G541R within the 4070A TM protein regulates fusion in murine leukemia viruses

The mutation G541R within the ectodomain of TM was isolated in three independent chimeric enveloped murine leukemia virus (MuLV) viral populations originally impaired in viral passage and in wild-type 4070A. Isolation of G541R in multiple populations suggested it played a critical role in viral envelope function. Using a viral vector system, the observed effects of the G541R mutation within MuLV envelope proteins were pleiotropic and included effects on the regulation of SU-TM interactions and membrane fusion. G541R suppresses enhanced cell-cell fusion events attributable to the absence of the R-peptide yet does not adversely affect virus titers. The ability to suppress cell-cell fusion is dependent on the presence of the C terminus of the amphotropic 4070A SU protein. Within the wild-type 4070A envelope background, the mutation results in a decreased level of Env at the cell surface that is mirrored in the virion. The TM mutation alters recognition of the SU C terminus by a monoclonal antibody, suggestive of an altered conformation. The presence of G541R allowed the virus to achieve a balance between cytopathogenicity and replication and restored productive viral entry.     

G100R mutation within 4070A murine leukemia virus Env increases virus receptor binding,kinetics of entry,and viral transduction efficiency

Passage of 4070A murine leukemia virus (MuLV) in D17 cells resulted in a G-to-R change at position 100 within the VRA of the envelope protein (Env). Compared with 4070A MuLV, virus with the G100R Env displayed enhanced binding on target cells, internalized the virus more rapidly, and increased the overall viral titer in multiple cell types. This provides a direct correlation between binding strength and efficiency of viral entry. Deletion of a His residue at the SU N terminus eliminated the transduction efficiency by the G100R virus, suggesting that the G100R virus maintains the regulatory characteristics of 4070A viral entry. The improved transduction efficiency of G100R Env would be an asset for gene delivery systems.     

Second-site changes affect viability of amphotropic/ecotropic chimeric enveloped murine leukemia viruses

Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells.     

A point mutation in the binding subunit of a retroviral envelope protein arrests virus entry at hemifusion

The transmembrane subunits of viral envelope proteins are thought to perform all of the functions required for membrane fusion during entry of enveloped viruses. However, changes in a conserved SPHQ motif near the N terminus of the receptor binding subunit of a murine leukemia virus (MLV) envelope protein block infection and induction of cell-cell fusion but not receptor binding. Here we report evidence that a histidine-to-arginine change at position 8 (H8R) in the SPHQ motif of Moloney MLV blocks infection by arresting virus-cell fusion at the hemifusion state. In cell-cell fusion assays, H8R envelope protein induced mixing of membrane outer leaflet lipids but did not lead to content mixing, a finding indicative of fusion pore formation. Kinetic studies of virus-cell fusion showed that lipid mixing of H8R virus membranes begins much later than for wild-type virus. The length of the delay in lipid mixing decreased upon addition of two second-site changes that increase H8R virus infection to 100-fold less than the wild-type virus. Finally, chlorpromazine, dibucaine, and trifluoperazine, agents that induce pores in an arrested hemifusion state, rescued infection by H8R virus to within 2.5-fold of the level of wild-type virus infection and cell-cell fusion to half that mediated by wild-type envelope protein. We interpret these results to indicate that fusion progressed to the hemifusion intermediate but fusion pore formation was inhibited. These results establish that membrane fusion of Moloney MLV occurs via a hemifusion intermediate. We also interpret these findings as evidence that histidine 8 is a key switch-point residue between the receptor-induced conformation changes that expose fusion peptide and those that lead to six-helix bundle formation.     

Distinct mechanisms of neutralization by monoclonal antibodies specific for sites in the N-terminal or C-terminal domain of murine leukemia virus SU

The epitope specificities and functional activities of monoclonal antibodies (MAbs) specific for the murine leukemia virus (MuLV) SU envelope protein subunit were determined. Neutralizing antibodies were directed towards two distinct sites in MuLV SU: one overlapping the major receptor-binding pocket in the N-terminal domain and the other involving a region that includes the most C-terminal disulfide-bonded loop. Two other groups of MAbs, reactive with distinct sites in the N-terminal domain or in the proline-rich region (PRR), did not neutralize MuLV infectivity. Only the neutralizing MAbs specific for the receptor-binding pocket were able to block binding of purified SU and MuLV virions to cells expressing the ecotropic MuLV receptor, mCAT-1. Whereas the neutralizing MAbs specific for the C-terminal domain did not interfere with the SU-mCAT-1 interaction, they efficiently inhibited cell-to-cell fusion mediated by MuLV Env, indicating that they interfered with a postattachment event necessary for fusion. The C-terminal domain MAbs displayed the highest neutralization titers and binding activities. However, the nonneutralizing PRR-specific MAbs bound to intact virions with affinities similar to those of the neutralizing receptor-binding pocket-specific MAbs, indicating that epitope exposure, while necessary, is not sufficient for viral neutralization by MAbs. These results identify two separate neutralization domains in MuLV SU and suggest a role for the C-terminal domain in a postattachment step necessary for viral fusion.     

Functional characterization of the N termini of murine leukemia virus envelope proteins

The function of the N terminus of the murine leukemia virus (MuLV) surface (SU) protein was examined. A series of five chimeric envelope proteins (Env) were generated in which the N terminus of amphotropic 4070A was replaced by equivalent sequences from ecotropic Moloney MuLV (M-MuLV). Viral titers of these chimeras indicate that exchange with homologous sequences could be tolerated, up to V17eco/T15ampho (crossover III). Constructs encoding the first 28 amino acids (aa) of ecotropic M-MuLV resulted in Env expression and binding to the receptor; however, the virus titer was reduced 5- to 45-fold, indicating a postbinding block. Additional exchange beyond the first 28 aa of ecotropic MuLV Env resulted in defective protein expression. These N-terminal chimeras were also introduced into the AE4 chimeric Env backbone containing the amphotropic receptor binding domain joined at the hinge region to the ecotropic SU C terminus. In this backbone, introduction of the first 17 aa of the ecotropic Env protein significantly increased the titer compared to that of its parental chimera AE4, implying a functional coordination between the N terminus of SU and the C terminus of the SU and/or transmembrane proteins. These data functionally dissect the N-terminal sequence of the MuLV Env protein and identify differential effects on receptor-mediated entry.     

Substitutions in the receptor-binding domain of the avian sarcoma and leukosis virus envelope uncouple receptor-triggered structural rearrangements in the surface and transmembrane subunits

The retrovirus avian sarcoma and leukosis virus (ASLV) enters cells via pH-independent membrane fusion. This reaction is catalyzed by the viral glycoprotein Env, composed of a membrane-distal subunit, SU, and a membrane-anchored subunit, TM. Previous mutational analysis of a variable region, central within the SU subunit, indicates that this region constitutes part of the receptor-binding domain for subgroup A envelope (EnvA) and furthermore that basic residues (R210, R213, R223, R224, and K227) within this region are critical determinants of efficient ASLV infection. Substitutions of these basic residues exert effects on both receptor binding and postbinding events in EnvA-mediated entry. In this study, we performed biochemical analysis of the EnvA protein from three of the receptor-binding domain mutants (R213A/K227A, R213A/R223A/R224A, and R213S) to define the role of this domain in early molecular events in the entry pathway. Protease sensitivity assays demonstrated that receptor binding was sufficient to trigger conformational changes in the SU subunit of mutants R213A/K227A and R213S similar to those in the wild-type EnvA, while R213A/R223A/R224A was constitutively sensitive to protease. In contrast, all three receptor-binding domain mutants disrupted receptor-triggered conversion of EnvA to an active, membrane-binding conformation as assessed by liposome flotation assays. Our results demonstrate that mutations in the receptor-binding site can dissociate receptor-triggered conformational changes in the SU subunit from membrane binding. Furthermore, they suggest that communication between the receptor-binding subunit, SU, and the fusogenic subunit, TM, is crucial for efficient activation of the fusogenic state of EnvA. Analysis of these mutants continues earlier observations that binding to the cellular receptor provides the trigger for efficient activation of this pH-independent viral envelope protein.     

Analysis of the cell fusion activities of chimeric simian immunodeficiency virus-murine leukemia virus envelope proteins: inhibitory effects of the R peptide.

It was previously reported that truncation or proteolytic removal of the C-terminal 16 amino acids (the R peptide) from the cytoplasmic tail of the murine leukemia virus (MuLV) envelope protein greatly increases its fusion activity. In this study, to investigate the specificity of the effect of the R peptide on the fusion activity of viral envelope proteins, we expressed simian immunodeficiency virus (SIV)-MuLV chimeric proteins in which the entire cytoplasmic tail of the SIV envelope protein was replaced by either the full-length MuLV cytoplasmic tail or a truncated MuLV cytoplasmic tail with the R peptide deleted. Extensive fusion of CD4-positive cells with the chimeric protein containing a truncated MuLV cytoplasmic tail was observed. In contrast, no cell fusion activity was found for the chimeric protein with a full-length MuLV cytoplasmic tail. We constructed another SIV-MuLV chimeric protein in which the MuLV R peptide was added to an SIV envelope protein cytoplasmic tail 17 amino acids from its membrane-spanning domain. No fusion activity was observed within this construct, while the corresponding truncated SIV envelope protein lacking the R peptide showed extensive fusion activity. No significant difference in the transport or surface expression was observed among the various SIV-MuLV chimeric proteins and the truncated SIV envelope protein. Our results thus demonstrate that the MuLV R peptide has profound inhibitory effects on virus-induced cell fusion, not only with MuLV but also in a distantly related retroviral envelope protein which utilizes a different receptor and fuses different cell types.     

Analysis of the unique hamster cell tropism of ecotropic murine leukemia virus PVC-211.

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic variant of Friend MuLV (F-MuLV). Previous studies from our laboratory demonstrated that unlike the parental F-MuLV, PVC-211 MuLV can infect rat brain capillary endothelial cells efficiently and that it has acquired genetic changes responsible for its expanded cellular tropism. To determine if PVC-211 MuLV also has expanded its host range, we tested its infectivity on Chinese hamster ovary-derived CHO-K1 cells, which are generally resistant to ecotropic MuLV. The results indicated that PVC-211 MuLV, but not F-MuLV, was highly infectious for CHO-K1 cells. Studies using glycosylation inhibitors and glycosylation mutants of CHO-K1 cells, as well as interference studies, suggested that PVC-211 MuLV has acquired the ability to interact with the ecotropic MuLV receptor on CHO-K1 cells that has undergone glycosylation-dependent modification. Using chimeric viruses between PVC-211 MuLV and F-MuLV, we were able to localize the viral genetic element crucial for CHO-K1 cell tropism within the env gene of PVC-211 MuLV and show that glycine at position 116 and lysine at position 129 of the envelope glycoprotein SU were important. These viral determinants also appear to confer tropism for other hamster cells resistant to ordinary ecotropic MuLVs. Further studies on the interaction between PVC-211 MuLV and the receptor on hamster cells may provide novel insights into the molecular mechanisms for receptor recognition and binding by viral envelope glycoproteins.     

Initial Binding of Murine Leukemia Virus Particles to Cells Does Not Require Specific Env-Receptor Interaction

The initial step of virus-cell interaction was studied by immunofluorescence microscopy. Single particles of murine leukemia virus (MLV) vectors and human immunodeficiency virus (HIV) were visualized by immunofluorescence. Fluorescent dots representing single virions could be localized by staining of capsid proteins (CA) or surface envelope proteins (SU) after fixation of virus supernatants. This technique can be used to determine particle concentration in viral supernatants and also to study virus-cell interaction. We investigated the role of the Env-receptor interaction for the initial binding event between the cell and the viral particles. Ecotropic MLV vector particles were shown to bind to human cells which do not express the specific viral receptor. In addition, MLV particles defective for Env were shown to bind the cells similarly to infectious MLV. Time course experiments of virus-cell binding and dissociation showed identical profiles for infectious and Env-defective MLV particles and suggested that MLV Env is not involved in the early phases of attachment of virus to cells. The possible implication of cellular factors in enhancing viral binding and infectivity is discussed.     

Suppression of a fusion defect by second site mutations in the ecotropic murine leukemia virus surface protein

Entry of ecotropic murine leukemia virus initiates when the envelope surface protein recognizes and binds to the virus receptor on host cells. The envelope transmembrane protein then mediates fusion of viral and host cell membranes and penetration into the cytoplasm. Using a genetic selection, we isolated an infectious retrovirus variant containing three changes in the surface protein-histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. Single replacement of histidine 8 with arginine (H8R) resulted in almost complete loss of infectivity, even though the mutant envelope proteins were stable and efficiently incorporated into virions. Virions carrying H8R envelope were proficient at binding cells expressing receptor but failed to induce cell-cell fusion of XC cells, indicating that the histidine at position 8 plays an essential role in fusion during penetration of the host cell membrane. Thus, there is at least one domain in SU that is involved in fusion; the fusion functions do not reside exclusively in TM. In contrast, envelope with all three changes induced cell-cell fusion of XC cells and produced virions that were 10,000-fold more infectious than those containing only the H8R substitution, indicating that changes at positions 227 and 243 can suppress a fusion defect caused by loss of histidine 8 function. Moreover, the other two changes acted synergistically, indicating that both compensate for the loss of the same essential function of histidine 8. The ability of these changes to suppress this fusion defect might provide a means for overcoming postbinding defects found in targeted retroviral vectors for use in human gene therapy.     

Lateral mobility of both envelope proteins (F and HN) of Sendai virus in the cell membrane is essential for cell-cell fusion

Fluorescence photobleaching recovery was employed to study the effects of specific immobilization of Sendai virus envelope glycoproteins (F, the fusion protein, and HN, the hemagglutinin-neuraminidase) on the virally mediated fusion of human erythrocytes. Lateral immobilization of varying fractions of F and/or HN (after virus adsorption and hemagglutination, but before fusion) was achieved by cross-linking them with succinyl concanavalin A (inhibiting both F and HN) or with specific rabbit IgG directed against either F or HN. Alternatively, agglutinated cells were treated with low concentrations of the above proteins (inducing only minor inhibition of either mobility or fusion), and immobilization of F and/or HN was induced by cross-linking with a secondary antibody; this protocol ensured a minimal contribution of direct binding to the viral proteins to the inhibition of fusion. Our results demonstrate that lateral immobilization of either F or HN results in a strong inhibition of cell-cell fusion and a much weaker inhibition of virus-cell fusion. The level of cell-cell fusion was directly correlated with the level of laterally mobile viral glycoproteins in the cell membrane (either F or HN). We conclude that lateral mobility of both F and HN in the red cell membrane is essential for cell-cell fusion and that not only F but also HN has a role in this fusion event. The possible reasons for the different dependence of cell-cell and virus-cell fusion on viral glycoprotein mobility are discussed.     

Identification of a Region in the Stalk Domain of the Nipah Virus Receptor Binding Protein That Is Critical for Fusion Activation

Paramyxoviruses, including the emerging lethal human Nipah virus (NiV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral and target cell membranes. For paramyxoviruses, membrane fusion is the result of the concerted action of two viral envelope glycoproteins: a receptor binding protein and a fusion protein (F). The NiV receptor binding protein (G) attaches to ephrin B2 or B3 on host cells, whereas the corresponding hemagglutinin-neuraminidase (HN) attachment protein of NDV interacts with sialic acid moieties on target cells through two regions of its globular domain. Receptor-bound G or HN via its stalk domain triggers F to undergo the conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We show that chimeric proteins containing the NDV HN receptor binding regions and the NiV G stalk domain require a specific sequence at the connection between the head and the stalk to activate NiV F for fusion. Our findings are consistent with a general mechanism of paramyxovirus fusion activation in which the stalk domain of the receptor binding protein is responsible for F activation and a specific connecting region between the receptor binding globular head and the fusion-activating stalk domain is required for transmitting the fusion signal.     

Characterization of chimeras between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A envelope proteins.

A series of 22 chimeric envelope (env) genes were generated between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A isolate. The chimeric envelopes were expressed within the complete, replication-competent provirus and tested for virus viability by transient expression assays. Eleven of the 22 viruses were viable. Five of these chimeric viruses showed an ecotropic host range, and six exhibited an amphotropic host range and viral interference. The host range determinants map to the first half of the surface (SU) protein. The N-terminal 72 amino acids of 4070A (42 of processed SU) are not required for amphotropic receptor usage. Ecotropic and amphotropic viruses differ in their ability to form large, multinucleated syncytia when cocultured with the rat XC cell line. Ecotropic murine leukemia virus forms large syncytia with XC cells, whereas no syncytia are reported for amphotropic virus. All chimeras which contained the N-terminal half of the ecotropic SU protein, encoding the receptor binding domain, formed the large multinucleated syncytia with XC cells.     

Receptor choice determinants in the envelope glycoproteins of amphotropic, xenotropic, and polytropic murine leukemia viruses.

The envelope glycoproteins (SU) of mammalian type C retroviruses possess an amino-terminal domain of about 200 residues, which is involved in binding a cell surface receptor. In this domain, highly conserved amino acid sequences are interrupted by two segments of variable length and sequence, VRA and VRB. We have studied the role of these variable regions in receptor recognition and binding by constructing chimeric molecules in which portions of the amino-terminal domains from amphotropic (4070A), xenotropic (NZB), and polytropic (MCF 247) murine leukemia virus SU proteins were permuted. These chimeras, which exchanged either one or two variable regions, were expressed at the surface of replication-defective viral particles by a pseudotyping assay. Wild-type or recombinant env genes were transfected into a cell line producing Moloney murine leukemia virus particles devoid of envelope glycoproteins in which a retrovirus vector genome carrying an Escherichia coli lacZ gene was packaged. The host range and sensitivity to interference of pseudotyped virions were assayed, and we observed which permutations resulted in receptor switch or loss of function. Our results indicate that the determinants of receptor choice are found within the just 120 amino acids of SU proteins. Downstream sequences contribute to the stabilization of the receptor-specific structure.     

Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induced syncytium formation

Amphotropic murine leukemia virus (A-MuLV) utilizes the PiT2 sodium-dependent phosphate transporter as its cell surface receptor to infect mammalian cells. The process of A-MuLV infection requires cleavage of the R peptide from the envelope protein. This occurs within virions thereby rendering them competent to fuse with target cells. Envelope proteins lacking the inhibitory R peptide (e.g. envelope (R-) proteins) induce viral envelope-mediated cell-cell fusion (syncytium). Here we have performed studies to determine if cell signaling through protein kinases is involved in the regulation of PiT2-mediated A-MuLV envelope (R-)-induced syncytium formation. Truncated A-MuLV retroviral envelope protein lacking the inhibitory R peptide (R-) was used to induce viral envelope-mediated cell-cell fusion. Signaling through cyclic AMP to activate PKA was found to inhibit envelope-induced cell-cell fusion, whereas treatment of cells with PKA inhibitors H89, KT5720, and PKA Catalpha siRNA all enhanced this cell fusion process. It was noted that activation of PKC, as well as overexpression of PKCepsilon, up-regulated A-MuLV envelope protein-induced cell-cell fusion, whereas exposure to PKC inhibitors and expression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) inhibited syncytium formation. v-ras transformed NIH3T3 cells were highly susceptible to A-MuLV envelope-induced cell-cell fusion, whereas expression of a dominant-negative mutant of Ras (N17Ras) inhibited this cell fusion process. Importantly, activation of Raf-1 protein kinase also is required for A-MuLV envelope-induced syncytium formation. Expression of constitutively active BXB Raf supported, whereas expression of a dominant-negative mutant of Raf-1 (Raf301) blocked, A-MuLV-induced cell-cell fusion. These results indicate that specific cell signaling components are involved in regulating PiT2-mediated A-MuLV-induced cell-cell fusion. Selective pharmacological modulation of these signaling components may be an effective means of altering cell susceptibility to viral-mediated cytopathic effects.     

Evidence for Cooperation between Murine Leukemia Virus Env Molecules in Mixed Oligomers

A retroviral Env molecule consists of a surface glycoprotein (SU) complexed with a transmembrane protein (TM). In turn, these complexes are grouped into oligomers on the surfaces of the cell and of the virion. In the case of murine leukemia viruses (MuLVs), the SU moieties are polymorphic, with SU proteins of different viral isolates directed towards different cell surface receptors. During maturation of the released virus particle, the 16 C-terminal residues of TM (the R peptide or p2E) are removed from the protein by the viral protease; this cleavage is believed to activate the membrane-fusing potential of MuLV Env. We have tested the possibility that different MuLV Env proteins in the same cell can interact with each other, both physically and functionally, in mixed oligomers. We found that coexpressed Env molecules can be precipitated out of cell lysates by antiserum which reacts with only one of them. Furthermore, they can evidently cooperate with each other: if one Env species lacks the R peptide, then it can apparently induce fusion if the SU protein of the other Env species encounters its cognate receptor on the surface of another cell. This functional interaction between different Env molecules has a number of implications with respect to the mechanism of induction of membrane fusion, for the genetic analysis of Env function, and for the design of targeted retroviral vectors for gene therapy.     

Role of basic residues in the subgroup-determining region of the subgroup A avian sarcoma and leukosis virus envelope in receptor binding and infection.

Receptor specificity in avian sarcoma and leukosis viruses (ASLV) maps to the central region of the envelope surface protein, SU. Two hypervariable regions, hr1 and hr2, within this region of SU are the principal determinants of receptor specificity. The cellular receptor for subgroup A ASLV, Tva, utilizes a 40-residue, acidic, cysteine-rich sequence for viral binding and entry. This domain in Tva is closely related to the ligand-binding domain of the low-density lipoprotein receptor (LDLR). Ligands bind to LDLR via the interaction of clustered basic residues in the ligand with the acidic cysteine-rich domains of the receptor. Analysis of the ASLV envelope sequences revealed a cluster of basic residues within hr2 that is unique to the subgroup A viruses, suggesting a possible role for these residues in receptor recognition. Therefore, the effects of altering these basic residues on subgroup A envelope expression, receptor binding, and infectivity were examined. Most of the mutant proteins were transported to the cell surface and processed normally. Receptor binding was diminished approximately 50% by alanine substitution at amino acid R213 or K227, whereas substitution by alanine at R210, R223, or R224 had no effect. However, when coupled with mutations at R213 or K227, changes at R223,R224 reduced envelope binding by 90%. Mutation of all five basic residues abrogated receptor binding. The effect of the hr2 mutations on ASLV envelope-mediated infection did not parallel the effect on receptor binding. Residues 210, 213, 223, and 224 were important for efficient infection, while mutations at residue 227 had little effect on infectivity. These results demonstrate that the basic residues in the ASLV envelope have roles in both receptor recognition and post-receptor binding events during viral entry.     

Actin cytoskeletal reorganizations and coreceptor-mediated activation of rac during human immunodeficiency virus-induced cell fusion

The membrane fusion events which initiate human immunodeficiency virus type 1 (HIV-1) infection and promote cytopathic syncytium formation in infected cells commence with the binding of the HIV envelope glycoprotein (Env) to CD4 and an appropriate coreceptor. Here, we show that HIV Env-coreceptor interactions activate Rac-1 GTPase and stimulate the actin filament network reorganizations that are requisite components of the cell fusion process. Disrupting actin filament dynamics with jasplakinolide or latrunculin A arrested fusion at a late step in the formation of Env-CD4-coreceptor complexes. Time-lapse confocal microscopy of living cells revealed vigorous activity of actin-based, target cell membrane extensions at the target cell-Env-expressing cell interface. The expression of dominant-negative forms of actin-regulating Rho-family GTPases established that HIV Env-mediated syncytium formation relies on Rac-1 but not on Cdc42 or Rho activation in target cells. Similar dependencies were found when cell fusion was induced by Env expressed on viral or cellular membranes. Additionally, Rac activity was specifically upregulated in a coreceptor-dependent manner in fusion reaction cell lysates. These results define a role for HIV Env-coreceptor interactions in activating the cellular factors essential for virus-cell and cell-cell fusion and provide evidence for the participation of pertussis toxin-insensitive signaling pathways in HIV-induced membrane fusion.     

Effects of fusion temperature on the lateral mobility of Sendai virus glycoproteins in erythrocyte membranes and on cell fusion indicate that glycoprotein mobilization is required for cell fusion

In order to investigate the requirement for lateral mobilization of viral envelope glycoproteins on the cell surface in the induction of cell-cell fusion, we employed fluorescence photobleaching recovery to study the effect of the fusion temperature on the lateral mobilization of Sendai virus glycoproteins in the human erythrocyte membrane. As the fusion temperature was reduced below 37 degrees C (to 31 or 25 degrees C), the rates of virus-cell fusion, the accompanying hemolysis, and cell-cell fusion were all slowed down. However, the plateau (final level) after the completion of fusion was significantly reduced at lower fusion temperatures only in the case of cell-cell fusion, despite the rather similar final levels of virus-cell fusion. A concomitant decrease as a function of the fusion temperature was observed in the fraction of cell-associated viral glycoproteins that became laterally mobile in the erythrocyte membrane during fusion, and a strict correlation was found between the level of laterally mobile viral glycoproteins in the cell membrane and the final extent of cell-cell fusion. The accompanying reduction in the lateral diffusion coefficients (D) of the viral glycoproteins (1.4-fold at 31 degrees C and 1.9-fold at 25 degrees C, as compared to 37 degrees C) does not appear to determine the final level of cell-cell fusion, since fusing the cells with a higher amount of virions at 25 degrees C increased the final level of cell-cell fusion while D remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)