Vaccinia Virus Protein Complex F12/E2 Interacts with Kinesin Light Chain Isoform 2 to Engage the Kinesin-1 Motor Complex

During vaccinia virus morphogenesis, intracellular mature virus (IMV) particles are wrapped by a double lipid bilayer to form triple enveloped virions called intracellular enveloped virus (IEV). IEV are then transported to the cell surface where the outer IEV membrane fuses with the cell membrane to expose a double enveloped virion outside the cell. The F12, E2 and A36 proteins are involved in transport of IEVs to the cell surface. Deletion of the F12L or E2L genes causes a severe inhibition of IEV transport and a tiny plaque size. Deletion of the A36R gene leads to a smaller reduction in plaque size and less severe inhibition of IEV egress. The A36 protein is present in the outer membrane of IEVs, and over-expressed fragments of this protein interact with kinesin light chain (KLC). However, no interaction of F12 or E2 with the kinesin complex has been reported hitherto. Here the F12/E2 complex is shown to associate with kinesin-1 through an interaction of E2 with the C-terminal tail of KLC isoform 2, which varies considerably between different KLC isoforms. siRNA-mediated knockdown of KLC isoform 1 increased IEV transport to the cell surface and virus plaque size, suggesting interaction with KLC isoform 1 is somehow inhibitory of IEV transport. In contrast, knockdown of KLC isoform 2 did not affect IEV egress or plaque formation, indicating redundancy in virion egress pathways. Lastly, the enhancement of plaque size resulting from loss of KLC isoform 1 was abrogated by removal of KLC isoforms 1 and 2 simultaneously. These observations suggest redundancy in the mechanisms used for IEV egress, with involvement of KLC isoforms 1 and 2, and provide evidence of interaction of F12/E2 complex with the kinesin-1 complex.     

Vaccinia Virus Protein F12 Associates with Intracellular Enveloped Virions through an Interaction with A36

Vaccinia virus is the prototypical member of the family Poxviridae. Three morphologically distinct forms are produced during infection: intracellular mature virions (IMV), intracellular enveloped virions (IEV), and extracellular enveloped virions (EEV). Two viral proteins, F12 and A36, are found exclusively on IEV but not on IMV and EEV. Analysis of membranes from infected cells showed that F12 was only associated with membranes and is not an integral membrane protein. A yeast two-hybrid assay revealed an interaction between amino acids 351 to 458 of F12 and amino acids 91 to 111 of A36. We generated a recombinant vaccinia virus that expresses an F12, which lacks residues 351 to 458. Characterization of this recombinant revealed a small-plaque phenotype and a subsequent defect in virus release similar to a recombinant virus that had F12L deleted. In addition, F12 lacking residues 351 to 458 was unable to associate with membranes in infected cells. These results suggest that F12 associates with IEV through an interaction with A36 and that this interaction is critical for the function of F12 during viral egress.     

The vaccinia virus A33R protein provides a chaperone function for viral membrane localization and tyrosine phosphorylation of the A36R protein

The products of the A33R and A36R genes of vaccinia virus are incorporated into the membranes of intracellular enveloped virions (IEV). When extracts of cells that had been infected with vaccinia virus and labeled with H(3)(32)PO(4) were immunoprecipitated with antibodies against the A33R protein, two prominent bands were resolved. The moderately and more intensely labeled bands were identified as phosphorylated A33R and A36R proteins, respectively. The immunoprecipitated complex contained disulfide-bonded dimers of A33R protein that were noncovalently linked to A36R protein. Biochemical analysis indicated that the two proteins were phosphorylated predominantly on serine residues, with lesser amounts on threonines. The A36R protein was also phosphorylated on tyrosine, as determined by specific binding to an anti-phosphotyrosine antibody. Serine phosphorylation and A33R-A36R protein complex formation occurred even when virus assembly was blocked at an early stage with the drug rifampin. Tyrosine phosphorylation was selectively reduced in cells infected with F13L or A34R gene deletion mutants that were impaired in the membrane-wrapping step of IEV formation. In addition, tyrosine phosphorylation was specifically inhibited in cells infected with an A33R deletion mutant that still formed IEV. Immunofluorescence and immunoelectron microscopy indicated that in the absence of the A33R protein, the A36R protein was localized in Golgi membranes but not in IEV. In the absence of the A36R protein, however, the A33R protein still localized to IEV membranes. These studies together with others suggest that the A33R protein guides the A36R protein to the IEV membrane, where it subsequently becomes tyrosine phosphorylated as a signal for actin tail formation.     

Visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-B5R membrane protein chimera

We produced an infectious vaccinia virus that expressed the B5R envelope glycoprotein fused to the enhanced green fluorescent protein (GFP), allowing us to visualize intracellular virus movement in real time. Previous transfection studies indicated that fusion of GFP to the C-terminal cytoplasmic domain of B5R did not interfere with Golgi localization of the viral protein. To determine whether B5R-GFP was fully functional, we started with a B5R deletion mutant that made small plaques and inserted the B5R-GFP gene into the original B5R locus. The recombinant virus made normal-sized plaques and acquired the ability to form actin tails, indicating reversal of the mutant phenotype. Moreover, immunogold electron microscopy revealed that both intracellular enveloped virions (IEV) and extracellular enveloped virions contained B5R-GFP. By confocal microscopy of live infected cells, we visualized individual fluorescent particles, corresponding to IEV in size and shape, moving from a juxtanuclear location to the periphery of the cell, where they usually collected prior to association with actin tails. The fluorescent particles could be seen emanating from cells at the tips of microvilli. Using a digital camera attached to an inverted fluorescence microscope, we acquired images at 1 frame/s. At this resolution, IEV movement appeared saltatory; in some frames there was no net movement, whereas in others movement exceeded 2 microm/s. Further studies indicated that IEV movement was reversibly arrested by the microtubule-depolymerizing drug nocodazole. This result, together with the direction, speed, and saltatory motion of IEV, was consistent with a role for microtubules in intracellular transport of IEV.     

Interactions between vaccinia virus IEV membrane proteins and their roles in IEV assembly and actin tail formation

The intracellular enveloped form of vaccinia virus (IEV) induces the formation of actin tails that are strikingly similar to those seen in Listeria and Shigella infections. In contrast to the case for Listeria and Shigella, the vaccinia virus protein(s) responsible for directly initiating actin tail formation remains obscure. However, previous studies with recombinant vaccinia virus strains have suggested that the IEV-specific proteins A33R, A34R, A36R, B5R, and F13L play an undefined role in actin tail formation. In this study we have sought to understand how these proteins, all of which are predicted to have small cytoplasmic domains, are involved in IEV assembly and actin tail formation. Our data reveal that while deletion of A34R, B5R, or F13L resulted in a severe reduction in IEV particle assembly, IEVs formed by the DeltaB5R and DeltaF13L deletion strains, but not DeltaA34R, were still able to induce actin tails. The DeltaA36R deletion strain produced normal amounts of IEV particles, although these were unable to induce actin tails. Using several different approaches, we demonstrated that A36R is a type Ib membrane protein with a large, 195-amino-acid cytoplasmic domain exposed on the surface of IEV particles. Finally, coimmunoprecipitation experiments demonstrated that A36R interacts with A33R and A34R but not with B5R and that B5R forms a complex with A34R but not with A33R or A36R. Using extracts from DeltaA34R- and DeltaA36R-infected cells, we found that the interaction of A36R with A33R and that of A34R with B5R are independent of A34R and A36R, respectively. We conclude from our observations that multiple interactions between IEV membrane proteins exist which have important implications for IEV assembly and actin tail formation. Furthermore, these data suggest that while A34R is involved in IEV assembly and organization, A36R is critical for actin tail formation.     

Mapping and functional analysis of interaction sites within the cytoplasmic domains of the vaccinia virus A33R and A36R envelope proteins

Incorporation of the vaccinia virus A36R protein into the outer membrane of intracellular enveloped virions (IEV) is dependent on expression of the A33R protein. Possible interactions of the 200-amino-acid cytoplasmic domain of the A36R protein with itself or with the cytoplasmic domain of the A33R, A34R, B5R, or F12L IEV membrane protein was investigated by using the yeast two-hybrid system. A strong interaction was detected only between the cytoplasmic domains of the A36R and A33R proteins. Upon further analyses, the interaction site was mapped to residues 91 to 111 of the A36R protein. To investigate the role of the A36R:A33R interaction during viral infection, five recombinant vaccinia viruses containing B5R-GFP as a marker were constructed. Four had the full-length A36R gene replaced with various-length C-terminal truncations of A36R, of which two contained residues 91 to 111 and two were missing this region. The fifth recombinant virus had an A33R gene with most of the 40-amino-acid cytoplasmic tail deleted. Residues 91 to 111 of A36R and the cytoplasmic tail of A33R were required for a strong interaction between the two proteins during viral infection and for maximal amounts of A36R protein on IEV. Mutants lacking these regions of A33R or A36R formed IEV that exhibited only short sporadic intracellular movement, displayed no actin tails, and formed small plaques on cell monolayers equivalent to those of an A36R deletion mutant and smaller than those formed by point mutations that specifically abrogate actin tail formation. The A33R interaction site of the A36R protein is highly conserved among orthopoxviruses and may overlap binding sites for cellular proteins needed for microtubular movement and actin tail formation.     

Lis1 is an initiation factor for dynein-driven organelle transport

The molecular motor cytoplasmic dynein is responsible for most minus-end-directed, microtubule-based transport in eukaryotic cells. It is especially important in neurons, where defects in microtubule-based motility have been linked to neurological diseases. For example, lissencephaly is caused by mutations in the dynein-associated protein Lis1. In this paper, using the long, highly polarized hyphae of the filamentous fungus Aspergillus nidulans, we show that three morphologically and functionally distinct dynein cargos showed transport defects in the genetic absence of Lis1/nudF, raising the possibility that Lis1 is ubiquitously used for dynein-based transport. Surprisingly, both dynein and its cargo moved at normal speeds in the absence of Lis1 but with reduced frequency. Moreover, Lis1, unlike dynein and dynactin, was absent from moving dynein cargos, further suggesting that Lis1 is not required for dynein-based cargo motility once it has commenced. Based on these observations, we propose that Lis1 has a general role in initiating dynein-driven motility.     

Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans Golgi network.

During the assembly of vaccinia virus, the intracellular mature virus becomes enwrapped by a cellular cisterna to form the intracellular enveloped virus (IEV), the precursor of the extracellular enveloped virus (EEV). In this study, we have characterized the origin of this wrapping cisterna by electron microscopic immunocytochemistry using lectins, antibodies against endocytic organelles, and recombinant vaccinia viruses expressing proteins which behave as Golgi resident proteins. No labelling for endocytic marker proteins could be detected on the wrapping membrane. However, the wrapping membrane labelled significantly for a trans Golgi network (TGN) marker protein. The recycling pathway from endosomes to the TGN appears to be greatly increased following vaccinia virus infection, since significant amounts of endocytic fluid-phase tracers were found in the lumen of the TGN, Golgi complex, and the wrapping cisternae. Using immunoelectron microscopy, we localized the vaccinia virus membrane proteins VV-p37, VV-p42, VV-p21, and VV-hemagglutinin (VV-HA) in large amounts in the wrapping cisternae, in the outer membranes of the IEV, and in the outermost membrane of the EEV. The bulk of the cellular VV-p37, VV-p21, and VV-p42 were in the TGN, whereas VV-HA was also found in large amounts on the plasma membrane and in endosomes. Collectively, these data argue that the TGN becomes enriched in vaccinia virus membrane proteins that facilitate the wrapping event responsible for the formation of the IEV.     

Characterization of a glutamine/proton cotransporter from Ricinus comnmnis roots using isolated plasma membrane vesicles

The mechanism of glutamine transport at the plasma membrane of sink tissue cells was investigated using isolated plasma membrane vesicles from roots of Ricinus communis L. var. sanguineous . Glutamine transport was found to be driven by both the pH gradient (ΔpH) and a membrane potential (ΔΨ) (alkaline and negative internal), which were created artificially across the plasma membrane. Glutamine wus accumulated 15–20-fold in the presence of both a ΔpH and Δ Ψ . There appeared to be a direct pH effect on Δ PS -driven transport, as a higher rate of transport was observed at pH 5.5 than at pH 7.5. The ΔpH +Δ Ψ -driven transport showed saturation kinetics with a K_m of 287 μ M . Altering the membrane potential changed the V_max but had no effect on the K_m of glutamine transport. These results are consistent with the presence of a proton-coupled, carrier-mediated system for glutamine uptake in Ricinus roots. A range of protein modifiers and transport inhibitors had limited effects on glutamine transport: highest inhibition uas observed with cytochalasin D. When glutamine transport was compared in plasma membrane vesicles isolated from the root lips of Ricinus and from the remainder of the root tissue a lower level of transport was observed in the root tips. A method for the solubilization and reconstitution of glutamine transport activity using the detergent CHAPS is also described.     

mRNA splicing regulates human papillomavirus type 11 E1 protein production and DNA replication

The papillomavirus replicative helicase E1 and the origin recognition protein E2 are required for efficient viral DNA replication. We fused the green fluorescent protein (GFP) to the human papillomavirus type 11 E1 protein either in a plasmid with the E1 coding region alone (nucleotides [nt] 832 to 2781) (pGFP-11E1) or in a plasmid containing both the E1 and E2 regions (nt 2723 to 3826) and the viral origin of replication (ori) (p11Rc). The former supported transient replication of an ori plasmid, whereas the latter was a self-contained replicon. Unexpectedly, these plasmids produced predominantly a cytoplasmic variant GFP or a GFP-E1 E4 protein, respectively. The majority of the mRNAs had an intragenic or intergenic splice from nt 847 to nt 2622 or from nt 847 to nt 3325, corresponding to the E2 or E1 E4 messages. pGFP-11E1dm and p11Rc-E1dm, mutated at the splice donor site, abolished these splices and increased GFP-E1 protein expression. Three novel, alternatively spliced, putative E2 mRNAs were generated in higher abundance from the mutated replicon than from the wild type. Relative to pGFP-11E1, low levels of pGFP-11E1dm supported more efficient replication, but high levels had a negative effect. In contrast, elevated E2 levels always increased replication. Despite abundant GFP-E1 protein, p11Rc-E1dm replicated less efficiently than the wild type. Collectively, these observations show that the E1/E2 ratio is as important as the E1 and E2 concentrations in determining the replication efficiency. These findings suggest that alternative mRNA splicing could provide a mechanism to regulate E1 and E2 protein expression and DNA replication during different stages of the virus life cycle.     

Herpesvirus Tegument Protein pUL37 Interacts with Dystonin/BPAG1 To Promote Capsid Transport on Microtubules during Egress

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that travels long distances through cells using the microtubule network. Its 125-nm-diameter capsid is a large cargo which efficiently recruits molecular motors for movement. Upon entry, capsids reach the centrosome by minus-end-directed transport. From there, they are believed to reach the nucleus by plus-end-directed transport. Plus-end-directed transport is also important during egress, when capsids leave the nucleus to reach the site of envelopment in the cytoplasm. Although capsid interactions with dynein and kinesins have been described in vitro, the actual composition of the cellular machinery recruited by herpesviruses for capsid transport in infected cells remains unknown. Here, we identify the spectraplakin protein, dystonin/BPAG1, an important cytoskeleton cross-linker involved in microtubule-based transport, as a binding partner of the HSV-1 protein pUL37, which has been implicated in capsid transport. Viral replication is delayed in dystonin-depleted cells, and, using video microscopy of living infected cells, we show that dystonin depletion strongly inhibits capsid movement in the cytoplasm during egress. This study provides new insights into the cellular requirements for HSV-1 capsid transport and identifies dystonin as a nonmotor protein part of the transport machinery.     

Vaccinia-induced epidermal growth factor receptor-MEK signalling and the anti-apoptotic protein F1L synergize to suppress cell death during infection

F1L is a functional Bcl-2 homologue that inhibits apoptosis at the mitochondria during vaccinia infection. However, the extent and timing of cell death during ΔF1L virus infection suggest that additional viral effectors cooperate with F1L to limit apoptosis. Here we report that vaccinia growth factor (VGF), a secreted virulence factor, promotes cell survival independently of its role in virus multiplication. Analysis of single and double knockout viruses reveals that VGF acts synergistically with F1L to protect against cell death during infection. Cell survival in the absence of F1L is dependent on VGF activation of the epidermal growth factor receptor. Furthermore, signalling through MEK kinases is necessary and sufficient for VGF-dependent survival. We conclude that VGF stimulates an epidermal growth factor receptor-MEK-dependent pro-survival pathway that synergizes with F1L to counteract an infection-induced apoptotic pathway that predominantly involves the BH3-only protein Bad.     

Sendai virus M protein binds independently to either the F or the HN glycoprotein in vivo.

We have analyzed the mechanism by which M protein interacts with components of the viral envelope during Sendai virus assembly. Using recombinant vaccinia viruses to selectively express combinations of Sendai virus F, HN, and M proteins, we have successfully reconstituted M protein-glycoprotein interaction in vivo and determined the molecular interactions which are necessary and sufficient to promote M protein-membrane binding. Our results showed that M protein accumulates on cellular membranes via a direct interaction with both F and HN proteins. Specifically, our data demonstrated that a small fraction (8 to 16%) of M protein becomes membrane associated in the absence of Sendai virus glycoproteins, while > 75% becomes membrane bound in the presence of both F and HN proteins. Selective expression of M protein together with either F or HN protein showed that each viral glycoprotein is individually sufficient to promote efficient (56 to 73%) M protein-membrane binding. Finally, we observed that M protein associates with cellular membranes in a time-dependent manner, implying a need for either maturation or transport before binding to glycoproteins.     

An ER membrane protein,Sop4, facilitates ER export of the yeast plasma membrane [H+]ATPase,Pma1

We have analyzed the mechanism by which Sop4, a novel ER membrane protein, regulates quality control and intracellular transport of Pma1–7, a mutant plasma membrane ATPase. At the restrictive temperature, newly synthesized Pma1–7 is targeted for vacuolar degradation instead of being correctly delivered to the cell surface. Loss of Sop4 at least partially corrects vacuolar mislocalization, allowing Pma1–7 routing to the plasma membrane. Ste2–3 is a mutant pheromone receptor which, like Pma1–7, is defective in targeting to the cell surface, resulting in a mating defect. sop4Δ suppresses the mating defect of ste2–3 cells as well as the growth defect of pma1–7 . Visualization of newly synthesized Pma1–7 in sop4Δ cells by indirect immunofluorescence reveals delayed export from the ER. Similarly, ER export of wild-type Pma1 is delayed in the absence of Sop4 although intracellular transport of Gas1 and CPY is unaffected. These observations suggest a model in which a selective increase in ER residence time for Pma1–7 may allow it to achieve a more favorable conformation for subsequent delivery to the plasma membrane. In support of this model, newly synthesized Pma1–7 is also routed to the plasma membrane upon release from a general block of ER-to-Golgi transport in sec13–1 cells.     

Sec6/8 complexes on trans-Golgi network and plasma membrane regulate late stages of exocytosis in mammalian cells.

Sec6/8 complex regulates delivery of exocytic vesicles to plasma membrane docking sites, but how it is recruited to specific sites in the exocytic pathway is poorly understood. We identified an Sec6/8 complex on trans-Golgi network (TGN) and plasma membrane in normal rat kidney (NRK) cells that formed either fibroblast- (NRK-49F) or epithelial-like (NRK-52E) intercellular junctions. At both TGN and plasma membrane, Sec6/8 complex colocalizes with exocytic cargo protein, vesicular stomatitis virus G protein (VSVG)-tsO45. Newly synthesized Sec6/8 complex is simultaneously recruited from the cytosol to both sites. However, brefeldin A treatment inhibits recruitment to the plasma membrane and other treatments that block exocytosis (e.g., expression of kinase-inactive protein kinase D and low temperature incubation) cause accumulation of Sec6/8 on the TGN, indicating that steady-state distribution of Sec6/8 complex depends on continuous exocytic vesicle trafficking. Addition of antibodies specific for TGN- or plasma membrane-bound Sec6/8 complexes to semiintact NRK cells results in cargo accumulation in a perinuclear region or near the plasma membrane, respectively. These results indicate that Sec6/8 complex is required for several steps in exocytic transport of vesicles between TGN and plasma membrane.     

Carboxypeptidase E cytoplasmic tail-driven vesicle transport is key for activity-dependent secretion of peptide hormones

Vesicular transport of peptide hormones from the cell body to the plasma membrane for activity-dependent secretion is important for endocrine function, but how it is achieved is unclear. Here we uncover a mechanism in which the cytoplasmic tail of transmembrane carboxypeptidase E (CPE) found in proopiomelanocotin (POMC)/ACTH vesicles interacts with microtubule-based motors to control transport of these vesicles to the release site in pituitary cells. Overexpression of the CPE tail in live cells significantly reduced the velocity and distance of POMC/ACTH- and CPE-containing vesicle movement into the cell processes. Biochemical studies showed that the CPE tail interacted with dynactin, which, in turn, recruited microtubule plus-end motors kinesin 2 and kinesin 3. Overexpression of the CPE tail inhibited the stimulated secretion of ACTH from AtT20 cells. Thus, the CPE cytoplasmic tail interaction with dynactin-kinesin 2/kinesin 3 plays an important role in the transport of POMC vesicles for activity-dependent secretion.     

Peroxisomes move by hitchhiking on early endosomes using the novel linker protein PxdA

Eukaryotic cells use microtubule-based intracellular transport for the delivery of many subcellular cargos, including organelles. The canonical view of organelle transport is that organelles directly recruit molecular motors via cargo-specific adaptors. In contrast with this view, we show here that peroxisomes move by hitchhiking on early endosomes, an organelle that directly recruits the transport machinery. Using the filamentous fungus Aspergillus nidulans we found that hitchhiking is mediated by a novel endosome-associated linker protein, PxdA. PxdA is required for normal distribution and long-range movement of peroxisomes, but not early endosomes or nuclei. Using simultaneous time-lapse imaging, we find that early endosome-associated PxdA localizes to the leading edge of moving peroxisomes. We identify a coiled-coil region within PxdA that is necessary and sufficient for early endosome localization and peroxisome distribution and motility. These results present a new mechanism of microtubule-based organelle transport in which peroxisomes hitchhike on early endosomes and identify PxdA as the novel linker protein required for this coupling.     

Cell cycle control of microtubule-based membrane transport and tubule formation in vitro

When higher eukaryotic cells enter mitosis, membrane organization changes dramatically and traffic between membrane compartments is inhibited. Since membrane transport along microtubules is involved in secretion, endocytosis, and the positioning of organelles during interphase, we have explored whether the mitotic reorganization of membrane could involve a change in microtubule-based membrane transport. This question was examined by reconstituting organelle transport along microtubules in Xenopus egg extracts, which can be converted between interphase and metaphase states in vitro in the absence of protein synthesis. Interphase extracts support the microtubule-dependent formation of abundant polygonal networks of membrane tubules and the transport of small vesicles. In metaphase extracts, however, the plus end- and minus end-directed movements of vesicles along microtubules as well as the formation of tubular membrane networks are all reduced substantially. By fractionating the extracts into soluble and membrane components, we have shown that the cell cycle state of the supernatant determines the extent of microtubule-based membrane movement. Interphase but not metaphase Xenopus soluble factors also stimulate movement of membranes from a rat liver Golgi fraction. In contrast to above findings with organelle transport, the minus end-directed movements of microtubules on glass surfaces and of latex beads along microtubules are similar in interphase and metaphase extracts, suggesting that cytoplasmic dynein, the predominant soluble motor in frog extracts, retains its force-generating activity throughout the cell cycle. A change in the association of motors with membranes may therefore explain the differing levels of organelle transport activity in interphase and mitotic extracts. We propose that the regulation of organelle transport may contribute significantly to the changes in membrane structure and function observed during mitosis in living cells.     

The pseudorabies virus VP1/2 tegument protein is required for intracellular capsid transport

Transport of capsids in cells is critical to alphaherpesvirus infection and pathogenesis; however, viral factors required for transport have yet to be identified. Here we provide a detailed examination of capsid dynamics during the egress phase of infection in Vero cells infected with pseudorabies virus. We demonstrate that the VP1/2 tegument protein is required for processive microtubule-based transport of capsids in the cytoplasm. A second tegument protein that binds to VP1/2, UL37, was necessary for wild-type transport but was not essential for this process. Both proteins were also required for efficient nuclear egress of capsids to the cytoplasm.     

Endocytosis plays a critical role in proteolytic processing of the Hendra virus fusion protein

The Hendra virus fusion (F) protein is synthesized as a precursor protein, F(0), which is proteolytically processed to the mature form, F(1) + F(2). Unlike the case for the majority of paramyxovirus F proteins, the processing event is furin independent, does not require the addition of exogenous proteases, is not affected by reductions in intracellular Ca(2+), and is strongly affected by conditions that raise the intracellular pH (C. T. Pager, M. A. Wurth, and R. E. Dutch, J. Virol. 78:9154-9163, 2004). The Hendra virus F protein cytoplasmic tail contains a consensus motif for endocytosis, YXXPhi. To analyze the potential role of endocytosis in the processing and membrane fusion promotion of the Hendra virus F protein, mutation of tyrosine 525 to alanine (Hendra virus F Y525A) or phenylalanine (Hendra virus F Y525F) was performed. The rate of endocytosis of Hendra virus F Y525A was significantly reduced compared to that of the wild-type (wt) F protein, confirming the functional importance of the endocytosis motif. An intermediate level of endocytosis was observed for Hendra virus F Y525F. Surprisingly, dramatic reductions in the rate of proteolytic processing were observed for Hendra virus F Y525A, although initial transport to the cell surface was not affected. The levels of surface expression for both Hendra virus F Y525A and Hendra virus F Y525F were higher than that of the wt protein, and these mutants displayed enhanced syncytium formation. These results suggest that endocytosis is critically important for Hendra virus F protein cleavage, representing a new paradigm for proteolytic processing of paramyxovirus F proteins.