Role of plasma membrane lipid microdomains in respiratory syncytial virus filament formation

The fusion protein (F) of respiratory syncytial virus (RSV) is the envelope glycoprotein responsible for the characteristic cytopathology of syncytium formation. RSV has been shown to bud from selective areas of the plasma membrane as pleomorphic virions, including both filamentous and round particles. With immunofluorescent microscopy, we demonstrated evidence of RSV filaments incorporating the fusion protein F and colocalizing with a lipid microdomain-specific fluorescent dye, 1,1-dihexadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate. Western blot analysis of Triton X-100 cold-extracted membrane fractions confirmed the presence of RSV proteins within the lipid microdomains. RSV proteins also colocalized with cellular proteins associated with lipid microdomains, caveolin-1, and CD44, as well as with RhoA, a small GTPase. ADP-ribosylation of RhoA by Clostridium botulinum exotoxin inactivated RhoA signaling and resulted in the absence of RSV-induced syncytia despite no significant change in viral titer. We demonstrated an overall decrease in both the number and length of the viral filaments and a shift in the localization of F to nonlipid microdomain regions of the membrane in the presence of C3 toxin. This suggests that the selective incorporation of RSV proteins into lipid microdomains during virus assembly may lead to critical interactions of F with cellular proteins, resulting in microvillus projections necessary for the formation of filamentous virus particles and syncytium formation. Thus, manipulation of membrane lipid microdomains may lead to alterations in the production of viral filaments and RSV pathogenesis and provide a new pharmacologic target for RSV therapy.     

RhoA signaling is required for respiratory syncytial virus-induced syncytium formation and filamentous virion morphology

Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threatening respiratory infections in infants, the elderly, and immunocompromised adults. RSV infection of HEp-2 cells induces the activation of RhoA, a small GTPase. We therefore asked whether RhoA signaling is important for RSV replication or syncytium formation. The treatment of HEp-2 cells with Clostridium botulinum C3, an enzyme that ADP-ribosylates and specifically inactivates RhoA, inhibited RSV-induced syncytium formation and cell-to-cell fusion, although similar levels of PFU were released into the medium and viral protein expression levels were equivalent. Treatment with another inhibitor of RhoA signaling, the Rho kinase inhibitor Y-27632, yielded similar results. Scanning electron microscopy of C3-treated infected cells showed reduced numbers of single blunted filaments, in contrast to the large clumps of long filaments in untreated infected cells. These data suggest that RhoA signaling is associated with filamentous virus morphology, cell-to-cell fusion, and syncytium formation but is dispensable for the efficient infection and production of infectious virus in vitro. Next, we developed a semiquantitative method to measure spherical and filamentous virus particles by using sucrose gradient velocity sedimentation. Fluorescence and transmission electron microscopy confirmed the separation of spherical and filamentous forms of infectious virus into two identifiable peaks. The C3 treatment of RSV-infected cells resulted in a shift to relatively more spherical virions than those from untreated cells. These data suggest that viral filamentous protuberances characteristic of RSV infection are associated with RhoA signaling, are important for filamentous virion morphology, and may play a role in initiating cell-to-cell fusion.     

RhoA Interacts with the Fusion Glycoprotein of Respiratory Syncytial Virus and Facilitates Virus-Induced Syncytium Formation

The fusion glycoprotein (F) of respiratory syncytial virus (RSV), which mediates membrane fusion and virus entry, was shown to bind RhoA, a small GTPase, in yeast two-hybrid interaction studies. The interaction was confirmed in vivo by mammalian two-hybrid assay and in RSV-infected HEp-2 cells by coimmunoprecipitation. Furthermore, the interaction of F with RhoA was confirmed in vitro by enzyme-linked immunosorbent assay and biomolecular interaction analysis. Yeast two-hybrid interaction studies with various deletion mutants of F and with RhoA indicate that the key binding domains of these proteins are contained within, or overlap, amino acids 146 to 155 and 67 to 110, respectively. The biological significance of this interaction was studied in RSV-infected HEp-2 cells that were stably transfected to overexpress RhoA. There was a positive correlation between RhoA expression and RSV syncytium formation, indicating that RhoA can facilitate RSV-induced syncytium formation.     

Mechanism of selective inhibition of respiratory syncytial virus by a benzodithiin compound (RD3-0028)

RD3-0028, a compound with a benzodithiin structure, was found to be a potent inhibitor of respiratory syncytial virus (RSV) replication. Its action is specific; no activity is seen against influenza A virus, measles virus, herpes simplex virus type 1 or 2, or human cytomegalovirus. A time-dependent drug addition experiment indicated that the antiviral activity occurs in the late stage of the RSV replication cycle, since this compound completely inhibited syncytium formation even when added up to 16 hr after the infection of cell monolayers at an MOI of 3. RD3-0028 had no direct virucidal effect on RSV. Western blotting analysis showed that RD3-0028 significantly decreased the amount of RSV proteins released into the cell culture medium. Moreover, five independent isolates of the RSV long strain were selected for growth in RD3-0028 (5-20 microg/ml). These resistant viruses were more than 80-fold less sensitive to RD3-0028 than the long strain. The F gene segment of each of these viruses was sequenced and in each case the mutant RNA segment contained at least one sequence alteration, converting asparagine 276 to tyrosine (F1 protein). These results suggest that RD3-0028 inhibits RSV replication by interfering with intracellular processing of the RSV fusion protein, or a step immediately thereafter, leading to loss of infectivity.     

Recombinant Sendai viruses expressing fusion proteins with two furin cleavage sites mimic the syncytial and receptor-independent infection properties of respiratory syncytial virus

Cell entry by paramyxoviruses requires fusion between viral and cellular membranes. Paramyxovirus infection also gives rise to the formation of multinuclear, fused cells (syncytia). Both types of fusion are mediated by the viral fusion (F) protein, which requires proteolytic processing at a basic cleavage site in order to be active for fusion. In common with most paramyxoviruses, fusion mediated by Sendai virus F protein (F(SeV)) requires coexpression of the homologous attachment (hemagglutinin-neuraminidase [HN]) protein, which binds to cell surface sialic acid receptors. In contrast, respiratory syncytial virus fusion protein (F(RSV)) is capable of fusing membranes in the absence of the viral attachment (G) protein. Moreover, F(RSV) is unique among paramyxovirus fusion proteins since F(RSV) possesses two multibasic cleavage sites, which are separated by an intervening region of 27 amino acids. We have previously shown that insertion of both F(RSV) cleavage sites in F(SeV) decreases dependency on the HN attachment protein for syncytium formation in transfected cells. We now describe recombinant Sendai viruses (rSeV) that express mutant F proteins containing one or both F(RSV) cleavage sites. All cleavage-site mutant viruses displayed reduced thermostability, with double-cleavage-site mutants exhibiting a hyperfusogenic phenotype in infected cells. Furthermore, insertion of both F(RSV) cleavage sites in F(SeV) reduced dependency on the interaction of HN with sialic acid for infection, thus mimicking the unique ability of RSV to fuse and infect cells in the absence of a separate attachment protein.     

Respiratory syncytial virus G and/or SH glycoproteins modify CC and CXC chemokine mRNA expression in the BALB/c mouse

Chemokine mRNA expression by pulmonary leukocytes following infection of BALB/c mice with two strains of respiratory syncytial virus (RSV) and one strain of parainfluenza virus type 3 (PIV-3) was determined. The results suggest that RSV G and/or SH proteins inhibit early MIP-1alpha, MIP-1beta, MIP-2, MCP-1, and IP-10 mRNA expression. TCA-3 mRNA expression was found to be increased during PIV-3 infection.     

Comparison of RNA synthesis initiation properties of non-segmented negative strand RNA virus polymerases

It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming.     

Characteristics of fusion of respiratory syncytial virus with HEp-2 cells as measured by R18 fluorescence dequenching assay.

The characteristics of fusion of respiratory syncytial virus (RSV) with HEp-2 cells were studied by the R18 fluorescence dequenching assay of membrane fusion. A gradual increase in fluorescence intensity indicative of virion-cell fusion was observed when R18-labeled RSV was incubated with HEp-2 cells. Approximately 35% dequenching of the probe fluorescence was observed in 1 h at 37 degrees C. Fusion showed a temperature dependence, with significant dequenching occurring above 18 degrees C. The dequenching was also dependent on the relative concentration of target membrane. Thus, increasing the concentration of target membrane resulted in increased levels of dequenching. In addition, viral glycoproteins were shown to be involved in this interaction, since dequenching was significantly reduced by pretreatment of labeled virus at 70 degrees C for 5 min or by trypsinization of R18-labeled virions prior to incubation with HEp-2 cells at 37 degrees C. The fusion of RSV with HEp-2 cells was unaffected over a pH range of 5.5 to 8.5, with some increase seen at lower pH values. Treatment of HEp-2 cells with ammonium chloride (20 and 10 mM), a lysosomotropic agent, during early stages of infection did not inhibit syncytium formation or progeny virion production by RSV. At the same concentrations of ammonium chloride, the production of vesicular stomatitis virus was reduced approximately 4 log10 units. These results suggest that fusion of the virus with the cell surface plasma membrane is the principal route of entry.     

Structural characterization of mumps virus fusion protein core

The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus, forms typical 3-4-4-4-3 spacing. The similar characterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins.     

Role of the fusogenic peptide sequence in syncytium induction and infectivity of human immunodeficiency virus type 2.

Syncytium induction is a characteristic feature of infection by human immunodeficiency virus (HIV) in vitro. The hydrophobic amino terminus of the transmembrane glycoprotein of HIV type 1 is an essential determinant of virus entry into the target cell population and the formation of syncytia in cell culture. To define the role of the HIV type 2 fusion peptide during infection and syncytium formation, we introduced 8 amino acid substitutions into the hydrophobic amino terminus of gp41, changing either the hydrophobicity, the charge, or the polarity of the amino acid. Viruses containing the envelope mutations were analyzed for their syncytium-inducing capacities, levels of infectivity, and envelope processing and expression. Mutations that increased the hydrophobic nature of the fusion peptide increased syncytium formation, whereas mutations which increased the charge and the polarity and/or decreased the hydrophobicity of the fusion domain severely reduced the capacity of the virus to induce syncytia. However, viruses severely compromised for syncytium formation exhibit only slightly lower levels of infectivity.     

Syncytium formation by recombinant vaccinia viruses carrying bovine parainfluenza 3 virus envelope protein genes.

The highly syncytium-inducing M strain and the weakly syncytium-inducing SC strain of bovine parainfluenza 3 virus differ by a single amino acid substitution in each of the hemagglutinin-neuraminidase (HN) and membrane (M) proteins, while their fusion (F) proteins are identical (T. Shioda, S. Wakao, S. Suzu, and H. Shibuta, Virology 162:388-396, 1988). We constructed recombinant vaccinia viruses which express separately the M virus HN (Vac-MHN), SC virus HN (Vac-SCHN), M virus M (Vac-MM), SC virus M (Vac-SCM), and common F (Vac-F) proteins. CV-1 cells were infected with the recombinants, singly or in combination, and implanted onto indicator MDBK cells for syncytium formation. Combinations of Vac-MHN plus Vac-F and Vac-SCHN plus Vac-F induced extensive and weak syncytium formation, respectively. Vac-F alone did not induce syncytium formation, and both Vac-MM and Vac-SCM had no effect on syncytium formation. These findings indicated that the syncytium formation by bovine parainfluenza 3 virus requires both the F and HN proteins and that the extensive syncytium formation by the M virus is due to the M virus HN protein. MSC, another weakly syncytium-inducing virus variant, newly isolated from the M virus, was identical to the M virus in the primary structure of the HN and M proteins but differed from the M virus by a single amino acid residue in the F protein. The combination of the recombinant vaccinia virus expressing the MSC virus F protein and Vac-MHN resulted in weak syncytium formation.     

Identification and characterization of fusion and processing domains of the human immunodeficiency virus type 2 envelope glycoprotein.

The envelope glycoprotein of the human immunodeficiency virus type 2 (HIV-2) is synthesized as a polyprotein precursor which is proteolytically processed to produce the mature surface and transmembrane envelope glycoproteins. The processed envelope glycoprotein species are responsible for the fusion between the viral envelope and the host cell membrane during the infection process. The envelope glycoprotein also induces syncytium formation between envelope-expressing cells and receptor-bearing cells. To characterize domains of the HIV-2 envelope glycoprotein involved in membrane fusion and in proteolytic processing, we introduced single amino acid mutations into the region of the HIV-2 surface glycoprotein corresponding to the principal neutralizing determinant (the V3 loop) of HIV-1, the putative HIV-2 envelope precursor-processing sequence, and the hydrophobic amino terminus of the HIV-2 transmembrane envelope glycoprotein. The effects of these mutations on syncytium formation, virus infectivity, envelope expression, envelope processing, and CD4 binding were analyzed. Our results suggest that the V3-like region of the HIV-2 surface glycoprotein and the hydrophobic amino terminus of the transmembrane glycoprotein are HIV-2 fusion domains and characterize the effects of mutations in the HIV-2 envelope glycoprotein precursor-processing sequence.     

Characterization of a novel parainfluenza virus,caviid parainfluenza virus 3, from laboratory guinea pigs (Cavia porcellus)

A novel Respirovirus was isolated from nasopharyngeal swab specimens from clinically normal laboratory guinea pigs, and was characterized and named caviid parainfluenza virus 3 (CavPIV-3). The CavPIV-3 is enveloped, is 100 to 300 nm in diameter, and has a characteristic 15-nm-diameter chevron-shaped virus ribonucleocapsid protein. Sequence analysis of the fusion glycoprotein of CavPIV-3 revealed it to be 94% identical to human and guinea pig parainfluenza 3 (PIV-3) viruses and 80% identical to bovine PIV-3. To determine whether CavPIV-3 causes clinical disease in laboratory guinea pigs and to compare the serologic response of guinea pigs to CavPIV-3 and to other paramyxoviruses, an infection study was performed, in which groups of guinea pigs were inoculated with CavPIV-3, Sendai virus, simian virus 5 (SV-5), murine pneumonia virus (PVM), or bovine PIV-3 virus. During the course of the study, guinea pigs were maintained in an infectious disease suite, housed in Micro-Isolator cages, and were only manipulated under a laminar flow hood. Clinical signs of disease were not observed in any of the paramyxovirus-inoculated guinea pigs during the eight-week course of the study, and histologic signs of disease were not evident at necropsy eight weeks after inoculation. Guinea pigs inoculated with CavPIV-3, Sendai virus, PVM, and bovine PIV-3 developed robust homologous or heterologous serologic responses. In contrast, guinea pigs inoculated with SV-5 developed modest or equivocal serologic responses, as assessed by use of an enzyme-linked immunosorbent assay. Further, use of the SV-5 enzyme-linked immunosorbent assay resulted in the highest degree of non-specific reactivity among all of the paramyxovirus assays. In summary, CavPIV-3 is a novel guinea pig Respirovirus that subclinically infects laboratory guinea pigs, resulting in a robust serologic response, but no observed clinical or histologic disease. The CavPIV-3 fusion glycoprotein gene sequence is available from GenBank as accession No. AF394241, and the CavPIV-3 virus is available from the American Type Culture Collection as accession No. DR-1547.     

Mapping of independent V3 envelope determinants of human immunodeficiency virus type 1 macrophage tropism and syncytium formation in lymphocytes.

The V3 region of the human immunodeficiency virus type 1 (HIV-1) envelope protein is known to have a major influence on macrophage tropism as well as the ability to cause syncytium formation or fusion in CD4-positive lymphocyte cultures. Using infectious molecular HIV-1 clones, a series of mutant clones was created which allowed detailed mapping of V3 amino acid positions involved in these properties. In these experiments the non-syncytium-inducing phenotype in T cells did not always correlate with macrophage tropism. Macrophage tropism appeared to depend on the presence of certain combinations of amino acids at five specific positions within and just outside of the V3 loop itself, whereas syncytium formation in lymphocytes was influenced by substitution of particular residues at two to four positions within V3. In most cases, different V3 amino acid positions were found to independently influence macrophage tropism and syncytium formation in T cells and position 13 was the only V3 location which appeared to simultaneously influence both macrophage tropism and syncytium formation in lymphocytes.     

CD4 immunoadhesin, but not recombinant soluble CD4, blocks syncytium formation by human immunodeficiency virus type 2-infected lymphoid cells.

Recombinant soluble CD4 (rCD4) has been shown to be an effective inhibitor of human immunodeficiency virus type 1 (HIV-1) and HIV-2 infection of lymphoid cells in vitro. In this report, we characterized the effects of rCD4, the V1V2 fragment of CD4, and the immunoadhesin CD4-immunoglobulin G on syncytium formation between lymphoid cells infected by HIV-1 or HIV-2 and uninfected cells. All three molecules blocked HIV-1-mediated syncytium formation, but only CD4-immunoglobulin G blocked HIV-2-mediated syncytium formation. rCD4 and the V1V2 fragment of CD4 enhanced HIV-2-mediated syncytium formation. These results suggest that the process of cell fusion is significantly different between HIV-1- and HIV-2-infected cells.     

A single amino acid mutation at position 170 of human parainfluenza virus type 1 fusion glycoprotein induces obvious syncytium formation and caspase-3-dependent cell death

An escape mutant of human parainfluenza virus type 1 (hPIV1), which was selected by serial passage in the presence of a sialidase inhibitor, 4-O-thiocarbamoylmethyl-2-deoxy-2,3-didehydro-N-acetylneur-aminic acid (TCM-Neu5Ac2en), exhibited remarkable syncytium formation and virus-induced cell death in LLC-MK2 cells but no difference in susceptibility for the sialidase inhibitor TCM-Neu5Ac2en from that of wild-type hPIV1 strain C35 (WT). The mutant virus also had higher replication and plaque formation abilities. The mutant virus acquired two amino acid mutations, Glu to Gly at position 170 and Ala to Glu 442 in fusion (F) glycoprotein, but no mutations in haemaggulutinin-neuraminidase (HN) glycoprotein. Using cells co-expressing F and HN genes with site-specific mutagenesis, we demonstrated that a point mutation of Glu to Gly at position 170, which was estimated to be located in hPIV1 F glycoprotein heptad repeat 1, was required for obvious syncytium formation and caspase-3-dependent cell death. In contrast, wild-type F glycoprotein induced no synctium formation or cell death. The findings suggest that a single amino acid mutation of hPIV1 F glycoprotein promotes syncytium formation that is followed by caspase-3-dependent cell death.     

The requirements for viral entry differ from those for virally induced syncytium formation in NIH 3T3/DTras cells exposed to Moloney murine leukemia virus.

Moloney murine leukemia virus (Mo-MuLV) has the unique ability to infect different cells via either a low-pH-dependent or a pH-independent entry pathway. Only the pH-independent mechanism of Mo-MuLV entry has been associated with Mo-MuLV-induced syncytium formation. We have now identified a transformed cell line (NIH 3T3/DTras) which efficiently forms syncytia when exposed to Mo-MuLV, yet is low pH dependent for Mo-MuLV entry. Treatment of NIH 3T3/DTras cells with chloroquine, an agent which raises endosomal pH, blocks Mo-MuLV entry, but not Mo-MuLV-induced syncytium formation. This demonstrates that fusion which accompanies viral entry and fusion which is responsible for syncytium formation occur as independent processes in these cells. In addition, we determined that neither inherent differences in the Mo-MuLV receptor nor reduced affinity for Mo-MuLV gp70 can account for resistance of NIH 3T3 cells to Mo-MuLV-induced syncytium formation.     

A CD4 domain important for HIV-mediated syncytium formation lies outside the virus binding site

HIV infection of chimpanzees results in a chronic viremia unaccompanied by the ultimately fatal immunodeficiency that marks HIV infection in man. We show here that expression of HIV envelope proteins allows syncytium formation between cells expressing human but not chimpanzee or macaque CD4. We find that the CD4 sequences regulating cell fusion lie outside the recognized virus binding site; in the simplest exchange, chimpanzee CD4 bearing human residue 87 supports syncytium formation, while human CD4 bearing chimpanzee residue 87 does not. Neither the equilibrium nor the forward rate constants for HIV-CD4 association are affected by substitution at position 87. Infection of human cells expressing chimpanzee CD4 is insensitive to lysosomotropic agents, suggesting that viral penetration under these circumstances does not require endocytosis. The benign course of HIV infection in chimpanzees may reflect the failure of the host to support direct cell to cell transmission of the virus.     

Inhibitory effect of interferons and tumor necrosis factor on syncytium formation by bovine leukemia virus

The treatments of the bovine spleen cells BESp with recombinant human leukocyte interferon (HuIFN-alpha A) and human fibroblast interferon (HuIFN-beta) inhibit the syncytium formation by bovine leukemia virus (BLV) and the virus yield, depending on the IFN doses. But HuIFN-gamma did not inhibit the syncytium formation. The tumor-necrosis factor (TNF) also inhibits the syncytium formation and the virus yield, depending on the TNF doses. Furthermore, the combination of the HuIFN-alpha A with the TNF inhibits the syncytium formation more strongly than the HuIFN-alpha A alone or the TNF alone.     

DC-SIGN facilitates fusion of dendritic cells with human T-cell leukemia virus type 1-infected cells

Interactions between the oncogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) and dendritic cells (DCs) are poorly characterized. We show here that monocyte-derived DCs form syncytia and are infected upon coculture with HTLV-1-infected lymphocytes. We examined the role of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a C-type lectin expressed in DCs, in HTLV-1-induced syncytium formation. DC-SIGN is known to bind with high affinity to various viral envelope glycoproteins, including human immunodeficiency virus (HIV) and hepatitis C virus, as well as to the cellular receptors ICAM-2 and ICAM-3. After cocultivating DCs and HTLV-1-infected cells, we found that anti-DC-SIGN monoclonal antibodies (MAbs) were able to decrease the number and size of HTLV-1-induced syncytia. Moreover, expression of the lectin in epithelial-cell lines dramatically enhanced the ability to fuse with HTLV-1-positive cells. Interestingly, in contrast to the envelope (Env) glycoproteins of HIV and other viruses, that of HTLV-1 does not bind directly to DC-SIGN. The facilitating role of the lectin in HTLV-1 syncytium formation is mediated by its interaction with ICAM-2 and ICAM-3, as demonstrated by use of MAbs directed against these adhesion molecules. Altogether, our results indicate that DC-SIGN facilitates HTLV-1 infection and fusion of DCs through an ICAM-dependent mechanism.