Upregulation of surface feline CXCR4 expression following ectopic expression of CCR5: implications for studies of the cell tropism of feline immunodeficiency virus

Feline CXCR4 and CCR5 were expressed in feline cells as fusion proteins with enhanced green fluorescent protein (EGFP). Expression of the EGFP fusion proteins was localized to the cell membrane, and surface expression of CXCR4 was confirmed by using a cross-species-reactive anti-CXCR4 monoclonal antibody. Ectopic expression of feline CCR5 enhanced expression of either endogenous feline CXCR4 or exogenous feline or human CXCR4 expressed from a retrovirus vector, indicating that experiments investigating the effect of CCR5 expression on feline immunodeficiency virus (FIV) infection must be interpreted with caution. Susceptibility to infection with cell culture-adapted strains of FIV or to syncytium formation following transfection with a eukaryotic vector expressing an env gene from a cell culture-adapted strain of virus correlated with expression of either human or feline CXCR4, whereas feline CCR5 had no effect. In contrast, neither CXCR4 nor CCR5 rendered cells permissive to either productive infection with primary strains of FIV or syncytium formation following transfection with primary env gene expression vectors. Screening a panel of Ghost cell lines expressing diverse human chemokine receptors confirmed that CXCR4 alone supported fusion mediated by the FIV Env from cell culture-adapted viruses. CXCR4 expression was upregulated in Ghost cells coexpressing CXCR4 and CCR5 or CXCR4, CCR5, and CCR3, and susceptibility to FIV infection could be correlated with the level of CXCR4 expression. The data suggest that beta-chemokine receptors may influence FIV infection by modulating the expression of CXCR4.     

A neutralizing antibody-inducing peptide of the V3 domain of feline immunodeficiency virus envelope glycoprotein does not induce protective immunity.

Specific-pathogen-free cats, immunized with a 22-amino-acid synthetic peptide designated V3.3 and derived from the third variable region of the envelope glycoprotein of the Petaluma isolate of feline immunodeficiency virus (FIV), developed high antibody titers to the V3.3 peptide and to purified virus, as assayed by enzyme-linked immunoassays, as well as neutralizing antibodies, as assayed by the inhibition of syncytium formation in Crandell feline kidney cells. V3.3-immunized animals and control cats were challenged with FIV and then monitored for 12 months; V3.3 immunization failed to prevent FIV infection, as shown by virus isolation, anti-whole virus and anti-p24 immunoglobulin G antibody responses, and positive PCRs for gag and env gene fragments. Sequence analysis of the V3 region showed no evidence for the emergence of escape mutants that might have contributed to the lack of protection. The sera of the V3.3-hyperimmunized cats and two anti-V3.3 monoclonal antibodies neutralized FIV infectivity for Crandell feline kidney cells at high antibody dilutions but paradoxically failed to completely neutralize FIV infectivity at low dilutions. Moreover, following FIV challenge, V3.3-immunized animals developed a faster and higher antiviral antibody response than control cats. This was probably due to enhanced virus replication, as also suggested by quantitative PCR data.     

Experimental vaccine protection against homologous and heterologous strains of feline immunodeficiency virus.

More than 90% of cats immunized with inactivated whole infected-cell or cell-free feline immunodeficiency virus (FIV) vaccines were protected against intraperitoneal infection with 10 50% animal infectious doses of either homologous FIV Petaluma (28 of 30 cats) or heterologous FIV Dixon strain (27 of 28 cats). All 15 control cats were readily infected with either strain of FIV. Protection appears to correlate with antiviral envelope antibody levels by a mechanism yet to be determined.     

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.     

Enhancement of feline immunodeficiency virus infection after immunization with envelope glycoprotein subunit vaccines.

Cats were immunized three times with different recombinant feline immunodeficiency virus (FIV) candidate vaccines. Recombinant vaccinia virus (rVV)-expressed envelope glycoprotein with (vGR657) or without (vGR657 x 15) the cleavage site and an FIV envelope bacterial fusion protein (beta-Galactosidase-Env) were incorporated into immune-stimulating complexes or adjuvanted with Quil A. Although all immunized cats developed antibodies against the envelope protein, only the cats vaccinated with the rVV-expressed envelope glycoproteins developed antibodies which neutralized FIV infection of Crandell feline kidney cells. These antibodies failed to neutralize infection of thymocytes with a molecularly cloned homologous FIV. After the third immunization the cats were challenged with homologous FIV. Two weeks after challenge the cell-associated viral load proved to be significantly higher in the cats immunized with vGR657 and vGR657 x 15 than in the other cats. The cats immunized with vGR657 and vGR657 x 15 also developed antibodies against the Gag proteins more rapidly than the cats immunized with beta-Galactosidase-Env or the control cats. This suggested that immunization with rVV-expressed glycoprotein of FIV results in enhanced infectivity of FIV. It was shown that the observed enhancement could be transferred to naive cats with plasma collected at the day of challenge.     

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.     

CXCR4 Is Required by a Nonprimate Lentivirus: Heterologous Expression of Feline Immunodeficiency Virus in Human, Rodent, and Feline Cells

A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors.     

A dormant internal ribosome entry site controls translation of feline immunodeficiency virus

The characterization of internal ribosome entry sites (IRESs) in virtually all lentiviruses prompted us to investigate the mechanism used by the feline immunodeficiency virus (FIV) to produce viral proteins. Various in vitro translation assays with mono- and bicistronic constructs revealed that translation of the FIV genomic RNA occurred both by a cap-dependent mechanism and by weak internal entry of the ribosomes. This weak IRES activity was confirmed in feline cells expressing bicistronic RNAs containing the FIV 5' untranslated region (UTR). Surprisingly, infection of feline cells with FIV, but not human immunodeficiency virus type 1, resulted in a great increase in FIV translation. Moreover, a change in the cellular physiological condition provoked by heat stress resulted in the specific stimulation of expression driven by the FIV 5' UTR while cap-dependent initiation was severely repressed. These results reveal the presence of a "dormant" IRES that becomes activated by viral infection and cellular stress.     

Probing the interaction between feline immunodeficiency virus and CD134 by using the novel monoclonal antibody 7D6 and the CD134 (Ox40) ligand

The feline immunodeficiency virus (FIV) targets activated CD4-positive helper T cells preferentially, inducing an AIDS-like immunodeficiency in its natural host species, the domestic cat. The primary receptor for FIV is CD134, a member of the tumor necrosis factor receptor superfamily, and all primary viral strains tested to date use CD134 for infection. We examined the expression of CD134 in the cat using a novel anti-feline CD134 monoclonal antibody (MAb), 7D6, and showed that as in rats and humans, CD134 expression is restricted tightly to CD4+, and not CD8+, T cells, consistent with the selective targeting of these cells by FIV. However, FIV is also macrophage tropic, and in chronic infection the viral tropism broadens to include B cells and CD8+ T cells. Using 7D6, we revealed CD134 expression on a B220-positive (B-cell) population and on cultured macrophages but not peripheral blood monocytes. Moreover, macrophage CD134 expression and FIV infection were enhanced by activation in response to bacterial lipopolysaccharide. Consistent with CD134 expression on human and murine T cells, feline CD134 was abundant on mitogen-stimulated CD4+ T cells, with weaker expression on CD8+ T cells, concordant with the expansion of FIV into CD8+ T cells with progression of the infection. The interaction between FIV and CD134 was probed using MAb 7D6 and soluble CD134 ligand (CD134L), revealing strain-specific differences in sensitivity to both 7D6 and CD134L. Infection with isolates such as PPR and B2542 was inhibited well by both 7D6 and CD134L, suggesting a lower affinity of interaction. In contrast, GL8, CPG, and NCSU were relatively refractory to inhibition by both 7D6 and CD134L and, accordingly, may have a higher-affinity interaction with CD134, permitting infection of cells where CD134 levels are limiting.     

Detection of feline immunodeficiency virus in semen from seropositive domestic cats (Felis catus).

Electroejaculates from experimentally infected domestic cats were evaluated for the presence of feline immunodeficiency virus (FIV). Virus was isolated from cell-free seminal plasma and seminal cells by cocultivation with a feline interleukin-2-dependent CD4+ T-cell line, in which productive infection was demonstrated by syncytium formation and FIV gag p26 antigen secretion. In addition, an 868-bp segment of the FIV gag provirus gene was identified in cocultured cells by PCR and Southern analysis. A 582-bp fragment of the FIV gag provirus genome was detected by nested PCR and Southern analysis in nonfractionated seminal cells and in sperm purified by a swim-up procedure. This is the first report describing the detection of replication-competent FIV in cell-free and cell-associated forms in domestic cat semen.     

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.     

Xenoinfection of nonhuman primates by feline immunodeficiency virus

New viral infections in humans usually result from viruses that have been transmitted from other species as zoonoses. For example, it is accepted widely that human immunodeficiency virus (HIV) is the result of the propagation and adaptation of a simian immunodeficiency virus (SIV) from nonhuman primates to man [1]. Previously, we reported productive infection of primary human cells in vitro by feline immunodeficiency virus (FIV) [2], a lentivirus that causes an immunodeficiency syndrome in cats similar to HIV in humans [3]. The present study extends these findings by demonstrating that cynomolgus macaques (Macaca fasicularis) infected with FIV exhibited clinical signs, including depletion of CD4+ cells and weight loss, that are consistent with FIV infection. The development of an antibody response to FIV gag-encoded proteins and detection of virus-specific sequences in sera, blood-derived cells, and necropsied tissue accompanied these changes. Moreover, the reactivation of FIV replication from latently infected cells was observed after stimulation in vitro with phorbol esters and in vivo with tetanus toxoid. The proposed use of lentiviruses in human gene therapy [4, 5] and of nonhuman cells and organs in xenotransplantation [6] has raised concerns about zoonoses as potential sources of new human pathogens. Therefore, the study of FIV infection of primate cells may provide insight into the principles underlying retroviral xenoinfections.     

Apoptosis induced by tumor necrosis factor in cells chronically infected with feline immunodeficiency virus.

Tumor necrosis factor alpha (TNF-alpha) induced morphologic changes such as chromatin condensation and cell shrinkage in a feline fibroblastic cell line (CRFK) chronically infected with feline immunodeficiency virus (FIV) but not in uninfected CRFK cells. DNA extracted from TNF-alpha-treated CRFK cells infected with FIV showed a ladder of nucleosomal DNA, indicating that this cytocidal effect by TNF-alpha was due to programmed cell death, or apoptosis. These findings may have implications for understanding the pathogenesis of FIV infection and for the design of specific therapeutic strategies for AIDS in humans as well as cats.     

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.     

Altered cell tropism and cytopathicity of feline immunodeficiency viruses in two different feline CD4-positive, CD8-negative cell lines.

Two interleukin-2-dependent feline CD4-positive and CD8-negative cell lines, MYA-1 and the newly established FeL-039, were used as host cells for infection with feline immunodeficiency virus (FIV). All FIV strains used, the Petaluma strain and several new isolates, were highly cytopathic to MYA-1. In contrast, the kinetics of FIV replication in FeL-039 differed greatly depending on the strain tested, i.e., noninfectious strain, highly cytopathic strain, and less cytopathic strain producing a persistent state for a long period. It appears, therefore, that cell tropism for FIV differed with each FIV strain tested even in T-cell lines showing similar cell surface phenotypes. Cytopathicity of FIV is evidently due to both the FIV strain and the host T cell.     

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.     

Differential cell tropism of feline immunodeficiency virus molecular clones in vivo

Independent studies have demonstrated different cell tropisms for molecular clones of feline immunodeficiency virus (FIV). In this report, we examined three clones, FIV-pF34, FIV-14, and FIV-pPPR, for replication in Crandell feline kidney (CrFK) cells, feline peripheral blood mononuclear cells (PBMC), and feline macrophage cultures. Importantly, cell tropism for these three clones was also examined in vivo. FIV-pF34 replication was efficient in CrFK cells but severely restricted in PBMC, whereas replication of FIV-pPPR was vigorous in PBMC but severely restricted in CrFK cells. FIV-14 replication was productive in both CrFK cells and PBMC. Interestingly, all three molecular clones replicated with similar efficiencies in primary feline monocyte-derived macrophages. In vivo, FIV-pF34 proved least efficient for establishing persistent infection, and proviral DNA when detectable, was localized predominately to nonlymphoid cell populations (macrophages). FIV-pPPR proved most efficient for induction of a persistent viremia in vivo, and proviral DNA was localized predominately in CD4(+) and CD8(+) lymphocyte subsets. FIV-14 inoculation of cats resulted in an infection characterized by seroconversion and localization of proviral DNA in CD4(+) lymphocytes only. Results of this study on diverse FIV molecular clones revealed that in vitro replication efficiency of an FIV isolate in PBMC directly correlated with replication efficiency in vivo, whereas proficiency for replication in macrophages in vitro was not predictive for replication potential in vivo. Also, infection of both CD4(+) and CD8(+) lymphocyte subsets was associated with higher virus load in vivo. Results of the studies on these three FIV clones, which exhibited differential cell tropism, indicated a correlation between in vitro and in vivo cell tropism and virus replication.     

Feline immunodeficiency virus infection phenotypically and functionally activates immunosuppressive CD4+CD25+ T regulatory cells

Disease progression of feline immunodeficiency virus (FIV) infection is characterized by up-regulation of B7.1 and B7.2 costimulatory molecules and their ligand CTLA4 on CD4(+) and CD8(+) T cells. The CD4(+)CTLA4(+)B7(+) phenotype described in FIV(+) cats is reminiscent of CD4(+)CD25(+)CTLA4(+) cells, a phenotype described for immunosuppressive T regulatory (Treg) cells. In the present study, we describe the phenotypic and functional characteristics of CD4(+)CD25(+) T cells in PBMC and lymph nodes (LN) of FIV(+) and control cats. Similar to Treg cells, feline CD4(+)CD25(+) but not CD4(+)CD25(-) T cells directly isolated from LN of FIV(+) cats do not produce IL-2 and fail to proliferate in response to mitogen stimulation. Unstimulated CD4(+)CD25(+) T cells from FIV(+) cats significantly suppress the proliferative response and the IL-2 production of Con A-stimulated autologous CD4(+)CD25(-) T cells compared with unstimulated CD4(+)CD25(+) T cells from FIV(-) cats. Flow-cytometric analysis confirmed the apparent activation phenotype of the CD4(+)CD25(+) cells in LN of chronically FIV(+) cats, because these cells showed significant up-regulation of expression of costimulatory molecules B7.1, B7.2, and CTLA4. These FIV-activated, anergic, immunosuppressive CD25(+)CTLA4(+)B7(+)CD4(+) Treg-like cells may contribute to the progressive loss of T cell immune function that is characteristic of FIV infection.