Herpes simplex virus dances with amyloid precursor protein while exiting the cell

Herpes simplex type 1 (HSV1) replicates in epithelial cells and secondarily enters local sensory neuronal processes, traveling retrograde to the neuronal nucleus to enter latency. Upon reawakening newly synthesized viral particles travel anterograde back to the epithelial cells of the lip, causing the recurrent cold sore. HSV1 co-purifies with amyloid precursor protein (APP), a cellular transmembrane glycoprotein and receptor for anterograde transport machinery that when proteolyzed produces A-beta, the major component of senile plaques. Here we focus on transport inside epithelial cells of newly synthesized virus during its transit to the cell surface. We hypothesize that HSV1 recruits cellular APP during transport. We explore this with quantitative immuno-fluorescence, immuno-gold electron-microscopy and live cell confocal imaging. After synchronous infection most nascent VP26-GFP-labeled viral particles in the cytoplasm co-localize with APP (72.8+/-6.7%) and travel together with APP inside living cells (81.1+/-28.9%). This interaction has functional consequences: HSV1 infection decreases the average velocity of APP particles (from 1.1+/-0.2 to 0.3+/-0.1 μm/s) and results in APP mal-distribution in infected cells, while interplay with APP-particles increases the frequency (from 10% to 81% motile) and velocity (from 0.3+/-0.1 to 0.4+/-0.1 μm/s) of VP26-GFP transport. In cells infected with HSV1 lacking the viral Fc receptor, gE, an envelope glycoprotein also involved in viral axonal transport, APP-capsid interactions are preserved while the distribution and dynamics of dual-label particles differ from wild-type by both immuno-fluorescence and live imaging. Knock-down of APP with siRNA eliminates APP staining, confirming specificity. Our results indicate that most intracellular HSV1 particles undergo frequent dynamic interplay with APP in a manner that facilitates viral transport and interferes with normal APP transport and distribution. Such dynamic interactions between APP and HSV1 suggest a mechanistic basis for the observed clinical relationship between HSV1 seropositivity and risk of Alzheimer's disease.     

A correlative approach at characterizing nanoparticle mobility and interactions after cellular uptake

The interactions of nanoparticles with human cells are of large interest in the context of nanomaterial safety. Here, we use live cell imaging and image‐based fluorescence correlation methods to determine colocalization of 88 nm and 32 nm silica nanoparticles with endocytotic vesicles derived from the cytoplasmic membrane and lysosomes, as well as to quantify intracellular mobility of internalized particles, in contrast to particle number quantification by counting techniques. In our study, A549 cells are used as a model for human type II alveolar epithelial cells. We present data supporting endocytotic uptake of the particles and subsequent active transport to the perinuclear region. The presence of particles in lamellar bodies is proposed as a potential exocytosis route. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

African swine fever virus interaction with microtubules

The role of microtubules in intracellular transport of African swine fever virus (ASFV) and virus-induced inclusions was studied by immunofluorescence using anti-ASFV and anti-tubulin antibodies, by electron microscopy of infected Vero cells and by in vitro binding of virions to purified microtubules. MTC, a reversible colchicine analogue, was used to depolymerize microtubules. In cells treated with MTC multiple large inclusions containing ASFV antigens and particles were observed in the cytoplasm. Removal of the drug lead to migration and fusion of the inclusions at a perinuclear location. To study the effect of microtubule repolymerization on virus particle distribution, the particles were counted in thin sections of MTC treated cells and at different times after removal of the drug. In cells treated with MTC 6.8% and 3.6% of the virus particles were found respectively in the cytoplasm and at the cell membrane while 38% of the particles were located around the virosome. With reversal of the drug effect the number of virus particles around the virosomes progressively decreased to 10% at 2 h while the number of particles in the cytoplasm and at the cell membrane increased. At 2 h after removal of the drug 33.5% of the particles were found budding from the cell membrane. Virus particles were found closely associated with microtubules in cytoskeletons obtained by Triton X-100 extraction of taxol treated cells. The association of virus particles with microtubules was also observed in vitro using purified microtubules and virus particles. The results show that microtubules are involved in the transport of African swine fever virus particles from the assembly site to the cell surface and in the movement and fusion of the virus inclusions.     

Movement of a karyophilic protein through the nuclear pores of oocytes

It has recently been shown that large karyophilic proteins are transported across the nuclear envelope in amphibian oocytes. In consideration of this, the present experiments were performed to identify the specific sites within the envelope through which transport occurs and determine if molecular size is a limiting factor in the transport process. The following experimental procedure was employed: Colloidal gold particles, varying in size from approximately 20 to 170 A in diameter were coated with nucleoplasmin, a 165,000-mol-wt karyophilic protein, which is known to be transported through the envelope. The coated gold particles were microinjected into the cytoplasm of Xenopus oocytes, and the cells were fixed 15 min and 1 h later. The intracellular localization of the gold was then determined with the electron microscope. It was found that nucleoplasmin-coated particles readily enter the nucleus. On the basis of the distribution of the particles associated with the envelope, we concluded that transport occurs through the nuclear pores. Furthermore, the size distributions of the gold particles present in the nucleus and cytoplasm were not significantly different, indicating that the envelope does not discriminate among particles with diameters ranging from 50 to 200 A (the dimensions including the nucleoplasmin coat). Colloidal gold coated with trypsin-digested nucleoplasmin (which lacks the polypeptide domain required for transport) or exogenous polyvinylpyrrolidone were largely excluded from the nucleus and showed no evidence of transport.     

Intracellular pathways of native iron in the maternal part of the porcine placenta

In the diffuse epitheliochorial porcine placenta iron is secreted as uteroferrin by the maternal epithelium of the areola-gland subunit of the placenta. To elucidate the intracellular pathways of physiological iron in uterine gland epithelium material from 10 sows at 15 to 111 days of gestation was processed for electron microscopy by different routine methods with or without postfixation in osmium tetroxide. Ferritin particles were identified by their size and shape and the content of iron was confirmed by X-ray energy dispersive microanalysis of accumulated ferritin particles. Distinct ferritin particles were not observed in the extracellular space either basal to or luminal to the epithelial cells. Intracellular ferritin was observed apparently free in the cytoplasm, but in variable amounts. Transfer tubules and dense bodies were located basally in the secretory cells. Both of these organelles contained ferritin particles, showed reaction sites for acid phosphatase and were stained by periodic acid-thiocarbohydrazide-silver proteinate. The ciliated cells differed by having apically located dense bodies containing numerous ferritin particles. Our finding of native ferritin in cells with hormonally regulated iron transport supports the concept that transfer tubules as part of the lysosomal complex are part of the endocytic pathway in secretory cells and indicate that ferritin here is an intracellular transport or storage intermediate.     

Defining the Subcellular Interface of Nanoparticles by Live-Cell Imaging

Understanding of nanoparticle-bio-interactions within living cells requires knowledge about the dynamic behavior of nanomaterials during their cellular uptake, intracellular traffic and mutual reactions with cell organelles. Here, we introduce a protocol of combined kinetic imaging techniques that enables investigation of exemplary fluorochrome-labelled nanoparticles concerning their intracellular fate. By time-lapse confocal microscopy we observe fast, dynamin-dependent uptake of polystyrene and silica nanoparticles via the cell membrane within seconds. Fluorescence recovery after photobleaching (FRAP) experiments reveal fast and complete exchange of the investigated nanoparticles at mitochondria, cytoplasmic vesicles or the nuclear envelope. Nuclear translocation is observed within minutes by free diffusion and active transport. Fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS) indicate diffusion coefficients of polystyrene and silica nanoparticles in the nucleus and the cytoplasm that are consistent with particle motion in living cells based on diffusion. Determination of the apparent hydrodynamic radii by FCS and RICS shows that nanoparticles exert their cytoplasmic and nuclear effects mainly as mobile, monodisperse entities. Thus, a complete toolkit of fluorescence fluctuation microscopy is presented for the investigation of nanomaterial biophysics in subcellular microenvironments that contributes to develop a framework of intracellular nanoparticle delivery routes.     

Colloidal gold labeling of intracellular ligands in dorsal root sensory neurons, visualized by scanning electron microscopy

We report a novel technique that combines high-resolution scanning electron microscopy (SEM) of intracellular structures with backscattered electron imaging (BEI) of colloidal gold-labeled intracellular ligands. Murine dorsal root ganglia were immersion-fixed, freeze-cleaved, labeled with gold complexes, and critical point-dried. Specimens were carbon-coated and viewed by BEI. They were then minimally sputter-coated with gold and previously identified cells relocated by secondary electron imaging (SEI). This permitted increased resolution of intracellular detail while gold particles remained detectable by BEI. Incubation with RNAse-gold and DNAse-gold complexes resulted in specific labeling of cytoplasm and nucleus, respectively. Immunolabeling of neurofilament (NF) and small nuclear ribonucleoproteins (snRNP) resulted in selective labeling of intracellular antigens. Nonspecific binding was abolished by use of 1% skin milk. Specifically, incubation with monoclonal anti-NF68 resulted in labeling of cytoplasm in 66% of neurons, notably of the large cells known to contain large amounts of NF. Satellite cells, which lack NF, showed low levels of background label. Human autoimmune anti-Sm serum recognizes snRNP particles, with the exception of the nucleolar U3 snRNP. Labeling with this serum resulted in specific labeling of 92% of nuclei, with only background labeling over nucleoli and cytoplasm. The results show that it is feasible to employ high-resolution SEM in conjunction with colloidal gold labeling to localize intracellular ligands in situ.     

Studies of nucleoside transporters using novel autofluorescent nucleoside probes

To better understand nucleoside transport processes and intracellular fates of nucleosides, we have developed a pair of fluorescent nucleoside analogues, FuPmR and dFuPmR, that differ only in the sugar moiety (ribofuranosyl versus 2'-deoxy, respectively), for real-time analysis of nucleoside transport into living cells by confocal microscopy. The binding and transportability of the two compounds were assessed for five recombinant human nucleoside transporters (hENT1/2, hCNT1/2/3) produced in Saccharomyces cerevisiae and/or oocytes of Xenopus laevis. The ribosyl derivative (FuPmR) was used to demonstrate proof of principle in live cell imaging studies in 11 cultured human cancer cell lines with different hENT1 activities. The autofluorescence emitted from FuPmR enabled direct visualization of its movement from the extracellular medium into the intracellular compartment of live cells, and this process was blocked by inhibitors of hENT1 (nitrobenzylmercaptopurine ribonucleoside, dipyridamole, and dilazep). Quantitative analysis of fluorescence signals revealed two stages of FuPmR uptake: a fast first stage that represented the initial uptake rate (i.e., transport rate) followed by a slow long-lasting second stage. The accumulation of FuPmR and/or its metabolites in nuclei and mitochondria was also visualized by live cell imaging. Measurements of fluorescence intensity increases in nuclei and mitochondria revealed rate-limited processes of permeant translocation across intracellular membranes, demonstrating for the first time the intracellular distribution of nucleosides and/or nucleoside metabolites in living cells. The use of autofluorescent nucleosides in time-lapse confocal microscopy is a novel strategy to quantitatively study membrane transport of nucleosides and their metabolites that will provide new knowledge of nucleoside biology.     

Simultaneous transport of different localized mRNA species revealed by live-cell imaging

Intracellular mRNA localization is a common mechanism to achieve asymmetric distributions of proteins. Previous studies have revealed that in a number of cell types, different mRNA species are localized by the same transport machinery. However, it has been unclear if these individual mRNA species are specifically sorted into separate or common ribonucleoprotein (RNP) particles before or during transport. Using budding yeast as a model system, we analyzed the intracellular movement of individual pairs of localized mRNA in live cells. Yeast cells localize more than 20 different mRNAs to the bud with the help of the Myo4p/She3p/She2p protein complex. For live cell imaging, mRNA pairs were tagged with tandem repeats of either bacteriophage MS2 or lambda boxB RNA sequences and fluorescently labeled by fusion protein constructs that bind to the RNA tag sequences. Using three-dimensional, single-particle tracking with dual-color detection, we have tracked the transport of two different localized mRNA species in real time. Our observations show that different localized mRNAs are coassembled into common RNP particles and cotransported in a directional manner to the target site. Nonlocalized mRNAs or mutant mRNAs that lack functional localization signals form separate particles that are not transported to the bud. This study reveals a high degree of co-ordination of mRNA trafficking in budding yeast.     

Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling

New surface-modified iron oxide nanoparticles were developed by precipitation of Fe(II) and Fe(III) salts with ammonium hydroxide and oxidation of the resulting magnetite with sodium hypochlorite, followed by the addition of poly( L-lysine) (PLL) solution. PLL of several molecular weights ranging from 146 ( L-lysine) to 579 000 was tested as a coating to boost the intracellular uptake of the nanoparticles. The nanoparticles were characterized by TEM, dynamic light scattering, FTIR, and ultrasonic spectrometry. TEM revealed that the particles were ca. 6 nm in diameter, while FTIR showed that their surfaces were well-coated with PLL. The interaction of PLL-modified iron oxide nanoparticles with DMEM culture medium was verified by UV-vis spectroscopy. Rat bone marrow stromal cells (rMSCs) and human mesenchymal stem cells (hMSC) were labeled with PLL-modified iron oxide nanoparticles or with Endorem (control). Optical microscopy and TEM confirmed the presence of PLL-modified iron oxide nanoparticles inside the cells. Cellular uptake was very high (more than 92%) for PLL-modified nanoparticles that were coated with PLL (molecular weight 388 00) at a concentration of 0.02 mg PLL per milliliter of colloid. The cellular uptake of PLL-modified iron oxide was facilitated by its interaction with the negatively charged cell surface and subsequent endosomolytic uptake. The relaxivity of rMSCs labeled with PLL-modified iron oxide and the amount of iron in the cells were determined. PLL-modified iron oxide-labeled rMSCs were imaged in vitro and in vivo after intracerebral grafting into the contralateral hemisphere of the adult rat brain. The implanted cells were visible on magnetic resonance (MR) images as a hypointense area at the injection site and in the lesion. In comparison with Endorem, nanoparticles modified with PLL of an optimum molecular weight demonstrated a higher efficiency of intracellular uptake by MSC cells.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: II. Inhibition of secretion of thyroglobulin in rat thyroid follicular cells as visualized by radioautography after 3H-fucose injection

Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for radioautography. In thyroid follicular cells of control animals, at this time interval, 57% of the total label was associated with colloid and secretory vesicles in the apical cytoplasm while 27% was localized in the Golgi apparatus and neighboring vesicles. In experimental animals, the proportion of label in colloid and apical vesicles was reduced by more than 69% after colchicine and more than 83% after vinblastine treatment. The proportion of label in the Golgi region, on the other hand, increased by more than 125% after colchicine and more than 179% after vinblastine treatment. Within the Golgi region, the great majority of the label was associated with secretory vesicles which accumulated adjacent to the trans face of the Golgi stacks. It is concluded that the drugs do not interfere with passage of newly synthesized thyroglobulin from the Golgi saccules to nearby secretory vesicles, but do inhibit intracellular migration of these vesicles to the cell apex. In most cells the number of vesicles in the apical cytoplasm diminished, but this was not always the case, suggesting that exocytosis may also be partially inhibited. The loss of microtubules in drug-treated cells suggests that the microtubules may be necessary for intracellular transport of thyroglobulin.     

An Intermittent Model for Intracellular Motions of Gold Nanostars by k-Space Scattering Image Correlation

Anisotropic metallic nanoparticles have been devised as powerful potential tools for in vivo imaging, photothermal therapy, and drug delivery thanks to plasmon-enhanced absorption and scattering cross sections, ease in synthesis and functionalization, and controlled cytotoxicity. The rational design of all these applications requires the characterization of the nanoparticles intracellular trafficking pathways. In this work, we exploit live-cell time-lapse confocal reflectance microscopy and image correlation in both direct and reciprocal space to investigate the intracellular transport of branched gold nanostars (GNSs). Different transport mechanisms, spanning from pure Brownian diffusion to (sub-)ballistic superdiffusion, are revealed by temporal and spatio-temporal image correlation spectroscopy on the tens-of-seconds timescale. According to these findings, combined with numerical simulations and with a Bayesian (hidden Markov model-based) analysis of single particle tracking data, we ascribe the superdiffusive, subballistic behavior characterizing the GNSs dynamics to a two-state switching between Brownian diffusion in the cytoplasm and molecular motor-mediated active transport. For the investigation of intermittent-type transport phenomena, we derive an analytical theoretical framework for Fourier-space image correlation spectroscopy (kICS). At first, we evaluate the influence of all the dynamic and kinetic parameters (the diffusion coefficient, the drift velocity, and the transition rates between the diffusive and the active transport regimes) on simulated kICS correlation functions. Then we outline a protocol for data analysis and employ it to derive whole-cell maps for each parameter underlying the GNSs intracellular dynamics. Capable of identifying even simpler transport phenomena, whether purely diffusive or ballistic, our intermittent kICS approach allows an exhaustive investigation of the dynamics of GNSs and biological macromolecules.     

Quantum dots for tracking cellular transport of lectin-functionalized nanoparticles

Successful drug delivery by functionalized nanocarriers largely depends on their efficient intracellular transport which has not yet been fully understood. We developed a new tracking technique by encapsulating quantum dots into the core of wheat germ agglutinin-conjugated nanoparticles (WGA-NP) to track cellular transport of functionalized nanocarriers. The resulting nanoparticles showed no changes in particle size, zeta potential or biobinding activity, and the loaded probe presented excellent photostability and tracking ability. Taking advantage of these properties, cellular transport profiles of WGA-NP in Caco-2 cells was demonstrated. The cellular uptake begins with binding of WGA to its receptor at the cell surface. The subsequent endocytosis happened in a cytoskeleton-dependent manner and by means of clathrin and caveolae-mediated mechanisms. After endosome creating, transport occurs to both trans-Golgi and lysosome. Our study provides new evidences for quantum dots as a cellular tracking probe of nanocarriers and helps understand intracellular transport profile of lectin-functionalized nanoparticles.     

Messenger ribonucleoproteins (informosomes) and RNA-binding proteins

Messenger ribonucleoproteins, first discovered in 1964 in our laboratory as free mRNA-containing particles of fish embryo cytoplasm and designated as informosomes, proved to have a universal occurrence in eukaryotic cells. Messenger ribonucleoproteins of different intracellular localization such as free cytoplasmic non-translatable informosomes, translatable messenger ribonucleoproteins in polyribosomes and nuclear pre-mRNA-containing particles are characterized by a number of features common for all of them. However, the transport from the nucleus into the cytoplasm as well as the transition from the free non-translatable state into the polyribosome-bound translatable state are accompanied by essential changes in the protein moiety of the particles. The existance of free RNA-binding proteins in eukaryotic cells has also been shown. These proteins seem to represent a pool for the formation of messenger ribonucleoproteins (informosomes).It has recently been demonstrated that the eukaryotic translation factors and, in particular, both the elongation factors and some initiation factors are among the cytoplasmic RNA-binding proteis. It is suggested that the mRNA in eukaryotic cells at different stages of its life time carries on itself the proteins which are required for its own biogenesis, processing and transport (nuclear informosomes), for its existence in a temporarily inactive state (free cytoplasmic informosomes) and for its functioning as a template (polyribosomal informosomes):omnia mea mecum porto.     

Toxicity and interaction of titanium dioxide nanoparticles with microtubule protein

Titanium dioxide (TiO2) nanoparticles (NPs) are widely used in several manufactured products. The small size of NPs facilitates their uptake into cells as well as transcytosis across epithelial cells into blood and lymph circulation to reach different sites, such as the central nervous system. Different studies have shown the risks that TiO2 NPs in the neuronal system and other organs present. As membrane-bound layer aggregates or single particles, TiO2 NPs can enter not only cells, but also mitochondria and nuclei. Therefore these particles can interact with cytoplasmic proteins such as microtubules (MTs). MTs are cytoskeletal proteins that are essential in eukaryotic cells for a variety of functions, such as cellular transport, cell motility and mitosis. MTs in neurons are used to transport substances such as neurotransmitters. Single TiO2 NPs in cytoplasm can interact with these proteins and affect their crucial functions in different tissues. In this study, we showed the effects of TiO2 NPs on MT polymerization and structure using ultraviolet spectrophotometer and fluorometry. The fluorescent spectroscopy showed a significant tubulin conformational change in the presence of TiO2 NPs and the ultraviolet spectroscopy results showed that TiO2 NPs affect tubulin polymerization and decrease it. The aim of this study was to find the potential risks that TiO2 NPs pose to human organs and cells.     

Cytoplasmic trafficking of the canine parvovirus capsid and its role in infection and nuclear transport

To begin a successful infection, viruses must first cross the host cell plasma membrane, either by direct fusion with the membrane or by receptor-mediated endocytosis. After release into the cytoplasm those viruses that replicate in the nucleus must target their genome to that location. We examined the role of cytoplasmic transport of the canine parvovirus (CPV) capsid in productive infection by microinjecting two antibodies that recognize the intact CPV capsid into the cytoplasm of cells and also by using intracellular expression of variable domains of a neutralizing antibody fused to green fluorescence protein. The two antibodies tested and the expressed scFv all efficiently blocked virus infection, probably by binding to virus particles while they were in the cytoplasm and before entering the nucleus. The injected antibodies were able to block most infections even when injected 8 h after virus inoculation. In control studies, microinjected capsid antibodies did not interfere with CPV replication when they were coinjected with an infectious plasmid clone of CPV. Cytoplasmically injected full and empty capsids were able to move through the cytosol towards the nuclear membrane in a process that could be blocked by nocodazole treatment of the cells. Nuclear transport of the capsids was slow, with significant amounts being found in the nucleus only 3 to 6 h after injection.     

Deformability and adhesive force of artificial platelets measured by atomic force microscopy

Platelet glycoprotein GPIaIIa is an adhesive protein that recognizes collagen. We have investigated polymerized albumin particles conjugated with recombinant GPIaIIa (rGPIaIIa-poly Alb) for their platelet-like function. To evaluate the feasibility of these particles to achieve the hemostatic process, we measured the deformability (Young’s modulus and spring constant) and the adhesive force of the particles using atomic force microscopy, which can measure the mechanical properties of individual cells. Our results showed that the Young’s modulus of these particles was 2.3-fold larger than that of natural platelets and 12-fold larger than that of human red blood cells. The Young’s modulus of the particles may have been determined by the properties of the polymerized albumin particle, although the glycoprotein of the platelet surface also contributed to the higher modulus. Our results also showed that the adhesive force of the rGPIaIIa-poly Alb with the collagen ligand was 52% of that of natural platelets. These two mechanical properties (deformability and adhesive force) of cells or particles, such as rGPIaIIa-poly Alb, are important specifications for the optimum design of platelet substitutes.     

Evidence that activation of platelet calpain is induced as a consequence of binding of adhesive ligand to the integrin, glycoprotein IIb-IIIa

Calpain (a Ca(2+)-dependent protease) is present in many cell types. Because it is present in the cytosol, the potential exists that it may regulate critical intracellular events by inducing crucial proteolytic cleavages. However, the concentrations of Ca2+ required to activate calpain are higher than those attained in the cytoplasm of most cells. Thus, the physiological importance of calpain and the mechanisms involved in its activation have remained elusive. In this study, we show that calpain rapidly moved to a peripheral location upon the addition of an agonist to suspensions of platelets, but it remained unactivated. We provide three lines of evidence that calpain was subsequently activated by a mechanism that required the binding of an adhesive ligand to the major platelet integrin, glycoprotein (GP) IIb- IIIa: calpain activation was prevented by RGDS, a tetrapeptide that inhibits the binding of adhesive ligand to GP IIb-IIIa; it was also prevented by monoclonal antibodies that inhibit adhesive ligand binding to GP IIb-IIIa; and its activation was markedly reduced in platelets from patients whose platelets have greatly reduced levels of functional GP IIb-IIIa. Thus, in platelets, binding of the extracellular domain of GP IIb-IIIa to its adhesive ligand can initiate a transmembrane signal that activates intracellular calpain. Because calpain is present in focal contacts of adherent cells, the interaction of integrins with adhesive ligands in the extracellular matrix may regulate activation of calpain in other cell types as well.     

Transport and assembly of gag proteins into Moloney murine leukemia virus.

We have studied the process of Moloney murine leukemia virus (M-MuLV) assembly by characterization of core (gag) protein mutants and analysis of wild-type (wt) gag proteins produced by cells in the presence of the ionophore monensin. Our genetic studies involved examination of linker insertion mutants of a Gag-beta-galactosidase (Gag-beta-gal) fusion protein, GBG2051, which is incorporated into virus particles when expressed in the presence of wt viral proteins. Analysis indicated that the amino-terminal two-thirds of the gag matrix domain is essential for targeting of proteins to the plasma membrane; mutant proteins localized to the cytoplasm or were trapped on intracellular membranes. Mutations through most of the coding region of the gag capsid domain generated proteins which were released from cells in membrane vesicles but not in virions. In contrast, linker insertions into p12gag or carboxy-terminal portions of the matrix or capsid coding regions did not affect assembly of fusion proteins into virus particles. Monensin, which blocks vesicular transport, inhibited gag protein intracellular transport and release from cells. Our results suggest that a significant proportion of M-MuLV myristylated gag proteins travel via vesicles to the cell surface. Specific matrix protein polypeptide regions and myristic acid modification are both necessary for appropriate gag protein transport, while capsid protein interactions appear to mediate the final phase of virion formation.