A single amino acid substitution in the transmembrane envelope glycoprotein of feline immunodeficiency virus alters cellular tropism.

The cellular tropism of the feline immunodeficiency virus (FIV) is affected by changes in variable region 3 (V3) of the surface (SU) envelope glycoprotein (Verschoor, E. J., et al., J. Virol. 69:4752-4757, 1995). By using high-dose DNA transfection, an FIV molecular clone with a non-CRFK-tropic V3 acquired the ability to replicate in CRFK cells. A single point mutation from a methionine to a threonine in the ectodomain of its transmembrane (TM) envelope glycoprotein was responsible for this change in viral tropism. This substitution is located in the putative SU interactive region, between the fusion peptide and the membrane-spanning region. Our results show that this region of the TM envelope glycoprotein constitutes an additional determinant for cell tropism.     

Bicyclams, Selective Antagonists of the Human Chemokine Receptor CXCR4, Potently Inhibit Feline Immunodeficiency Virus Replication

Bicyclams are low-molecular-weight anti-human immunodeficiency virus (HIV) agents that have been shown to act as potent and selective CXC chemokine receptor 4 (CXCR4) antagonists. Here, we demonstrate that bicyclams are potent inhibitors of feline immunodeficiency virus (FIV) replication when evaluated in Crandell feline kidney (CRFK) cells. With a series of bicyclam derivatives, 50% inhibitory concentrations (IC50s) against FIV were obtained in this cell system that were comparable to those obtained for HIV-1 IIIB replication in the human CD4(+) MT-4 T-cell line. The bicyclams were also able to block FIV replication in feline thymocytes, albeit at higher concentrations than in the CRFK cells. The prototype bicyclam AMD3100, 1-1'-[1,4-phenylene-bis(methylene)]-bis(1,4,8, 11-tetraazacyclotetradecane), was only fourfold less active in feline thymocytes (IC50, 62 ng/ml) than in CRFK cells (IC50, 14 ng/ml). AMD2763, 1,1'-propylene-bis(1,4,8, 11-tetraazacyclotetradecane), which is a less potent CXCR4 antagonist, was virtually inactive against FIV in feline thymocytes (IC50, >66.5 microgram/ml), while it was clearly active in CRFK cells (IC50, 0.9 microgram/ml). The CXC chemokine stromal-cell-derived factor 1alpha had anti-FIV activity in CRFK cells (IC50, 200 ng/ml) but not in feline thymocytes (IC50, >2.5 microgram/ml). When primary FIV isolates were evaluated for their drug susceptibility in feline thymocytes, the bicyclams AMD3100 and its Zn2+ complex, AMD3479, inhibited all six primary isolates at equal potency. The marked susceptibility of FIV to the bicyclams suggests that FIV predominantly uses feline CXCR4 for entering its target cells.     

Isolation and characterization of human immunodeficiency virus type 1 variants infectious to brain-derived cells: detection of common point mutations in the V3 region of the env gene of the variants.

T-cell-line-tropic human immunodeficiency virus type 1 cannot infect CD4-positive, brain-derived cells. We isolated several new variants that readily infected brain-derived cells. Mutation of proline to serine, to alanine, or to threonine in the well-conserved GPGR sequence in the V3 region of the envelope glycoprotein was found in all these variants. This indicates the importance of amino acid sequences at the tip of the V3 region for brain cell tropism of human immunodeficiency virus type 1.     

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.     

Adaptation to persistent growth in the H9 cell line renders a primary isolate of human immunodeficiency virus type 1 sensitive to neutralization by vaccine sera.

Seven diverse primary isolates of human immunodeficiency virus type 1 (HIV-1) were examined and found to be refractory to neutralization by antisera to recombinant gp120 (rgp120) protein from HIV-1 MN. This stands in marked contrast to the sensitivity exhibited by certain laboratory-adapted viruses. To understand the difference between primary and laboratory-adapted viruses, we adapted the primary virus ACH 168.10 to growth in the FDA/H9 cell line. ACH 168.10 was chosen because the V3 region of gp120 closely matches that of MN. After 4 weeks, infection became evident. The virus (168A) replicated in FDA/H9 cells with extensive cytopathic effect but was unchanged in sensitivity to antibody-mediated neutralization. Thus, growth in cell lines is not sufficient to render primary virus sensitive to neutralization. The 168A virus was, however, partially sensitive to CD4 immunoadhesin (CD4-Ig). Adaptation was continued to produce a persistently infected FDA/H9 culture that displayed minimal cytopathic effect. The virus (168C) was now sensitive to neutralization by MN rgp120 vaccine sera and by MN-specific monoclonal antibodies and showed increased sensitivity to HIVIG and CD4-Ig. 168C encoded three amino acid changes in gp120, including one within the V3 loop (I-166-->R, I-282-->N, G-318-->R). MN-specific monoclonal antibodies bound equally to the surface of cells infected with either neutralization-resistant or -sensitive virus. The coincidence of changes in neutralization sensitivity with changes in cell tropism and cytopathic effect suggests a common underlying mechanism(s) acting through the whole of the envelope protein complex.     

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.     

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.     

An epitope in the V1 domain of the simian immunodeficiency virus (SIV) gp120 protein is recognized by CD8+ cytotoxic T lymphocytes from an SIV-infected rhesus macaque.

Cytotoxic T-lymphocyte (CTL) responses against the external envelope glycoprotein (gp120) of the simian immunodeficiency virus (SIV) were studied in a rhesus macaque infected with SIVmac/239. CD8+ T cells enriched from concanavalin A-stimulated peripheral blood mononuclear cells lysed autologous target cells infected with recombinant vaccinia virus vectors expressing the SIVmac/239 or SIVsm/H4 envelope protein, which share approximately 80% identity in amino acid sequence. A CD8+ CTL line derived by limiting dilution culture of the concanavalin A-stimulated lymphocytes was also specific for the envelope proteins of both SIV isolates. Mapping studies revealed that this cell line recognized an epitope between amino acids 113 and 121 (CNKSETDRW) in the V1 domain of gp120. Amino acid substitutions are observed at positions 116 and 120 among viruses of the SIVsm/mac/human immunodeficiency virus type 2 group, and thus synthetic peptides representing these variants were tested for the ability to sensitize target cells for lysis by the CTL line. Autologous target cells sensitized with a synthetic peptide representing the SIVmac/239 sequence were efficiently killed. In contrast, recognition of target cells was reduced or abolished when peptides representing the amino acid substitutions at position 116 or 120 of other SIVmac, SIVsm, SIVmne, or SIVstm strains were tested. Further studies of CTL responses against this epitope could provide insights into mechanisms of variability within the gp120 V1 domain and its importance in evasion of immunity in infected or vaccinated monkeys.     

Frequent perinatal transmission of feline immunodeficiency virus by chronically infected cats.

Vertical transmission of feline immunodeficiency virus (FIV) was studied in cats infected with either of two FIV clinical isolates (FIV-B-2542 or FIV-AB-2771) prior to breeding and conception. Queens infected 4 to 30 months (mean = 14 months) prior to conception transmitted FIV to 59 of 83 (71%) kittens; 50.6% were virus positive on the day of birth. To examine potential routes of FIV transmission from mother to offspring, kittens were delivered via either vaginal or cesarean birth and nursed by either their virus-infected natural mothers or uninfected surrogate mothers. Comparison of FIV infection rates at birth with those at 6 months of age in kittens delivered by cesarean and surrogate raised demonstrated that late in utero transmission occurred in approximately 20% of kittens. Comparison of kittens nursed by FIV mothers with those by uninfected surrogate mothers demonstrated a 13.5% increase in infection rate of kittens exposed to milk-borne virus. Isolation of virus from 40% of maternal vaginal wash samples and the slightly greater infection rate in vaginally versus cesarean-delivered surrogate-nursed kittens suggested that intrapartum transmission may occur. In addition, we found that low maternal CD4 count (<200 cells per microl), longer duration of maternal infection (>15 months), and maternal symptoms of clinical immunodeficiency correlated with increased rates of mother-to-kitten FIV transmission, paralleling observations in human immunodeficiency virus-infected women. We conclude that FIV infection provides a model in which to explore aspects of human immunodeficiency virus vertical transmission and intervention difficult to address in human patients.     

Feline immunodeficiency virus decreases cell-cell communication and mitochondrial membrane potential.

The in vitro effects of viral replication on mitochondrial membrane potential (MMP) and gap junctional intercellular communication (GJIC) were evaluated as two parameters of potential cellular injury. Two distinct cell types were infected with the Petaluma strain of feline immunodeficiency virus (FIV). Primary astroglia supported acute FIV infection, resulting in syncytia within 3 days of infection, whereas immortalized Crandell feline kidney (CRFK) cells of epithelial origin supported persistent FIV infection in the absence of an obvious cytopathic effect. An examination of cells under conditions that included an infection rate of more than 90% for either population revealed that the astroglia produced about four times more virus than the CRFK cells. The mitochondrial uptake of the cationic fluorescent dye rhodamine 123 in infected astroglia was less than 45% of that of normal control cells, whereas the MMP of the CRFK cells, which produced about one-fourth as much virus, was 80.8% of that of the normal cells. Cell-cell communication between adjacent cells was determined by the recovery of fluorescence following photobleaching of a single cell. In spite of the lower level of innate cell-cell communication among cultured CRFK cells than among astroglia, viral replication resulted in a 30% decrease in the GJIC of both astroglia and CRFK cells. These studies indicate that cell injury, as defined by an inhibition of MMP and GJIC, can occur as a result of persistent and acute infection with the Petaluma strain of FIV.     

Phylogenetic analysis of feline immunodeficiency virus in feral and companion domestic cats of New Zealand

Nested PCR was used to amplify envelope V3-V6 gene fragments of feline immunodeficiency virus (FIV) from New Zealand cats. Phylogenetic analyses established that subtypes A and C predominate among New Zealand cats, with clear evidence of intersubtype recombination. In addition, 17 sequences were identified that were distinct from all known FIV clades, and we tentatively suggest these belong to a novel subtype.     

Differential regulation of cellular tropism and sensitivity to soluble CD4 neutralization by the envelope gp120 of human immunodeficiency virus type 1.

Using recombinant and mutant viruses generated between two human immunodeficiency virus type 1 isolates that display differences in cell tropism and sensitivity to soluble CD4 neutralization, we show that these two properties of the virus are regulated by different mechanisms. Whereas there is an association between V3 loop conformation and a particular cellular tropism, soluble CD4 neutralization sensitivity appears to be determined by amino acid differences in the C2 domain of the envelope gp120 that modulate the stability of gp120-gp41 association. Our findings further illustrate the importance of functional interactions among different regions of the envelope gp120 in regulating the biological phenotypes of human immunodeficiency virus and suggest that additional probing of the V3 loop with monoclonal antibodies may identify specific structural features of this loop that determine cell tropism.     

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.     

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.     

Persistent gene expression in mouse nasal epithelia following feline immunodeficiency virus-based vector gene transfer

Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.     

Identification of amino acid residues important for heparan sulfate proteoglycan interaction within variable region 3 of the feline immunodeficiency virus surface glycoprotein

Heparan sulfate proteoglycans (HSPGs) act as binding receptors or attachment factors for the viral envelope of many viruses, including strains of HIV and feline immunodeficiency virus (FIV). The FIV gp95 glycoprotein (SU) from laboratory-adapted strains (tissue culture adapted [TCA]) such as FIV-34TF10 can bind to HSPG, whereas SU from field strains (FS) such as FIV-PPR cannot. Previous studies indicate that SU-HSPG interactions occur within the V3 loop. We utilized a series of nested V3 peptides to further map the HSPG binding sites and found that both sides of the predicted V3 loop stem were critical for the binding but not the CXCR4 binding domain near the predicted tip of the V3 loop. Neutralization assays for TCA strain entry using the same set of V3 peptides showed that peptides targeting CXCR4 or HSPG binding sites can block infection, supporting the V3 loop as a critical neutralization target. Site-directed mutagenesis identified two highly conserved arginines, R379 and R389, on the N-terminal side of the V3 stem as critical for the contact between SU and HSPG. Residues K407, K409, K410, and K412 on the C-terminal side of the V3 stem form a second nonconserved domain necessary for HSPG binding, consistent with the observed specificity distinctions with FS FIV. Our findings discriminate structural determinants important for HSPG and CXCR4 binding by FIV SU and thus further define the importance of the V3 loop for virus entry and infection.     

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.     

Functional role of the V1/V2 region of human immunodeficiency virus type 1 envelope glycoprotein gp120 in infection of primary macrophages and soluble CD4 neutralization.

We have examined the influence of the V1/V2 region of the human immunodeficiency virus type 1 (HIV-1) gp120 on certain biologic properties of the virus. We observed that on the genomic background of the T-cell-line-tropic strain, HIV-1SF2mc, both the V1 and V2 domains of the macrophage-tropic strain, HIV-1SF162mc, in addition to the required V3 domain, are necessary to attain full macrophage tropism. Furthermore, the V2 domain modulates the sensitivity of HIV-1 to soluble CD4 neutralization. Structural studies of recombinant and mutant envelope glycoproteins suggest that the function of the V1/V2 region is to interact with the V3 domain and confer on the envelope gp120 of HIV-1SF2mc a conformation more similar to that of the macrophage-tropic strain HIV-1SF162mc. The conformation of the envelope gp120 appears to be strain specific and plays an important role in determining HIV-1 tissue tropism and sensitivity to soluble CD4 neutralization.     

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.