may be responsible for the cellular responses induced by uPA binding.     

A Species-Specific Amino Acid Difference in the Macaque CD4 Receptor Restricts Replication by Global Circulating HIV-1 Variants Representing Viruses from Recent Infection

HIV-1 replicates poorly in macaque cells, and this had hindered the advancement of relevant nonhuman primate model systems for HIV-1 infection and pathogenesis. Several host restriction factors have been identified that contribute to this species-specific restriction to HIV-1 replication, but these do not fully explain the poor replication of most strains of HIV-1 in macaque cells. Only select HIV-1 envelope variants, typically those derived from viruses that have been adapted in cell culture, result in infectious chimeric SIVs encoding HIV-1 envelope (SHIVs). Here we demonstrate that most circulating HIV-1 variants obtained directly from infected individuals soon after virus acquisition do not efficiently mediate entry using the macaque CD4 receptor. The infectivity of these viruses is ca. 20- to 50-fold lower with the rhesus and pig-tailed macaque versus the human CD4 receptor. In contrast, culture-derived HIV-1 envelope variants that facilitate efficient replication in macaques showed similar infectivity with macaque and human CD4 receptors (within ∼2-fold). The ability of an envelope to mediate entry using macaque CD4 correlated with its ability to mediate entry of cells expressing low levels of the human CD4 receptor and with soluble CD4 sensitivity. Species-specific differences in the functional capacity of the CD4 receptor to mediate entry mapped to a single amino acid difference at position 39 that is under strong positive selection, suggesting that the evolution of CD4 may have been influenced by its function as a viral receptor. These results also suggest that N39 in human CD4 may be a critical residue for interaction of transmitted HIV-1 variants. These studies provide important insights into virus-host cell interactions that have hindered the development of relevant nonhuman primate models for HIV-1 infection and provide possible markers, such as sCD4 sensitivity, to identify potential HIV-1 variants that could be exploited for development of better SHIV/macaque model systems.     

The role of envelope glycoprotein processing in murine leukemia virus infection.

The murine leukemia virus envelope protein is synthesized as a precursor molecule, Pr85env, which is proteolytically cleaved at an arginine residue to produce two mature envelope proteins, gp70 and p15(E). The results presented here indicate that mutation to lysine of the arginine found at the envelope precursor cleavage site results in a precursor which is cleaved with an efficiency at least 10-fold lower than the efficiency with which the wild-type protein is cleaved. This mutation has been used to investigate the requirement for envelope protein processing in various aspects of retroviral infection. Viruses produced by cells transfected with mutant proviral clones are approximately 10-fold less infectious than wild-type viruses. Mutant viruses are incapable of inducing XC cell syncytium formation and are 100-fold less efficient than wild-type viruses at rendering cells resistant to superinfection. Envelope glycoproteins bearing the lysine mutation are found in reduced amounts on the surface of infected cells, and as a result mutant virions contain significantly less envelope protein than do wild-type virions. The phenotypic effects of the processing mutation described here are most likely the result of this paucity of envelope glycoproteins in virions carrying the mutation.     

Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain.

The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored three-domain membrane protein playing a central role in pericellular plasminogen activation. We have found that urokinase (uPA) can cleave its receptor between domains 1 and 2 generating a cell-associated uPAR variant without ligand-binding properties. In extracts of U937 cells there are two uPAR variants which after complete deglycosylation have apparent molecular masses of 35,000 and 27,000. Analysis with monoclonal antibodies showed that these variants represented the intact uPAR and a two-domain form, uPAR(2+3), lacking ligand-binding domain 1. Trypsin treatment showed that both variants are present on the outside of the cells. Addition to the culture medium of an anticatalytic monoclonal antibody to uPA inhibited the formation of the uPAR(2+3), indicating that uPA is involved in its generation. Purified uPAR can be cleaved directly by uPA as well as by plasmin. The uPA-catalyzed cleavage does not require binding of the protease to the receptor through its epidermal growth factor-like receptor-binding domain, since low molecular weight uPA that lacks this domain also cleaves uPAR. This unusual reaction in which a specific binding protein is proteolytically inactivated by its own ligand may represent a regulatory step in the plasminogen activation cascade.     

Production of a urokinase plasminogen activator-IgG fusion protein (uPA-IgG) in the baculovirus expression system

Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPAIgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 μg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 μg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA.     

Dimerization controls the lipid raft partitioning of uPAR/CD87 and regulates its biological functions

The urokinase-type plasminogen activator receptor (uPAR/CD87) is a glycosylphosphatidylinositol-anchored membrane protein with multiple functions in extracellular proteolysis, cell adhesion, cell migration and proliferation. We now report that cell surface uPAR dimerizes and that dimeric uPAR partitions preferentially to detergent-resistant lipid rafts. Dimerization of uPAR did not require raft partitioning as the lowering of membrane cholesterol failed to reduce dimerization and as a transmembrane uPAR chimera, which does not partition to lipid rafts, also dimerized efficiently. While uPA bound to uPAR independently of its membrane localization and dimerization status, uPA-induced uPAR cleavage was strongly accelerated in lipid rafts. In contrast to uPA, the binding of Vn occurred preferentially to raft- associated dimeric uPAR and was completely blocked by cholesterol depletion.     

Heparan sulfate-independent cell binding and infection with furin-precleaved papillomavirus capsids

Papillomavirus infection normally involves virion binding to cell surface heparan sulfate proteoglycans (HSPGs). However, we found that human papillomavirus type 16 pseudovirions efficiently bound and infected cells lacking HSPGs if their L2 capsid protein was precleaved by furin, a cellular protease required for infection. The inability of pseudovirions to efficiently bind and infect cultured primary keratinocytes was also overcome by furin precleavage, suggesting that the defect involves altered HSPG modification. We conclude that the primary function of HSPG binding is to enable cell surface furin cleavage of L2 and that binding to a distinct cell surface receptor(s) is a subsequent step of papillomavirus infection.     

Rhabdovirus-Based Vectors with Human Immunodeficiency Virus Type 1 (HIV-1) Envelopes Display HIV-1-Like Tropism and Target Human Dendritic Cells

We describe replication-competent, vaccine strain-based rabies viruses (RVs) that lack their own single glycoprotein and express, instead, a chimeric RV-human immunodeficiency virus type 1 (HIV-1) envelope protein composed of the ectodomain and transmembrane domains of HIV-1 gp160 and the cytoplasmic domain of RV G. The envelope proteins from both X4 (NL4-3)- and R5X4 (89.6)-tropic HIV-1 strains were utilized. These recombinant viruses very closely mimicked an HIV-1- like tropism, as indicated by blocking experiments. Infection was inhibited by SDF-1 on cells expressing CD4 and CXCR4 for both viruses, whereas RANTES abolished infection of cells expressing CCR5 in addition to CD4 in studies of the RV expressing HIV-1(89.6) Env. In addition, preincubation with soluble CD4 or monoclonal antibodies directed against HIV-1 gp160 blocked the infectivity of both G-deficient viruses but did not affect the G-containing RVs. Our results also indicated that the G-deficient viruses expressing HIV-1 envelope protein, in contrast to wild-type RV but similar to HIV-1, enter cells by a pH-independent pathway. As observed for HIV-1, the surrogate viruses were able to target human peripheral blood mononuclear cells, macrophages, and immature and mature human dendritic cells (DC). Moreover, G-containing RV-based vectors also infected mature human DC, indicating that infection of these cells is also supported by RV G. The ability of RV-based vectors to infect professional antigen-presenting cells efficiently further emphasizes the potential use of recombinant RVs as vaccines.     

Human immunodeficiency virus type 1 V1-V2 envelope loop sequences expand and add glycosylation sites over the course of infection, and these modifications affect antibody neutralization sensitivity

Over the course of infection, human immunodeficiency virus type 1 (HIV-1) continuously adapts to evade the evolving host neutralizing antibody responses. Changes in the envelope variable loop sequences, particularly the extent of glycosylation, have been implicated in antibody escape. To document modifications that potentially influence antibody susceptibility, we compared envelope variable loops 1 and 2 (V1-V2) from multiple sequences isolated at the primary phase of infection to those isolated around 2 to 3 years into the chronic phase of infection in nine women with HIV-1 subtype A. HIV-1 sequences isolated during chronic infection had significantly longer V1-V2 loops, with a significantly higher number of potential N-linked glycosylation sites, than the sequences isolated early in infection. To assess the effects of these V1-V2 changes on antibody neutralization and infectivity, we created chimeric envelope sequences, which incorporated a subject's V1-V2 sequences into a common subtype A envelope backbone and then used them to generate pseudotyped viruses. Compared to the parent virus, the introduction of a subject's early-infection V1-V2 envelope variable loops rendered the chimeric envelope more sensitive to that subject's plasma samples but only to plasma samples collected >6 months after the sequences were isolated. Neutralization was not detected with the same plasma when the early-infection V1-V2 sequences were replaced with chronic-infection V1-V2 sequences, suggesting that changes in V1-V2 contribute to antibody escape. Pseudotyped viruses with V1-V2 segments from different times in infection, however, showed no significant difference in neutralization sensitivity to heterologous pooled plasma, suggesting that viruses with V1-V2 loops from early in infection were not inherently more neutralization sensitive. Pseudotyped viruses bearing chimeric envelopes with early-infection V1-V2 sequences showed a trend in infecting cells with low CD4 concentrations more efficiently, while engineered viruses with V1-V2 sequences isolated during chronic infection were moderately better at infecting cells with low CCR5 concentrations. These studies suggest that changes within the V1-V2 envelope domains over the course of an infection influence sensitivity to autologous neutralizing antibodies and may also impact host receptor/coreceptor interactions.     

The very low density lipoprotein receptor regulates urokinase receptor catabolism and breast cancer cell motility in vitro

The very low density lipoprotein receptor (VLDLr) binds diverse ligands, including urokinase-type plasminogen activator (uPA) and uPA-plasminogen activator inhibitor-1 (PAI-1) complex. In this study, we characterized the effects of the VLDLr on the internalization, catabolism, and function of the uPA receptor (uPAR) in MCF-7 and MDA-MB-435 breast cancer cells. When challenged with uPA.PAI-1 complex, MDA-MB-435 cells internalized uPAR; this process was inhibited by 80% when the activity of the VLDLr was neutralized with receptor-associated protein (RAP). To determine whether internalized uPAR is degraded, we studied the catabolism of [35S]methionine-labeled uPAR. In the absence of exogenous agents, the uPAR catabolism t(1)/(2) was 8.2 h. uPA.PAI-1 complex accelerated uPAR catabolism (t(1)/(2) to 1.8 h), while RAP inhibited uPAR catabolism in the presence (t(1)/(2) of 7.8 h) and absence (t(1)/(2) of 16.9 h) of uPA.PAI-1 complex, demonstrating a critical role for the VLDLr. When MCF-7 cells were cultured in RAP, cell surface uPAR levels increased gradually, reaching a new steady-state in 3 days. The amount of uPA which accumulated in the medium also increased. Culturing in RAP for 3 days increased MCF-7 cell motility by 2.2 +/- 0.1-fold and by 4.4 +/- 0.3-fold when 1.0 nM uPA was added. The effects of RAP on MCF-7 cell motility were entirely abrogated by an antibody which binds uPA and prevents uPA binding to uPAR. MCF-7 cells that were cultured in RAP demonstrated increased levels of activated mitogen-activated protein kinases. Furthermore, the MEK inhibitor, PD098059, decreased the motility of RAP-treated cells without affecting control cultures. These studies suggest a model in which the VLDLr regulates autocrine uPAR-initiated signaling and thereby regulates cellular motility.     

Viral and cellular factors governing hamster cell infection by murine and gibbon ape leukemia viruses.

Hamster cells are resistant to infection by most retroviruses, including Moloney murine leukemia virus (MoMLV) and gibbon ape leukemia viruses (GaLVs). We have constructed MoMLV-GaLV hybrid virions to identify viral and cellular determinants responsible for the inability of GaLV and MoMLV to infect hamster cells. The substitution of MoMLV core components for GaLV core components circumvents the resistance of hamster cells to infection by GaLV, demonstrating that hamster cells have receptors for GaLV but are not efficiently infected by this primate retrovirus because of a postpenetration block. In contrast, hamster cells are apparently resistant to MoMLV infection because although they bear a receptor for MoMLV, the receptor is nonfunctional. Treatment of CHO K1 or BHK 21 hamster cells with the glycosylation inhibitor tunicamycin allows the cells to be infected by MoMLV. The construction of MoMLV-GaLV hybrid virions that can efficiently infect resistant cells has allowed the identification of viral and cellular factors responsible for restricting infection of hamster cells by MoMLV and GaLV.     

Crystal structure of the urokinase receptor in a ligand-free form

The urokinase receptor urokinase-type plasminogen activator receptor (uPAR) is a surface receptor capable of not only focalizing urokinase-type plasminogen activator (uPA)-mediated fibrinolysis to the pericellular micro-environment but also promoting cell migration and chemotaxis. Consistent with this multifunctional role, uPAR binds several extracellular ligands, including uPA and vitronectin. Structural studies suggest that uPAR possesses structural flexibility. It is, however, not clear whether this flexibility is an inherent property of the uPAR structure per se or whether it is induced upon ligand binding. The crystal structure of human uPAR in its ligand-free state would clarify this issue, but such information remains unfortunately elusive. We now report the crystal structures of a stabilized, human uPAR (H47C/N259C) in its ligand-free form to 2.4 Å and in complex with amino-terminal fragment (ATF) to 3.2 Å. The structure of uPAR^H47C/N259C in complex with ATF resembles the wild-type uPAR·ATF complex, demonstrating that these mutations do not perturb the uPA binding properties of uPAR. The present structure of uPAR^H47C/N259C provides the first structural definition of uPAR in its ligand-free form, which represents one of the biologically active conformations of uPAR as defined by extensive biochemical studies. The domain boundary between uPAR DI–DII domains is more flexible than the DII–DIII domain boundary. Two important structural features are highlighted by the present uPAR structure. First, the DI–DIII domain boundary may face the cell membrane. Second, loop 130–140 of uPAR plays a dynamic role during ligand loading/unloading. Together, these studies provide new insights into uPAR structure–function relationships, emphasizing the importance of the inter-domain dynamics of this modular receptor.     

Changes in the Extracellular Envelope Glycoprotein of Variants That Evolve during the Course of Simian Immunodeficiency Virus SIVMne Infection Affect Neutralizing Antibody Recognition, Syncytium Formation, and Macrophage Tropism but Not Replication, Cytopathicity, or CCR-5 Coreceptor Recognition

Simian immunodeficiency virus SIVMne, like human immunodeficiency virus, evolves from a macrophage-tropic, non-syncytium-inducing virus at early times in infection to a T-cell-tropic, syncytium-inducing, cytopathic virus population over the course of progression to AIDS. Because the viruses isolated late in SIVMne infection of macaques include a complex mixture of variants, the viral determinants of such phenotypic changes have not been defined. To identify genetic changes that are important to virus evolution in the host, we constructed chimeric viruses by introducing variant envelope genes representative of proviruses throughout the course of infection and disease into the SIVMne parental clone (SIVMneCL8) that infected the macaque. The chimeric viruses expressed sequences encoding the surface unit of the envelope glycoprotein (Env-SU) of variants cloned between 35 and 170 weeks postinfection. The chimera with Env-SU from 35 weeks postinfection encoded only four changes in V1 compared to SIVMneCL8, whereas the chimeras encoding Env-SU from variants isolated later in infection encoded progressively more mutations both in V1 and elsewhere. Like SIVMneCL8, the chimeras were infectious for CEMx174 cells and macaque peripheral blood mononuclear cells. However, in contrast to SIVMneCL8, the chimeric viruses did not infect macaque macrophages, although each retained the ability to recognize the CCR-5 coreceptor. Thus, these data provide direct evidence that changes which evolve in Env-SU during the course of SIVMne infection do not alter CCR-5 interactions. Viruses encoding Env-SU from the latest times in infection (121 to 170 weeks postinfection), after disease was apparent, were syncytium inducing. However, these viruses were not highly cytopathic, suggesting that additional viral determinants may be required for the rapidly replicating, cytopathic phenotype of the uncloned mixed variant population. Changes in Env-SU did allow the virus to escape serum neutralizing antibodies that recognized the SIVMneCL8 parent. Moreover, the chimera encoding the Env-SU of a virus from 35 weeks postinfection, which differed from SIVMneCL8 only in V1, was not sensitive to neutralization by infected macaque sera, suggesting that V1 may define a portion of the principal neutralizing determinant for SIVMne. Together, these data suggest that SIV variants with changes in the Env-SU may be selected primarily by virtue of their ability to escape neutralizing antibody recognition.