Selective Interaction of Heparin with the Variable Region 3 within Surface Glycoprotein of Laboratory-Adapted Feline Immunodeficiency Virus

Heparan sulfate proteoglycans (HSPG) can act as binding receptors for certain laboratory-adapted (TCA) strains of feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV). Heparin, a soluble heparin sulfate (HS), can inhibit TCA HIV and FIV entry mediated by HSPG interaction in vitro. In the present study, we further determined the selective interaction of heparin with the V3 loop of TCA of FIV. Our current results indicate that heparin selectively inhibits infection by TCA strains, but not for field isolates (FS). Heparin also specifically interferes with TCA surface glycoprotein (SU) binding to CXCR4, by interactions with HSPG binding sites on the V3 loop of the FIV envelope protein. Peptides representing either the N- or C-terminal side of the V3 loop and containing HSPG binding sites were able to compete away the heparin block of TCA SU binding to CXCR4. Heparin does not interfere with the interaction of SU with anti-V3 antibodies that target the CXCR4 binding region or with the interaction between FS FIV and anti-V3 antibodies since FS SU has no HSPG binding sites within the HSPG binding region. Our data show that heparin blocks TCA FIV infection or entry not only through its competition of HSPG on the cell surface interaction with SU, but also by its interference with CXCR4 binding to SU. These studies aid in the design and development of heparin derivatives or analogues that can inhibit steps in virus infection and are informative regarding the HSPG/SU interaction.     

Mapping of the CXCR4 binding site within variable region 3 of the feline immunodeficiency virus surface glycoprotein

Feline immunodeficiency virus (FIV) shares with T-cell tropic strains of human immunodeficiency virus type 1 (HIV-1) the use of the chemokine receptor CXCR4 for cellular entry. In order to map the interaction of the FIV envelope surface unit (SU) with CXCR4, full-length FIV SU-Fc as well as constructs with deletions of extended loop L2, V3, V4, or V5 were produced in stable CHO cell lines. Binding studies were performed using these proteins on 3201 cells (CXCR4(hi) CD134(-)), with or without the CXCR4 inhibitor AMD3100. The findings established that SU binding to CXCR4 specifically requires the V3 region of SU. Synthetic peptides spanning the V3 region as well as a panel of monoclonal antibodies (MAbs) to SU were used to further map the site of CXCR4 interaction. Both the SU V3-specific antibodies and the full-length V3 peptide potently blocked binding of SU to CXCR4 and virus entry. By using a set of nested peptides overlapping a region of SU specifically recognized by CD134-dependent neutralizing V3 MAbs, we showed that the neutralizing epitope and the region required for CXCR4 binding are within the same contiguous nine-amino-acid sequence of V3. Site-directed mutagenesis was used to reveal that serine 393 and tryptophan 394 at the predicted tip of V3 are required to facilitate entry into the target cell via CXCR4. Although the amino acid sequences are not identical between FIV and HIV, the ability of FIV to bind and utilize both feline and human CXCR4 makes the feline model an attractive venue for development of broad-based entry antagonists.     

Structural mapping of CD134 residues critical for interaction with feline immunodeficiency virus

CD134 is a primary binding receptor for feline immunodeficiency virus (FIV), and with CXCR4 facilitates infection of CD4(+) T cells. Human CD134 fails to support FIV infection. To delineate the regions important for defining virus specificity of CD134, we exchanged domains between human and feline CD134. The binding site for FIV surface glycoprotein (SU) is located in domain 1, in a region distinct from the natural ligand (CD134L)-binding site. Mutagenesis showed that Asp60 and Asp62 are required for interaction with FIV, and modeling studies localized these two residues to the outer edge of domain 1. Substitutions S60D and N62D, in conjunction with H45S, R59G and V64K, imparted both FIV SU binding and receptor function to human CD134. Finally, we demonstrated that soluble CD134 facilitates infection of CD134(-) CXCR4(+) target cells in a manner analogous to CD4 augmentation of HIV infection.     

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.

Several members of the chemokine receptor family have recently been identified as coreceptors, with CD4, for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. In this report, we show that the envelope glycoproteins of several strains of HIV-2 and simian immunodeficiency virus (SIV) employ the same chemokine receptors for infection. Envelope glycoproteins from HIV-2 use CCR5 or CXCR4, while those from several strains of SIV use CCR5. Our data indicate also that some viral envelopes can use more than one coreceptor for entry and suggest that some of these coreceptors remain to be identified. To further understand how different envelope molecules use CCR5 as an entry cofactor, we show that soluble purified envelope glycoproteins (SU component) from CCR5-tropic HIV-1, HIV-2, and SIV can compete for binding of iodinated chemokine to CCR5. The competition is dependent on binding of the SU glycoprotein to cell surface CD4 and implies a direct interaction between envelope glycoproteins and CCR5. This interaction is specific since it is not observed with SU glycoprotein from a CXCR4-tropic virus or with a chemokine receptor that is not competent for viral entry (CCR1). For HIV-1, the interaction can be inhibited by antibodies specific for the V3 loop of SU. Soluble CD4 was found to potentiate binding of the HIV-2 ST and SIVmac239 envelope glycoproteins to CCR5, suggesting that a CD4-induced conformational change in SU is required for subsequent binding to CCR5. These data suggest a common fundamental mechanism by which structurally diverse HIV-1, HIV-2, and SIV envelope glycoproteins interact with CD4 and CCR5 to mediate viral entry.     

The Second Extracellular Loop of CXCR4 Determines Its Function as a Receptor for Feline Immunodeficiency Virus

The feline homolog of the α-chemokine receptor CXCR4 has recently been shown to support cell-cell fusion mediated by CXCR4-dependent strains of human immunodeficiency virus (HIV) and strains of feline immunodeficiency virus (FIV) that have been selected for growth in the Crandell feline kidney (CrFK) cell line. In this report we demonstrate that expression of CXCR4 alone is sufficient to render cells from diverse species permissive for fusion with FIV-infected cells, suggesting that CXCR4 is the sole receptor for CrFK-tropic strains of FIV, analogous to CD4-independent strains of HIV-2. To identify the regions of CXCR4 involved in fusion mediated by FIV, we screened panels of chimeric CXCR4 molecules for the ability to support fusion with FIV-infected cells. Human CXCR4 supported fusion more efficiently than feline CXCR4 and feline/human CXCR4 chimeras, suggesting that the second and third extracellular loops of human CXCR4 contain a critical determinant for receptor function. Rat/human CXCR4 chimeras suggested that the second extracellular loop contained the principal determinant for receptor function; however, chimeras constructed between human CXCR2 and CXCR4 revealed that the first and third loops of CXCR4 contribute to the FIV Env binding site, as replacement of these domains with the corresponding domains of CXCR2 rendered the molecule nonfunctional in fusion assays. Mutation of the DRY motif and the C-terminal cytoplasmic tail of CXCR4 did not affect the ability of the molecule to support fusion, suggesting that neither signalling via G proteins nor receptor internalization was required for fusion mediated by FIV; similarly, truncation of the N terminus of CXCR4 did not affect the function of the molecule as a receptor for FIV. CXCR4-transfected feline cells were rendered permissive for infection with both the CrFK-tropic PET isolate of FIV and the CXCR4-dependent RF strain of HIV-1, and susceptibility to infection correlated well with ability to support fusion. The data suggest that the second extracellular loop of CXCR4 is the major determinant of CXCR4 usage by FIV.     

Shared usage of the chemokine receptor CXCR4 by the feline and human immunodeficiency viruses.

Feline immunodeficiency virus (FIV) induces a disease state in the domestic cat that is similar to AIDS in human immunodeficiency virus (HIV)-infected individuals. As with HIV, FIV can be divided into primary and cell culture-adapted isolates. Adaptation of FIV to replicate and form syncytia in the Crandell feline kidney (CrFK) cell line is accompanied by an increase in the net charge of the V3 loop of the envelope glycoprotein, mirroring the changes observed in the V3 loop of HIV gp120 with the switch from a non-syncytium-inducing phenotype to a syncytium-inducing phenotype. These data suggest a common mechanism of infection with FIV and HIV. In this study, we demonstrate that cell culture-adapted strains of FIV are able to use the alpha-chemokine receptor CXCR4 for cell fusion. Following ectopic expression of human CXCR4 on nonpermissive human cells, the cells are able to fuse with FIV-infected feline cells. Moreover, fusion between FIV-infected feline cells and CXCR4-transfected human cells is inhibited by both anti-CXCR4 and anti-FIV antibodies. cDNAs encoding the feline CXCR4 homolog were cloned from both T-lymphoblastoid and kidney cell lines. Feline CXCR4 displayed 94.9% amino acid sequence identity with human CXCR4 and was found to be expressed widely on cell lines susceptible to infection with cell culture-adapted strains FIV. Ectopic expression of feline CXCR4 on human cells rendered the cells susceptible to FIV-dependent fusion. Moreover, feline CXCR4 was found to be as efficient as human CXCR4 in supporting cell fusion between CD4-expressing murine fibroblast cells and either HIV type 1 (HIV-1) or HIV-2 Env-expressing human cells. Previous studies have demonstrated that feline cells expressing human CD4 are not susceptible to infection with HIV-1; therefore, further restrictions to HIV-1 Env-dependent fusion may exist in feline cells. As feline and human CXCR4 support both FIV- and HIV-dependent cell fusion, these results suggest a close evolutionary link between FIV and HIV and a common mechanism of infection involving an interaction between the virus and a member of the seven-transmembrane domain chemokine receptor family of molecules.     

Engineered CD4- and CXCR4-using simian immunodeficiency virus from African green monkeys is neutralization sensitive and replicates in nonstimulated lymphocytes

During human immunodeficiency virus type 1 (HIV-1) infection, disease progression correlates with the occurrence of variants using the coreceptor CXCR4 for cell entry. In contrast, apathogenic simian immunodeficiency virus (SIV) from African green monkeys (SIVagm), specifically the molecular virus clone SIVagm3mc, uses CCR5, Bob, and Bonzo as coreceptors throughout the course of infection. The influence of an altered coreceptor usage on SIVagm3mc replication was studied in vitro and in vivo. The putative coreceptor binding domain, the V3 region of the surface envelope (SU) glycoprotein, was replaced by the V3 loop of a CD4- and CXCR4-tropic HIV-1 strain. The resulting virus, termed SIVagm3-X4mc, exclusively used CD4 and CXCR4 for cell entry. Consequently, its in vitro replication was inhibited by SDF-1, the natural ligand of CXCR4. Surprisingly, SIVagm3-X4mc was able to replicate in vitro not only in interleukin-2- and phytohemagglutinin-stimulated but also in nonstimulated peripheral blood mononuclear cells (PBMCs) from nonhuman primates. After experimental infection of two pig-tailed macaques with either SIVagm3-X4mc or SIVagm3mc, the coreceptor usage was maintained during in vivo replication. Cell-associated and plasma viral loads, as well as viral DNA copy numbers, were found to be comparable between SIVagm3mc and SIVagm 3-X4mc infections, and no pathological changes were observed up to 14 months postinfection. Interestingly, the V3 loop exchange rendered SIVagm3-X4mc susceptible to neutralizing antibodies present in the sera of SIVagm3-X4mc- and SIVagm3mc-infected pig-tailed macaques. Our study describes for the first time a successful exchange of a V3 loop in nonpathogenic SIVagm resulting in CD4 and CXCR4 usage and modulation of virus replication in nonstimulated PBMCs as well as sensitivity toward neutralization.     

Factors that increase the effective concentration of CXCR4 dictate feline immunodeficiency virus tropism and kinetics of replication

The surface glycoprotein (gp95) of the feline immunodeficiency virus (FIV) binds in a strain-specific manner to several cell surface molecules, including CXCR4, heparan sulfate proteoglycans (HSPGs), DC-SIGN, and a 43-kDa cell surface receptor on T cells recently identified as CD134 by M. Shimojima et al. (Science 303:1192-1195, 2004). CXCR4 is the entry receptor in all known cases, and the other molecules act as binding receptors to help facilitate infection. In this report, we confirm and extend the findings regarding CD134 as a primary receptor for FIV. In addition, we show that temperature critically influences the binding properties of FIV gp95 to CXCR4 and HSPGs. The data show that gp95 of the field strain FIV-PPR bound to CXCR4 at 22 degrees C, whereas binding was not detected at 4 degrees C. In contrast, binding of the laboratory adapted FIV-34TF10 gp95 was observed at either 4 degrees C or 22 degrees C, albeit at increased levels at the higher temperature. The level of CXCR4 increased after the temperature was switched from 4 to 22 degrees C, whereas the level of HSPGs decreased, resulting in higher binding of gp95 from both strains to CXCR4 and lower binding of gp95 of FIV-34TF10 to HSPGs (FIV-PPR gp95 does not bind to these molecules). The findings also show that HSPGs facilitate the CXCR4-mediated infectivity of CrFK and G355-5 cells by FIV-34TF10. These two nonlymphoid cell lines express very low levels of CXCR4 and are permissive to FIV-34TF10 but not to productive infection by FIV-PPR. However, overexpression of human CXCR4 in CrFK or G-355-5 cells resulted in extensive cell fusion and infection by FIV-PPR. Taken together, these findings indicate that factors that increase the effective concentration of CXCR4 enhance FIV infectivity and may involve (i) temperature or ligand-induced conformational changes in CXCR4 that enhance SU binding, (ii) coreceptor interactions with gp95 that either alter gp95 conformation to enhance CXCR4 binding and/or raise the localized concentration of receptor or ligand, or (iii) direct increase in CXCR4 concentration via overexpression.     

Alternative coreceptor requirements for efficient CCR5- and CXCR4-mediated HIV-1 entry into macrophages

Macrophage tropism of human immunodeficiency virus type 1 (HIV-1) is distinct from coreceptor specificity of the viral envelope glycoproteins (Env), but the virus-cell interactions that contribute to efficient HIV-1 entry into macrophages, particularly via CXCR4, are not well understood. Here, we characterized a panel of HIV-1 Envs that use CCR5 (n = 14) or CXCR4 (n = 6) to enter monocyte-derived macrophages (MDM) with various degrees of efficiency. Our results show that efficient CCR5-mediated MDM entry by Env-pseudotyped reporter viruses is associated with increased tolerance of several mutations within the CCR5 N terminus. In contrast, efficient CXCR4-mediated MDM entry was associated with reduced tolerance of a large deletion within the CXCR4 N terminus. Env sequence analysis and structural modeling identified amino acid variants at positions 261 and 263 within the gp41-interactive region of gp120 and a variant at position 326 within the gp120 V3 loop that were associated with efficient CXCR4-mediated MDM entry. Mutagenesis studies showed that the gp41 interaction domain variants exert a significant but strain-specific influence on CXCR4-mediated MDM entry, suggesting that the structural integrity of the gp120-gp41 interface is important for efficient CXCR4-mediated MDM entry of certain HIV-1 strains. However, the presence of Ile326 in the gp120 V3 loop stem, which we show by molecular modeling is located at the gp120-coreceptor interface and predicted to interact with the CXCR4 N terminus, was found to be critical for efficient CXCR4-mediated MDM entry of divergent CXCR4-using Envs. Together, the results of our study provide novel insights into alternative mechanisms of Env-coreceptor engagement that are associated with efficient CCR5- and CXCR4-mediated HIV-1 entry into macrophages.     

Shared usage of the chemokine receptor CXCR4 by primary and laboratory-adapted strains of feline immunodeficiency virus

Strains of the feline immunodeficiency virus (FIV) presently under investigation exhibit distinct patterns of in vitro tropism. In particular, the adaptation of FIV for propagation in Crandell feline kidney (CrFK) cells results in the selection of strains capable of forming syncytia with cell lines of diverse species origin. The infection of CrFK cells by CrFK-adapted strains appears to require the chemokine receptor CXCR4 and is inhibited by its natural ligand, stromal cell-derived factor 1alpha (SDF-1alpha). Here we found that inhibitors of CXCR4-mediated infection by human immunodeficiency virus type I (HIV-1), such as the bicyclam AMD3100 and short peptides derived from the amino-terminal region of SDF-1alpha, also blocked infection of CrFK by FIV. Nevertheless, we observed differences in the ranking order of the peptides as inhibitors of FIV and HIV-1 and showed that such differences are related to the species origin of CXCR4 and not that of the viral envelope. These results suggest that, although the envelope glycoproteins of FIV and HIV-1 are substantially divergent, FIV and HIV-1 interact with CXCR4 in a highly similar manner. We have also addressed the role of CXCR4 in the life cycle of primary isolates of FIV. Various CXCR4 ligands inhibited infection of feline peripheral blood mononuclear cells (PBMC) by primary FIV isolates in a concentration-dependent manner. These ligands also blocked the viral transduction of feline PBMC by pseudotyped viral particles when infection was mediated by the envelope glycoprotein of a primary FIV isolate but not by the G protein of vesicular stomatitis virus, indicating that they act at an envelope-mediated step and presumably at viral entry. These findings strongly suggest that primary and CrFK-adapted strains of FIV, despite disparate in vitro tropisms, share usage of CXCR4.     

Sequential CD134-CXCR4 interactions in feline immunodeficiency virus (FIV): soluble CD134 activates FIV Env for CXCR4-dependent entry and reveals a cryptic neutralization epitope

Recombinant soluble CD134 (sCD134) facilitated feline immunodeficiency virus (FIV) entry into CXCR4-positive, cell surface CD134-negative target cells. sCD134-activated entry was dose dependent and CXCR4 dependent. We used the sCD134 activation system to explore the neutralization by four anti-V3 monoclonal antibodies (MAbs). V3 MAbs weakly neutralized FIV infection using target cells expressing both CD134 and CXCR4 but potently inhibited sCD134-activated entry into target cells expressing CXCR4 alone. These findings provide direct evidence for a sequential interaction of FIV Env with CD134 and CXCR4 and reveal the presence of a cryptic epitope in V3 that is masked in the mature envelope oligomers.     

R5X4 viruses are evolutionary, functional, and antigenic intermediates in the pathway of a simian-human immunodeficiency virus coreceptor switch

To examine the pathway of the coreceptor switching of CCR5-using (R5) virus to CXCR4-using (X4) virus in simian-human immunodeficiency virus SHIV(SF162P3N)-infected rhesus macaque BR24, analysis was performed on variants present at 20 weeks postinfection, the time when the signature gp120 V3 loop sequence of the X4 switch variant was first detected by PCR. Unexpectedly, circulating and tissue variants with His/Ile instead of the signature X4 V3 His/Arg insertions predominated at this time point. Phylogenetic analysis of the sequences of the C2 conserved region to the V5 variable loop of the envelope (Env) protein showed that viruses bearing HI insertions represented evolutionary intermediates between the parental SHIV(SF162P3N) and the final X4 HR switch variant. Functional analyses demonstrated that the HI variants were phenotypic intermediates as well, capable of using both CCR5 and CXCR4 for entry. However, the R5X4 intermediate virus entered CCR5-expressing target cells less efficiently than the parental R5 strain and was more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 or the final X4 virus. It was also more sensitive than the parental R5 virus to antibody neutralization, especially to agents directed against the CD4 binding site, but not as sensitive as the late X4 virus. Significantly, the V3 loop sequence that determined CXCR4 use also conferred soluble CD4 neutralization sensitivity. Collectively, the data illustrate that, similar to human immunodeficiency virus type 1 (HIV-1) infection in individuals, the evolution from CCR5 to CXCR4 usage in BR24 transitions through an intermediate phase with reduced virus entry and coreceptor usage efficiencies. The data further support a model linking an open envelope gp120 conformation, better CD4 binding, and expansion to CXCR4 usage.     

Binding of recombinant feline immunodeficiency virus surface glycoprotein to feline cells: role of CXCR4, cell-surface heparans, and an unidentified non-CXCR4 receptor

To address the role of CXCR4 in the cell-surface attachment of the feline immunodeficency virus (FIV), a soluble fusion protein, gp95-Fc, consisting of the surface glycoprotein (SU, gp95) of either a primary (PPR) or cell line-adapted (34TF10) FIV strain was fused in frame with the Fc domain of human immunoglobulin G1. The recombinant SU-immunoadhesins were used as probes to investigate the cellular binding of FIV SU. In agreement with the host cell range properties of both viruses, binding of 34TF10 gp95-Fc was observed for all cell lines tested, whereas PPR gp95-Fc bound only to primary feline T cells. 34TF10 gp95-Fc also bound to Jurkat and HeLa cells, consistent with the ability of FIV to use human CXCR4 as a fusion receptor. As expected, 34TF10 gp95-Fc binding to Jurkat cells was blocked by addition of stromal cell-derived factor 1alpha (SDF-1alpha), as was binding to the 3201 feline lymphoma cell line. However, SDF-1alpha, RANTES, macrophage inflammatory protein 1beta, and heparin all failed to inhibit the binding of either gp95-Fc to primary T cells, suggesting that a non-CXCR4 receptor is involved in the binding of FIV SU. In this regard, an unidentified 40-kDa protein species from the surface of primary T cells but not Jurkat and 3201 cells specifically coprecipitated with both gp95-Fc. Yet another type of binding of 34TF10 gp95-Fc to adherent kidney cells was noted. SDF-1alpha failed to block the binding of 34TF10 gp95-Fc to either HeLa, Crandel feline leukemia, or G355-5 cells. However, binding was severely impaired in the presence of soluble heparin, as well as after enzymatic removal of surface heparans or on cells deficient in heparan expression. These overall findings suggest that in addition to CXCR4, a non-CXCR4 receptor and cell-surface heparans also play an important role in FIV gp95 cell surface interactions on specific target cells.     

Cooperation of the V1/V2 and V3 domains of human immunodeficiency virus type 1 gp120 for interaction with the CXCR4 receptor

Human immunodeficiency virus type 1 (HIV-1) entry is triggered by the interaction of the gp120 envelope glycoprotein with a cellular chemokine receptor, either CCR5 or CXCR4. We have identified different mutations in human CXCR4 that prevent efficient infection by one HIV-1 strain (NDK) but not another (LAI) and sought to define these strain-dependent effects at the gp120 level. The lack of activity toward the NDK strain of the HHRH chimeric CXCR4 in which the second extracellular loop (ECL2) derived from the rat CXCR4 and of CXCR4 with mutations at an aspartic acid in ECL2 (D193A and D193R) was apparently due to the sequence of the third variable loop (V3) of gp120, more precisely, to its C-terminal part. Indeed, substitution of the LAI V3 loop or only its C-terminal part in the NDK gp 120 context was sufficient to restore usage of the HHRH, D193A, and D193R receptors. The same result was achieved upon mutation of a single lysine residue of the NDK V3 loop to alanine (K319A) but not to arginine (K319R). These results provide a strong case for a direct interaction between the gp120 V3 loop and the ECL2 domain of CXCR4. By contrast, V3 substitutions had no effect on the inability of NDK to infect cells via a mutant CXCR4 in which the amino-terminal extracellular domain (NT) is deleted. In experiments with a set of chimeric NDK-LAI gp120s, the V1/V2 region from LAI gp120 was both necessary and sufficient for usage of the NT-deleted CXCR4. Different variable domains of gp120 can therefore cooperate for a functional interaction with CXCR4.     

Feline immunodeficiency virus cell entry

The process of feline immunodeficiency virus (FIV) cell entry was examined using assays for virus replication intermediates. FIV subtype B was found to utilize the chemokine receptor CXCR4, but not CCR5, as a cellular receptor. Zidovudine blocked formation of late viral replication products most effectively, including circular DNA genome intermediates. Our findings extend the role of CXCR4 as a primary receptor for CD4-independent cell entry by FIV.     

Feline immunodeficiency virus xenoinfection: the role of chemokine receptors and envelope diversity

The use of chemokine receptors as cell recognition signals is a property common to several lentiviruses, including feline, human, and simian immunodeficiency viruses. Previously, two feline immunodeficiency virus (FIV) isolates, V1CSF and Petaluma, were shown to use chemokine receptors in a strain-dependent manner to infect human peripheral blood mononuclear cells (PBMC) (J. Johnston and C. Power, J. Virol. 73:2491-2498, 1999). Since the sequences of these viruses differed primarily in regions of the FIV envelope gene implicated in receptor use and cell tropism, envelope chimeras of V1CSF and Petaluma were constructed to investigate the role of envelope diversity in the profiles of chemokine receptors used by FIV to infect primate cells. By use of a receptor-blocking assay, all viruses were found to infect human and macaque PBMC through a mechanism involving the CXCR4 receptor. However, infection by viruses encoding the V3-to-V5 region of the V1CSF surface unit was also inhibited by blockade of the CCR3 or CCR5 receptor. Similar results were obtained with GHOST cells, human osteosarcoma cells expressing specific combinations of chemokine receptors. CXCR4 was required for infection by all FIV strains, but viruses expressing the V3-to-V5 region of V1CSF required the concurrent presence of either CCR3 or CCR5. In contrast, CXCR4 alone was sufficient to allow infection of GHOST cells by FIV strains possessing the V3-to-V5 region of Petaluma. To assess the role of primate chemokine receptors in productive infection, Crandell feline kidney (CrFK) cells that expressed human CXCR4, CCR3, or CCR5 in addition to feline CXCR4 were generated. Sustained infection by viruses encoding the V3-to-V5 region of V1CSF was detected in CrFK cells expressing human CCR3 or CCR5 but not in cells expressing CXCR4 alone, while all CrFK cell lines were permissive to viruses encoding the V3-to-V5 region of Petaluma. These results indicate that FIV uses chemokine receptors to infect both human and nonhuman primate cells and that the profiles of these receptors are dependent on envelope sequence, and they provide insights into the mechanism by which xenoinfections may occur.     

Feline immunodeficiency virus tropism

Feline immunodeficiency virus (FIV) induces a disease similar to acquired immunodeficiency syndrome (AIDS) in cats, yet in contrast to human immunodeficiency virus (HIV), CD4 is not the viral receptor. We identified a primary receptor for FIV as CD134 (OX40), a T cell activation antigen and costimulatory molecule. CD134 expression promotes viral binding and renders cells permissive for viral entry, productive infection, and syncytium formation. Infection is CXCR4-dependent, analogous to infection with X4 strains of HIV. Thus, despite the evolutionary divergence of the feline and human lentiviruses, both viruses use receptors that target the virus to a subset of cells that are pivotal to the acquired immune response. Further, we applied the new method for FIV receptor to Ebola virus entry factors with some modifications, and identified receptor-type tyrosine kinases, Axl and Dtk (members of Tyro3 family). Distribution of the molecules matches well with the Ebola virus tropism.     

The crown and stem of the V3 loop play distinct roles in human immunodeficiency virus type 1 envelope glycoprotein interactions with the CCR5 coreceptor

Human immunodeficiency virus type 1 envelope glycoprotein gp120 interacts with CD4 and the CCR5 coreceptor in order to mediate viral entry. A CD4-induced surface on gp120, primarily composed of residues in the V3 loop and the C4 domain, interacts with CCR5. In the present study, we generated envelope glycoproteins comprising chimeric V3 loops and/or V3 loops with deletions and studied their binding to CCR5 amino-terminal domain (Nt)-based sulfopeptides and cell surface CCR5, as well as their ability to mediate viral entry. We thus delineated two functionally distinct domains of the V3 loop, the V3 stem and the V3 crown. The V3 stem alone mediates soluble gp120 binding to the CCR5 Nt. In contrast, both the V3 stem and crown are required for soluble gp120 binding to cell surface CCR5. Within the context of a virion, however, the V3 crown alone determines coreceptor usage. Our data support a two-site gp120-CCR5 binding model wherein the V3 crown and stem interact with distinct regions of CCR5 in order to mediate viral entry.     

Evolution of replication efficiency following infection with a molecularly cloned feline immunodeficiency virus of low virulence

The development of an effective vaccine against human immunodeficiency virus is considered to be the most practicable means of controlling the advancing global AIDS epidemic. Studies with the domestic cat have demonstrated that vaccinal immunity to infection can be induced against feline immunodeficiency virus (FIV); however, protection is largely restricted to laboratory strains of FIV and does not extend to primary strains of the virus. We compared the pathogenicity of two prototypic vaccine challenge strains of FIV derived from molecular clones; the laboratory strain PET(F14) and the primary strain GL8(414). PET(F14) established a low viral load and had no effect on CD4(+)- or CD8(+)-lymphocyte subsets. In contrast, GL8(414) established a high viral load and induced a significant reduction in the ratio of CD4(+) to CD8(+) lymphocytes by 15 weeks postinfection, suggesting that PET(F14) may be a low-virulence-challenge virus. However, during long-term monitoring of the PET(F14)-infected cats, we observed the emergence of variant viruses in two of three cats. Concomitant with the appearance of the variant viruses, designated 627(W135) and 628(W135,) we observed an expansion of CD8(+)-lymphocyte subpopulations expressing reduced CD8 beta-chain, a phenotype consistent with activation. The variant viruses both carried mutations that reduced the net charge of the V3 loop (K409Q and K409E), giving rise to a reduced ability of the Env proteins to both induce fusion and to establish productive infection in CXCR4-expressing cells. Further, following subsequent challenge of na?ve cats with the mutant viruses, the viruses established higher viral loads and induced more marked alterations in CD8(+)-lymphocyte subpopulations than did the parent F14 strain of virus, suggesting that the E409K mutation in the PET(F14) strain contributes to the attenuation of the virus.