Fusion function of the Semliki Forest virus spike is activated by proteolytic cleavage of the envelope glycoprotein precursor p62.

The precursor protein p62 of the prototype alphavirus Semliki Forest virus (SFV) undergoes during transport to the cell surface a proteolytic cleavage to form the mature envelope glycoprotein E2. To investigate the biological significance of this cleavage event, single amino acid substitutions were introduced at the cleavages site through mutagenesis of cDNA corresponding to the structural region of the SFV genome. The phenotypes of the cleavage site mutants were studied in BHK cells by using recombinant vaccinia virus vectors. Nonconservative substitutions completely abolished p62 cleavage. Uncleaved p62 was transported with normal kinetics to the cell surface, where it became accessible to low concentrations of exogenous trypsin. The proteolytic cleavage of envelope glycoprotein precursors has been shown to activate the membrane fusion potential of viral spikes in several virus families. Here we demonstrate that the fusion function of the SFV spike is activated by the cleavage of p62. Cleavage-deficient p62 expressed at the cell surface did not function in low-pH-triggered (pH 5.5) cell-cell membrane fusion; however, cleavage of the mutated p62 with exogenous trypsin restored the fusion function. We discuss a model for SFV assembly and fusion where p62 cleavage plays a crucial role in the stability of the multimeric association of the viral envelope glycoproteins.     

Furin processing and proteolytic activation of Semliki Forest virus

The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed "p62," which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.     

Membrane fusion process of Semliki Forest virus. II: Cleavage-dependent reorganization of the spike protein complex controls virus entry

The envelope of the Semliki Forest virus (SFV) contains two transmembrane proteins, E2 and E1, in a heterodimeric complex. The E2 subunit is initially synthesized as a precursor protein p62, which is proteolytically processed to the mature E2 form before virus budding at the plasma membrane. The p62 (E2) protein mediates binding of the heterodimer to the nucleocapsid during virus budding, whereas E1 carries the entry functions of the virus, that is, cell binding and low pH-mediated membrane fusion activity. We have investigated the significance of the cleavage event for the maturation and entry of the virus. To express SFV with an uncleaved p62 phenotype, BHK-21 cells were transfected by electroporation with infectious viral RNA transcribed from a full-length SFV cDNA clone in which the p62 cleavage site had been changed. The uncleaved p62E1 heterodimer was found to be used for the formation of virus particles with an efficiency comparable to the wild type E2E1 form. However, in contrast to the wild type virus, the mutant virus was virtually noninfectious. Noninfectivity resulted from impaired uptake into cells, as well as from the inability of the virus to promote membrane fusion in the mildly acidic conditions of the endosome. This inability could be reversed by mild trypsin treatment, which converted the viral p62E1 form into the mature E2E1 form, or by treating the virus with a pH 4.5 wash, which in contrast to the more mild pH conditions of endosomes, effectively disrupted the p62E1 subunit association. We conclude that the p62 cleavage is not needed for virus budding, but regulates entry functions of the E1 subunit by controlling the heterodimer stability in acidic conditions.     

Processing of the p62 envelope precursor protein of Semliki Forest virus

The spike protein of Semliki Forest virus is composed of three subunits, E1, E2, and E3, which mediate the fusion of the virus membrane with that of the host cell. E2 and E3 are synthesized as a precursor, p62, which is cleaved post-translationally after an Arg-His-Arg-Arg sequence. In vitro mutagenesis of a cDNA clone of the spike proteins was used to specifically alter amino acids in this cleavage site. Cleavage of p62 was completely blocked by mutation of the proximal Arg residue to Phe, without affecting transport or surface expression of the spike protein. The cleavage mutation resulted in the loss of spike protein fusion activity within the physiological pH range. Fusion activity was restored by cleavage with exogenous chymotrypsin and showed the same low pH dependence as that of wild type. The cleavage sensitivity of newly synthesized p62 was investigated by pulse-chase analysis and chymotrypsin treatment in detergent solution. p62 was sensitive to cleavage immediately following its synthesis. Protein trapped in the rough endoplasmic reticulum or Golgi apparatus by carbonyl cyanide m-chlorophenylhydrazone, monensin, or Brefeldin A treatment was also fully sensitive to cleavage. These results suggest that p62 does not require an organelle-mediated conformational change for processing. Thus, in vivo, the site of p62 processing is probably controlled by the location or activity of the cleavage enzyme, rather than the sensitivity of the p62 substrate.     

In vivo generation and characterization of a soluble form of the Semliki forest virus fusion protein

During infection of host cells, a number of enveloped animal viruses are known to produce soluble forms of viral membrane glycoproteins lacking the transmembrane domain. The roles of such soluble glycoproteins in viral life cycles are incompletely understood, but in several cases they are believed to modulate host immune response and viral pathogenesis. Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells through low-pH-dependent fusion and buds from the plasma membrane. Fusion is mediated by the E1 subunit of the SFV spike protein. Previous studies described the in vivo generation of E1s, a truncated soluble form of E1, under conditions in which budding is inhibited in mammalian host cells. We have here examined the properties of E1s generation and the biological activity of E1s. E1s cleavage required spike protein transport out of the endoplasmic reticulum and was independent of virus infection. Cell surface E1 efficiently acted as a precursor for E1s. E1s generation was strongly pH dependent in BHK cells, with optimal cleavage at a pH of < or =7.0, conditions that inhibited the budding of SFV but not the budding of the rhabdovirus vesicular stomatitis virus. The pH dependence of E1s production and SFV budding was unaffected by the stability of the spike protein dimer but was a function of the host cell. Similar to the intact virus and in vitro-generated E1 ectodomain, treatment of E1s at low pH in the presence of target membranes triggered specific acid-dependent conformational changes. Thus, under a variety of conditions, SFV-infected cells can produce a soluble form of E1 that is biologically active.     

The nucleocapsid-binding spike subunit E2 of Semliki Forest virus requires complex formation with the E1 subunit for activity.

Alphaviruses, such as Semliki Forest virus (SFV), mature by budding at the plasma membrane (PM) of infected cells and enter uninfected ones by a membrane fusion process in the endosomes. Both processes are directed by the p62/E2-E1 membrane protein heterodimer of the virus. The p62 protein, or its mature form E2, provides a cytoplasmic protein domain for interaction with the nucleocapsid (NC) of the virus, and the E1 protein functions as a membrane fusogen. We have previously shown that the p62/E2 protein of SFV controls the membrane fusion activity of E1 through its complex formation with the latter (A. Salminen, J. M. Wahlberg, M. Lobigs, P. Liljeström, and H. Garoff, J. Cell Biol. 116:349-357, 1992). In the present work, we show that the E1 protein controls the NC-binding activity of p62/E2. We have studied E1 expression-deficient SFV variants and shown that although the p62/E2 proteins can be transported to the PM they cannot establish stable NC associations.     

Activation of the alphavirus spike protein is suppressed by bound E3

Alphaviruses are taken up into the endosome of the cell, where acidic conditions activate the spikes for membrane fusion. This involves dissociation of the three E2-E1 heterodimers of the spike and E1 interaction with the target membrane as a homotrimer. The biosynthesis of the heterodimer as a pH-resistant p62-E1 precursor appeared to solve the problem of premature activation in the late and acidic parts of the biosynthetic transport pathway in the cell. However, p62 cleavage into E2 and E3 by furin occurs before the spike has left the acidic compartments, accentuating the problem. In this work, we used a furin-resistant Semliki Forest virus (SFV) mutant, SFV(SQL), to study the role of E3 in spike activation. The cleavage was reconstituted with proteinase K in vitro using free virus or spikes on SFV(SQL)-infected cells. We found that E3 association with the spikes was pH dependent, requiring acidic conditions, and that the bound E3 suppressed spike activation. This was shown in an in vitro spike activation assay monitoring E1 trimer formation with liposomes and a fusion-from-within assay with infected cells. Furthermore, the wild type, SFV(wt), was found to bind significant amounts of E3, especially if produced in dense cultures, which lowered the pH of the culture medium. This E3 also suppressed spike activation. The results suggest that furin-cleaved E3 continues to protect the spike from premature activation in acidic compartments of the cell and that its release in the neutral extracellular space primes the spike for low-pH activation.     

Membrane fusion process of Semliki Forest virus. I: Low pH-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells

The Semliki Forest virus (SFV) directs the synthesis of a heterodimeric membrane protein complex which is used for virus membrane assembly during budding at the surface of the infected cell, as well as for low pH-induced membrane fusion in the endosomes when particles enter new host cells. Existing evidence suggests that the E1 protein subunit carries the fusion potential of the heterodimer, whereas the E2 subunit, or its intracellular precursor p62, is required for binding to the nucleocapsid. We show here that during virus uptake into acidic endosomes the original E2E1 heterodimer is destabilized and the E1 proteins form new oligomers, presumably homooligomers, with altered E1 structure. This altered structure of E1 is specifically recognized by a monoclonal antibody which can also inhibit penetration of SFV into host cells as well as SFV-mediated cell-cell fusion, thus suggesting that the altered E1 structure is important for the membrane fusion. These results give further support for a membrane protein oligomerization-mediated control mechanism for the membrane fusion potential in alphaviruses.     

Assembly of the semliki forest virus membrane glycoproteins in the membrane of the endoplasmic reticulum in vitro

A cell-free system has been constructed to study the mechanism by which a single messenger RNA directs the synthesis of proteins destined for two different cellular locations. The Semliki Forest virus (SFV) 26 S mRNA codes for the viral capsid protein (C protein) and the membrane proteins p62 and E1. The three virus proteins are read in this order from the messenger RNA using one initiation site. The C protein is left on the cytoplasmic side and the p62 and the El proteins are inserted into the endoplasmic reticulum membrane. Translation of 26 S mRNA in a HeLa cell-free system in the presence of microsomes from dog pancreas reproduced the segregation, and proteolytic processing and glycosylation observed in infected cells. The signal for membrane binding was in the amino-terminal end of p62. The results indicate that the membrane proteins become inserted in the nascent state. The cleavage between p62 and El was coupled to membrane insertion. If the membranes were added after a period corresponding to the synthesis of about 100 amino acids of the p62 protein, segregation, glycosylation and cleavage between p62 and E1 failed to occur.     

Reconstitution of transcytosis in SLO-permeabilized MDCK cells: existence of an NSF-dependent fusion mechanism with the apical surface of MDCK cells.

Recently, it was demonstrated that delivery from the trans-Golgi network (TGN) to the basolateral surface of Madin-Darby canine kidney (MDCK) cells required N-ethylmaleimide-sensitive factor (NSF)-alpha soluble NSF attachment protein (SNAP)-SNAP receptor (SNARE) complexes, while delivery from the TGN to the apical surface was independent of NSF-alpha SNAP-SNARE. To determine if all traffic to the apical surface of this cell line was NSF independent, we reconstituted the transcytosis of pre-internalized IgA to the apical surface and recycling to the basolateral surface. Transcytosis and the recycling of IgA required ATP and cytosol, and both were inhibited by treatment with N-ethylmaleimide. This inhibition was reversed by the addition of recombinant NSF. Botulinum neurotoxin serotype E, which is known to cleave the 25,000 Da synaptosomal associated protein, inhibited both transcytosis and recycling, although incompletely. We conclude that membrane traffic to a target membrane is not determined by utilizing a single molecular mechanism for fusion. Rather, a target membrane, e.g. the apical plasma membrane of MDCK cells, may use multiple molecular mechanisms to fuse with incoming vesicle.     

A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface

We have isolated a membrane fraction enriched in a class of transport carriers that form at the trans Golgi network (TGN) and are destined for the cell surface in HeLa cells. Protein kinase D (PKD) is required for the biogenesis of these carriers that contain myosin II, Rab6a, Rab8a, and synaptotagmin II, as well as a number of secretory and plasma membrane‐specific cargoes. Our findings reveal a requirement for myosin II in the migration of these transport carriers but not in their biogenesis per se. Based on the cargo secreted by these carriers we have named them CARTS for CAR riers of the T GN to the cell S urface. Surprisingly, CARTS are distinct from the carriers that transport vesicular stomatitis virus (VSV)‐G protein and collagen I from the TGN to the cell surface. Altogether, the identification of CARTS provides a valuable means to understand TGN to cell surface traffic.     

Mutants of the membrane-binding region of Semliki Forest virus E2 protein. I. Cell surface transport and fusogenic activity

Three mutations of the membrane-binding region of the Semliki Forest virus (SFV) p62 polypeptide (the precursor for virion E3 and E2) have been made by oligonucleotide-directed mutagenesis of a cDNA clone encoding the SFV structural proteins. One of the mutations (A2) substitutes a Glu for an Ala in the middle of the hydrophobic stretch which spans the bilayer. A1 and A3 alter the two basic charged amino acids in the cytoplasmic domain next to the hydrophobic region. The wild-type charge cluster of Arg-Ser-Lys (+2) has been changed to Gly-Ser-Met (0;A3) or to Gly-Ser-Glu (-1;A1). The mutant p62 proteins have been analyzed both in the presence and the absence of E1, the other half of the heterodimer spike complex of SFV. The mutant proteins expressed in COS-7 cells are glycosylated and are of the expected sizes. When co-expressed with E1, all three mutants are cleaved to yield the E2 protein and transported to the surface of COS-7 cells. When expressed in the absence of E1, the mutant p62 proteins remain uncleaved but still reach the cell surface. Once at the cell surface, all three mutants, when co-expressed with E1, can promote low pH-triggered cell-cell fusion. These results show that the three mutant p62/E2 proteins are still membrane associated in a functionally unaltered way.     

Interactions between the transmembrane segments of the alphavirus E1 and E2 proteins play a role in virus budding and fusion

The alphavirus envelope is built by heterodimers of the membrane proteins E1 and E2. The complex is formed as a p62E1 precursor in the endoplasmic reticulum. During transit to the plasma membrane (PM), it is cleaved into mature E1-E2 heterodimers, which are oligomerized into trimeric complexes, so-called spikes that bind both to each other and, at the PM, also to nucleocapsid (NC) structures under the membrane. These interactions drive the budding of new virus particles from the cell surface. The virus enters new cells by a low-pH-induced membrane fusion event where both inter- and intraheterodimer interactions are reorganized to establish a fusion-active membrane protein complex. There are no intact heterodimers left after fusion activation; instead, an E1 homotrimer remains in the cellular (or viral) membrane. We analyzed whether these transitions depend on interactions in the transmembrane (TM) region of the heterodimer. We observed a pattern of conserved glycines in the TM region of E1 and made two mutants where either the glycines only (SFV/E1(4L)) or the whole segment around the glycines (SFV/E1(11L)) was replaced by leucines. We found that both mutations decreased the stability of the heterodimer and increased the formation of the E1 homotrimer at a suboptimal fusion pH, while the fusion activity was decreased. This suggested that TM interactions play a role in virus assembly and entry and that anomalous or uncoordinated protein reorganizations take place in the mutants. In addition, the SFV/E1(11L) mutant was completely deficient in budding, which may reflect an inability to form multivalent NC interactions at the PM.     

A cytosolic complex of p62 and rab6 associates with TGN38/41 and is involved in budding of exocytic vesicles from the trans-Golgi network

TGN38/41, an integral membrane protein predominantly localized to the trans-Golgi network, has been shown to cycle to the plasma membrane and return to the TGN within 30 min. (Ladinsky, M. S., and K. E. Howell. 1992. Eur. J. Cell Biol. 59:92-105). In characterizing the proteins which associate with TGN38/41, a peripheral 62-kD protein, two forms of rab6 and two other small GTP-binding proteins were identified by coimmunoprecipitation. However, approximately 90% of the 62-kD protein is cytosolic and is associated with the same subset of small GTP- binding proteins. Both the membrane and cytoplasmic complexes were characterized by sizing column fractionation and velocity sedimentation. The membrane complex was approximately 250 kD (11.6 S) consisting of the cytosolic complex and a heterodimer of TGN38/41 (160 kD). The cytosolic complex was approximately 86 kD (6.1 S) consisting of p62 and one small GTP-binding protein. Preliminary evidence indicates that phosphorylation of the p62 molecule regulates the dissociation of the cytosolic complex from TGN38/41. Functionally the cytosolic p62 complex must bind to TGN38/41 for the budding of exocytic transport vesicles from the TGN as assayed in a cell-free system (Salamero, J., E. S. Sztul, and K. E. Howell. 1990. Proc. Natl. Acad. Sci. USA. 87:7717-7721). Interference with p62, rab6 or TGN38, and TGN41 cytoplasmic domains by immunodepletion or competing peptides completely inhibited the budding of exocytic transport vesicles. These results support an essential role for interaction of the cytosolic p62/rab6 complex with TGN38/41 in budding of exocytic vesicles from the TGN.     

In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release.

We report on the construction of a full-length cDNA clone of Semliki Forest virus (SFV). By placing the cDNA under the SP6 promoter, infectious RNA can be produced in vitro and used to transfect cells to initiate virus infection. To achieve efficient transfections, a new protocol for electroporation of RNA was developed. This method gave up to 500-fold improvement over the traditional DEAE-dextran transfection procedure. Since virtually 100% of the cells can be transfected by electroporation, this method is a useful tool for detailed biochemical studies of null mutations of SFV that abolish production of infections virus particles. We used the cDNA clone of SFV to study what effects a deletion of the 6,000-molecular-weight membrane protein (6K membrane protein) had on virus replication. The small 6K protein is part of the structural precursor molecule (C-p62-6K-E1) of the virus. Our results conclusively show that the 6K protein is not needed for the heterodimerization of the p62 and E1 spike membrane proteins in the endoplasmic reticulum, nor is it needed for their transport out to the cell surface. The absence of the 6K protein did, however, result in a dramatic reduction in virus release, suggesting that the protein exerts its function late in the assembly pathway, possibly during virus budding.     

Alphavirus assembly and entry: role of the cytoplasmic tail of the E1 spike subunit.

The alphavirus Semliki Forest virus (SFV) matures by budding at the cell surface. This process is driven by interactions of its membrane protein heterodimer E2-E1 and the nucleocapsid. The virus penetrates into new cells by an E1-mediated membrane fusion event. The E1 subunit has a short, strongly positively charged cytoplasmic tail peptide (Arg-Arg) which is very conserved among different alphavirus E1 proteins. In this work, we have used in vitro mutagenesis of a full-size cDNA clone of SFV to study the role of the tail peptide of the E1 subunit in virus budding and fusion processes in baby hamster kidney cell culture. Our results suggest that the E1 tail plays no major role in SFV multiplication in animal cell culture.     

Functional and physical interactions of the herpes simplex virus type 1 UL20 membrane protein with glycoprotein K

Herpes simplex virus type 1 glycoprotein K (gK) and the UL20 protein (UL20p) are coordinately transported to the trans-Golgi network (TGN) and cell surfaces and are required for cytoplasmic virion envelopment at the TGN. In addition, cell surface expression of gK and UL20p is required for virus-induced cell fusion. Previously, confocal microscopy colocalization and intracellular transport experiments strongly suggested direct protein-protein interactions between gK and UL20p. Direct protein-protein interactions between gK and UL20p were demonstrated through reciprocal coimmunoprecipitation experiments, as well as with glutathione S-transferase (GST) pull-down experiments. A fusion protein consisting of the amino-terminal 66 amino acids of UL20p fused in-frame with GST was expressed in Escherichia coli and purified via glutathione column chromatography. Precipitation of GST-UL20p from mixtures of GST-UL20p fusion protein with cellular extracts containing gK specifically coprecipitated gK but not other viral glycoproteins. The purified UL20p-GST fusion protein reacted with all gK-associated protein species. It was concluded that the amino terminus of UL20p, most likely, interacted with gK domain III, which is predicted to lie intracellularly. UL20p-gK domain-specific interactions must serve important functions in the coordinate transport of UL20p and gK to the TGN, because retention of UL20p in the endoplasmic reticulum (ER) via the addition of an ER retention signal at the carboxyl terminus of UL20p forced the ER retention of gK and drastically inhibited intracellular virion envelopment and virus-induced cell fusion.     

Cholesterol is required in the exit pathway of Semliki Forest virus

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction triggered by low pH. For fusion to occur cholesterol is required in the target membrane, as demonstrated both in in vitro fusion assays and in vivo for virus infection of a host cell. In this paper we examine the role of cholesterol in postfusion events in the SFV life cycle. Cholesterol-depleted insect cells were transfected with SFV RNA or infected at very high multiplicities to circumvent the fusion block caused by the absence of cholesterol. Under these conditions, the viral spike proteins were synthesized and transported to the site of p62 cleavage with normal kinetics. Surprisingly, the subsequent exit of virus particles was dramatically slowed compared to cholesterol-containing cells. The inhibition of virus production could be reversed by the addition of cholesterol to depleted cells. In contrast to results with SFV, no cholesterol requirement for virus exit was observed for the production of either the unrelated vesicular stomatitis virus or a cholesterol-independent SFV fusion mutant. Thus, cholesterol was only critical in the exit pathway of viruses that also require cholesterol for fusion. These results demonstrate a specific and unexpected lipid requirement in virus exit, and suggest that in addition to its role in fusion, cholesterol is involved in the assembly or budding of SFV.     

Fusion induced by a class II viral fusion protein, semliki forest virus E1, is dependent on the voltage of the target cell

Cells expressing the low pH-triggered class II viral fusion protein E1 of Semliki Forest virus (SFV) were fused to target cells. Fusion was monitored by electrical capacitance and aqueous dye measurements. Electrical voltage-clamp measurements showed that SFV E1-induced cell-cell fusion occurred quickly after acidification for a trans-negative potential across the target membrane (i.e., negative potential inside the target cell) but that a trans-positive potential eliminated all fusion. Use of an ionophore to control potentials for a large population of cells confirmed the dependence of fusion on voltage polarity. In contrast, fusion induced by the class I fusion proteins of human immunodeficiency virus, avian sarcoma leukosis virus, and influenza virus was independent of the voltage polarity across the target cell. Initial pore size and pore growth were also independent of voltage polarity for the class I proteins. An intermediate of SFV E1-induced fusion was created by transient acidification at low temperature. Membranes were hemifused at this intermediate state, and raising the temperature at neutral pH allowed full fusion to occur. Capacitance measurements showed that maintaining a trans-positive potential definitely blocked fusion at steps following the creation of the hemifusion intermediate and may have inhibited fusion at prior steps. It is proposed that the trans-negative voltage across the endosomal membrane facilitates fusion after low-pH-induced conformational changes of SFV E1 have occurred.     

The Spir actin organizers are involved in vesicle transport processes

The p150-Spir protein, which was discovered as a phosphorylation target of the Jun N-terminal kinase, is an essential regulator of the polarization of the Drosophila oocyte. Spir proteins are highly conserved between species and belong to the family of Wiskott-Aldrich homology region 2 (WH2) proteins involved in actin organization. The C-terminal region of Spir encodes a zinc finger structure highly homologous to FYVE motifs. A region with high homology between the Spir family proteins is located adjacent (N-terminal) to the modified FYVE domain and is designated as "Spir-box." The Spir-box has sequence similarity to a region of rabphilin-3A, which mediates interaction with the small GTPase Rab3A. Coexpression of p150-Spir and green fluorescent protein-tagged Rab GTPases in NIH 3T3 cells revealed that the Spir protein colocalized specifically with the Rab11 GTPase, which is localized at the trans-Golgi network (TGN), post-Golgi vesicles, and the recycling endosome. The distinct Spir localization pattern was dependent on the integrity of the modified FYVE finger motif and the Spir-box. Overexpression of a mouse Spir-1 dominant interfering mutant strongly inhibited the transport of the vesicular stomatitis virus G (VSV G) protein to the plasma membrane. The viral protein was arrested in membrane structures, largely colocalizing with the TGN marker TGN46. Our findings that the Spir actin organizer is targeted to intracellular membrane structures by its modified FYVE zinc finger and is involved in vesicle transport processes provide a novel link between actin organization and intracellular transport.