Vesicular stomatitis virus nucleocapsids diffuse through cytoplasm by hopping from trap to trap in random directions.

Within 2-6 hours after infection by vesicular stomatitis virus (VSV), newly assembled VSV particles are released from the surface of infected cells. In that time, viral ribonucleoprotein (RNP) particles (nucleocapsids) travel from their initial sites of synthesis near the nucleus to the edge of the cell, a distance of 5-10 µm. The hydrodynamic radius of RNP particles (86 nm) precludes simple diffusion through the mesh of cytoskeletal fibers. To reveal the relative importance of different transport mechanisms, movement of GFP-labeled RNP particles in live A549 cells was recorded within 3 to 4 h postinfection at 100 frames/s by fluorescence video microscopy. Analysis of more than 200 RNP particle tracks by Bayesian pattern recognition software found that 3% of particles showed rapid, directional motion at about 1 µm/s, as previously reported. 97% of the RNP particles jiggled within a small, approximately circular area with Gaussian width σ = 0.06 µm. Motion within such "traps" was not directional. Particles stayed in traps for approximately 1 s, then hopped to adjacent traps whose centers were displaced by approximately 0.17 µm. Because hopping occurred much more frequently than directional motion, overall transport of RNP particles was dominated by hopping over the time interval of these experiments.

3D rotating wall vessel and 2D cell culture of four veterinary virus pathogens: A comparison of virus yields, portions of infectious particles and virus growth curves.

Only very few comparative studies have been performed that evaluate general trends of virus growth under 3D in comparison with 2D cell culture conditions. The aim of this study was to investigate differences when four animal viruses are cultured in 2D and 3D. Suid herpesvirus 1 (SuHV-1), Vesicular stomatitis virus (VSIV), Bovine adenovirus (BAdV) and Bovine parainfluenza 3 virus (BPIV-3) were cultivated in 3D rotating wall vessels (RWVs) and conventional 2D cultures. The production of virus particles, the portion of infectious particles, and the infectious growth curves were compared. For all viruses, the production of virus particles (related to cell density), including the non-infectious ones, was lower in 3D than in 2D culture. The production of only infectious particles was significantly lower in BAdV and BPIV-3 in 3D cultures in relation to cell density. The two cultivation approaches resulted in significantly different virus particle-to-TCID50 ratios in three of the four viruses: lower in SuHV-1 and BPIV-3 and higher in BAdV in 3D culture. The infectious virus growth rates were not significantly different in all viruses. Although 3D RWV culture resulted in lower production of virus particles compared to 2D systems, the portion of infectious particles was higher for some viruses.

Asymmetric packaging of polymerases within vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) is a prototypic negative sense single-stranded RNA virus. The bullet-shape appearance of the virion results from tightly wound helical turns of the nucleoprotein encapsidated RNA template (N-RNA) around a central cavity. Transcription and replication require polymerase complexes, which include a catalytic subunit L and a template-binding subunit P. L and P are inferred to be in the cavity, however lacking direct observation, their exact position has remained unclear. Using super-resolution fluorescence imaging and atomic force microscopy (AFM) on single VSV virions, we show that L and P are packaged asymmetrically towards the blunt end of the virus. The number of L and P proteins varies between individual virions and they occupy 57 ± 12 nm of the 150 nm central cavity of the virus. Our finding positions the polymerases at the opposite end of the genome with respect to the only transcriptional promoter.

Self-organization of the vesicular stomatitis virus nucleocapsid into a bullet shape.

The typical bullet shape of Rhabdoviruses is thought to rely on the matrix protein for stabilizing the nucleocapsid coil. Here we scrutinize the morphology of purified and recombinant nucleocapsids of vesicular stomatitis virus in vitro. We elucidate pH and ionic strength conditions for their folding into conical tips and further growth into whole bullets, and provide cryo-electron microscopy reconstructions of the bullet tip and the helical trunk. We address conformational variability of the reconstituted nucleocapsids and the issue of constraints imposed by the binding of matrix protein. Our findings bridge the gap between the isolated nucleoprotein-RNA string in its form of an undulating ribbon, and the tight bullet-shaped virion skeleton.

The nucleocapsid of vesicular stomatitis virus.

The nucleocapsid of vesicular stomatitis virus serves as the genomic template for transcription and replication. The viral genomic RNA is sequestered in the nucleocapsid in every step of the virus replication cycle. The structure of the nucleocapsid and the entire virion revealed how the viral genomic RNA is encapsidated and packaged in the virus. A unique mechanism for viral RNA synthesis is derived from the structure of the nuleocapsid and its interactions with the viral RNA-dependent RNA polymerase.

Conserved eukaryotic fusogens can fuse viral envelopes to cells.

Caenorhabditis elegans proteins AFF-1 and EFF-1 [C. elegans fusion family (CeFF) proteins] are essential for developmental cell-to-cell fusion and can merge insect cells. To study the structure and function of AFF-1, we constructed vesicular stomatitis virus (VSV) displaying AFF-1 on the viral envelope, substituting the native fusogen VSV glycoprotein. Electron microscopy and tomography revealed that AFF-1 formed distinct supercomplexes resembling pentameric and hexameric "flowers" on pseudoviruses. Viruses carrying AFF-1 infected mammalian cells only when CeFFs were on the target cell surface. Furthermore, we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinella spiralis and the chordate Branchiostoma floridae could also fuse mammalian cells. Thus, FF proteins are part of an ancient family of cellular fusogens that can promote fusion when expressed on a viral particle.

Current good manufacturing practice production of an oncolytic recombinant vesicular stomatitis viral vector for cancer treatment.

Vesicular stomatitis virus (VSV) is an oncolytic virus currently being investigated as a promising tool to treat cancer because of its ability to selectively replicate in cancer cells. To enhance the oncolytic property of the nonpathologic laboratory strain of VSV, we generated a recombinant vector [rVSV(MΔ51)-M3] expressing murine gammaherpesvirus M3, a secreted viral chemokine-binding protein that binds to a broad range of mammalian chemokines with high affinity. As previously reported, when rVSV(MΔ51)-M3 was used in an orthotopic model of hepatocellular carcinoma (HCC) in rats, it suppressed inflammatory cell migration to the virus-infected tumor site, which allowed for enhanced intratumoral virus replication leading to increased tumor necrosis and substantially prolonged survival. These encouraging results led to the development of this vector for clinical translation in patients with HCC. However, a scalable current Good Manufacturing Practice (cGMP)-compliant manufacturing process has not been described for this vector. To produce the quantities of high-titer virus required for clinical trials, a process that is amenable to GMP manufacturing and scale-up was developed. We describe here a large-scale (50-liter) vector production process capable of achieving crude titers on the order of 10(9) plaque-forming units (PFU)/ml under cGMP. This process was used to generate a master virus seed stock and a clinical lot of the clinical trial agent under cGMP with an infectious viral titer of approximately 2 × 10(10) PFU/ml (total yield, 1 × 10(13) PFU). The lot has passed all U.S. Food and Drug Administration-mandated release testing and will be used in a phase 1 clinical translational trial in patients with advanced HCC.

Distinct structural rearrangements of the VSV glycoprotein drive membrane fusion.

The entry of enveloped viruses into cells requires the fusion of viral and cellular membranes, driven by conformational changes in viral glycoproteins. Many studies have shown that fusion involves the cooperative action of a large number of these glycoproteins, but the underlying mechanisms are unknown. We used electron microscopy and tomography to study the low pH-induced fusion reaction catalyzed by vesicular stomatitis virus glycoprotein (G). Pre- and post-fusion crystal structures were observed on virions at high and low pH, respectively. Individual fusion events with liposomes were also visualized. Fusion appears to be driven by two successive structural rearrangements of G at different sites on the virion. Fusion is initiated at the flat base of the particle. Glycoproteins located outside the contact zone between virions and liposomes then reorganize into regular arrays. We suggest that the formation of these arrays, which have been shown to be an intrinsic property of the G ectodomain, induces membrane constraints, achieving the fusion reaction.

Virion disruption by ozone-mediated reactive oxygen species.

It is well documented in the scientific literature that ozone-oxygen mixtures inactivate microorganisms including bacteria, fungi and viruses (Hoff, J.C., 1986. Inactivation of microbial agents by chemical disinfectants. EPA 600 S2-86 067. Office of Water, U.S. Environmental Protection Agency, Washington, DC; Khadre, M.A., Yousef, A.E., Kim, J.-G., 2001. Microbiological aspects of ozone applications in food: a review. J. Food Sci. 66, 1242-1252). In the current study, delivery and absorption of precisely known concentrations of ozone (in liquid media) were used to inactivate virus infectivity. An ozone-oxygen delivery system capable of monitoring and recording ozone concentrations in real time was used to inactivate a series of enveloped and non-enveloped viruses including herpes simplex virus type-1 (HHV-1, strain McIntyre), vesicular stomatitis Indiana virus (VSIV), vaccinia virus (VACV, strain Elstree), adenovirus type-2 (HAdV-2), and the PR8 strain of influenza A virus (FLUAVA/PR/8/34/H1N1; FLUAV). The results of the study showed that ozone exposure reduced viral infectivity by lipid peroxidation and subsequent lipid envelope and protein shell damage. These data suggest that a wide range of virus types can be inactivated in an environment of known ozone exposure.

Plasma membrane microdomains containing vesicular stomatitis virus M protein are separate from microdomains containing G protein and nucleocapsids.

Immunogold electron microscopy and analysis were used to determine the organization of the major structural proteins of vesicular stomatitis virus (VSV) during virus assembly. We determined that matrix protein (M protein) partitions into plasma membrane microdomains in VSV-infected cells as well as in transfected cells expressing M protein. The sizes of the M-protein-containing microdomains outside the virus budding sites (50 to 100 nm) were smaller than those at sites of virus budding (approximately 560 nm). Glycoprotein (G protein) and M protein microdomains were not colocalized in the plasma membrane outside the virus budding sites, nor was M protein colocalized with microdomains containing the host protein CD4, which efficiently forms pseudotypes with VSV envelopes. These results suggest that separate membrane microdomains containing either viral or host proteins cluster or merge to form virus budding sites. We also determined whether G protein or M protein was colocalized with VSV nucleocapsid protein (N protein) outside the budding sites. Viral nucleocapsids were observed to cluster in regions of the cytoplasm close to the plasma membrane. Membrane-associated N protein was colocalized with G protein in regions of plasma membrane of approximately 600 nm. In contrast to the case for G protein, M protein was not colocalized with these areas of nucleocapsid accumulation. These results suggest a new model of virus assembly in which an interaction of VSV nucleocapsids with G-protein-containing microdomains is a precursor to the formation of viral budding sites.

The 24-angstrom structure of respiratory syncytial virus nucleocapsid protein-RNA decameric rings.

Respiratory syncytial virus (RSV), a nonsegmented, negative-sense RNA-containing virus, is a common cause of lower respiratory tract disease. Expression of RSV nucleocapsid protein (N) in insect cells using the baculovirus expression system leads to the formation of N-RNA complexes that are morphologically indistinguishable from viral nucleocapsids. When imaged in an electron microscope, three distinct types of structures were observed: tightly wound short-pitch helices, highly extended helices, and rings. Negative stain images of N-RNA rings were used to calculate a three-dimensional reconstruction at 24 A resolution, revealing features similar to those observed in nucleocapsids from other viruses of the order Mononegavirales. The reconstructed N-RNA rings comprise 10 N monomers and have an external radius of 83 A and an internal radius of 40 A. Comparison of this structure with crystallographic data from rabies virus and vesicular stomatitis virus N-RNA rings reveals striking morphological similarities.

Species differences in transgene DNA uptake in hepatocytes after adenoviral transfer correlate with the size of endothelial fenestrae.

Sinusoidal fenestrae may restrict the transport of gene transfer vectors according to their size. Using Vitrobot technology and cryo-electron microscopy, we show that the diameter of human adenoviral serotype 5 vectors is 93 nm with protruding fibers of 30 nm. Thus, a diameter of fenestrae of 150 nm or more is likely to be sufficient for passage of vectors from the sinusoidal lumen to the space of Disse and subsequent uptake of vectors in hepatocytes. The average diameter of fenestrae in New Zealand White rabbits (103+/-1.3 nm) was 1.4-fold (P<0.0001) lower than in C57BL/6 mice (141+/-5.4 nm). The percentage of sinusoidal fenestrae with a diameter larger than 150 nm was 10-fold (P<0.01) lower in rabbits (3.2+/-0.24%) than in C57BL/6 mice (32+/-5%), and this resulted in 8.8-fold (P=0.01) lower transgene DNA levels in hepatocytes in rabbits after adenoviral transfer. Injection of N-acetylcysteine combined with transient liver ischemia preceding intraportal transfer in rabbits increased the percentage of sinusoidal fenestrae above 150 nm 2.0-fold (P<0.001) and increased transgene DNA levels in hepatocytes 6.6-fold (P<0.05). In conclusion, species differences in transgene DNA uptake in hepatocytes after adenoviral transfer correlate with the diameter of fenestrae.

Isolation and crystallization of a unique size category of recombinant Rabies virus Nucleoprotein-RNA rings.

In order to study the packaging of rabies virus RNA inside the viral nucleocapsid, rabies nucleoprotein was expressed in insect cells. In the cells, it binds to cellular RNA to form long, helical or short circular complexes, depending on the length of the bound RNA. The circular complexes contained from 9 up to 13 N-protomers per ring. Separation of the rings into defined size classes was impossible through regular column chromatographies or gradient centrifugation. The size classes could be separated by native polyacrylamide gel electrophoresis. A large-scale separation was achieved with a 4% native gel using a preparative electrophoresis apparatus. Crystallization trials were set up with N-RNA rings from three size classes and crystals were obtained in all cases. The best diffracting crystals, diffracting up to 6A, contained rings with 11 N-protomers plus an RNA molecule of 99 nucleotides. The diffraction limit was improved to 3.5A by air dehydration prior to flash freezing.

Compatibility of lyotropic liquid crystals with viruses and mammalian cells that support the replication of viruses.

We report a study that investigates the biocompatibility of materials that form lyotropic liquid crystals (LCs) with viruses and mammalian cells that support the replication of viruses. This study is focused on aqueous solutions of tetradecyldimethyl-amineoxide (C(14)AO) and decanol (D), or disodium cromoglycate (DSCG; C(23)H(14)O(11)Na(2)), which can form optically birefringent, liquid crystalline phases. The influence of these materials on the ability of vesicular stomatitis virus (VSV) to infect human epitheloid cervical carcinoma (HeLa) cells was examined by two approaches. First, VSV was dispersed in aqueous C(14)AO+ D or DSCG, and then HeLa cells were inoculated by contacting the cells with the aqueous C(14)AO + D or DSCG containing VSV. The infectivity of VSV to the HeLa cells was subsequently determined. Second, VSV was incubated in LC phases of either C(14)AO + D or DSCG for 4 h, and the concentration (titer) of infectious virus in the LC was determined by dilution into cell culture medium and subsequent inoculation of HeLa cells. Using these approaches, we found that the LC containing C(14)AO + D caused inactivation of virus as well as cell death. In contrast, we determined that VSV retained its infectivity in the presence of aqueous DSCG, and that greater than 74-82% of the HeLa cells survived contact with aqueous DSCG (depending on concentration of DSCG). Because VSV maintained its function (and we infer structure) in LCs formed from DSCG, we further explored the influence of the virus on the ordering of the LC. Whereas the LC formed from DSCG was uniformly aligned on surfaces prepared from self-assembled monolayers (SAMs) of HS(CH(2))(11)(OCH(2)CH(2))(4)OH on obliquely deposited films of gold in the absence of VSV, the introduction of 10(7)-10(8) infectious virus particles per milliliter caused the LC to assume a non-uniform orientation and a colorful appearance that was readily distinguished from the uniformly aligned LCs. Control experiments using cell lysates with equivalent protein concentrations but no virus did not perturb the uniform alignment of the LC.

Pseudotypes of vesicular stomatitis virus with CD4 formed by clustering of membrane microdomains during budding.

Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.

Propagation of viruses on micropatterned host cells.

We have developed a technique to characterize the in vitro propagation of viruses. Microcontact printing was used to generate linear arrays of alkanethiols on gold surfaces, which served as substrates for the patterned culture of baby hamster kidney (BHK-21) cells. Vesicular stomatitis virus (VSV) was added to unpatterned cell reservoirs adjacent to the patterned cells and incubated, setting in motion a continuously advancing viral infection into the patterned cells. At different incubation times, multiple arrays were chemically fixed to stop the viral propagation. Viral propagation distances into the patterned cells were determined by indirect immunofluorescent labeling and visualization of the VSV surface glycoprotein (G). The infection spread at approximately 50 microm/h in the 140-microm lines. Moreover, different temporal stages of the infection process were simultaneously visualized along individual lines. These stages included initiation of infection, based on G protein expression; cell-cell fusion, based on virus-induced clustering of cell nuclei; and cytoskeletal degradation, based on localized release of cells from the surface. This work sets a foundation for parallel, high-throughput characterization of viral and cellular processes.

Relative neurotropism of a recombinant rhabdovirus expressing a green fluorescent envelope glycoprotein.

A new recombinant vesicular stomatitis virus (rVSV) that expresses green fluorescent protein (GFP) on the cytoplasmic domain of the VSV glycoprotein (G protein) was used in the mouse as a model for studying brain infections by a member of the Mononegavirales order that can cause permanent changes in behavior. After nasal administration, virus moved down the olfactory nerve, first to periglomerular cells, then past the mitral cell layer to granule cells, and finally to the subventricular zone. Eight days postinoculation, rVSV was eliminated from the olfactory bulb. Little sign of infection could be found outside the olfactory system, suggesting that anterograde or retrograde axonal transport of rVSV was an unlikely mechanism for movement of rVSV out of the bulb. When administered intracerebrally by microinjection, rVSV spread rapidly within the brain, with strong infection at the site of injection and at some specific periventricular regions of the brain, including the dorsal raphe, locus coeruleus, and midline thalamus; the ventricular system may play a key role in rapid rVSV dispersion within the brain. Thus, the lack of VSV movement out of the olfactory system was not due to the absence of potential for infections in other brain regions. In cultures of both mouse and human central nervous system (CNS) cells, rVSV inoculations resulted in productive infection, expression of the G-GFP fusion protein in the dendritic and somatic plasma membrane, and death of all neurons and glia, as detected by ethidium homodimer nuclear staining. Although considered a neurotropic virus, rVSV also infected heart, skin, and kidney cells in dispersed cultures. rVSV showed a preference for immature neurons in vitro, as shown by enhanced viral infection in developing hippocampal cultures and in the outer granule cell layer in slices of developing cerebellum. Together, these data suggest a relative affinity of rVSV for some neuronal types in the CNS, adding to our understanding of the long-lasting changes in rodent behavior found after transient VSV infection.

Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives.

Inactivation of enveloped viruses (VSV, SFV, and SHV-1) by surfactin lipopeptides was dependent on the hydrophobicity, i.e. the number of carbon atoms of the fatty acid, and on the charge of the peptide moiety as well as on the virus species. Surfactins with fatty acid chains of 13 carbon atoms showed very low antiviral activity in comparison to C14 and C15 isoforms. C15 surfactin monomethyl ester also inactivated SFV which was resistant to the mixture of surfactin isoforms as produced by Bacillus subtilis. In contrast, the dimethyl ester showed no virus-inactivation capacity. Disintegration of viral structures as determined by electron microscopy after inactivation of VSV and SFV was comparable to the titer reduction. The effect of the surfactin isoforms and methyl esters on erythrocyte hemolysis correlated with the virus-inactivation capacity. Surfactins with a fatty acid chain moiety of 15 carbon atoms and one negative charge showed the highest antiviral activity.

Efficient transduction of mammalian cells by a recombinant baculovirus having the vesicular stomatitis virus G glycoprotein.

Baculovirus vectors recently have been shown to be capable of efficient transduction of human hepatoma cells and primary hepatocytes in culture. This paper describes the generation of a novel recombinant baculovirus (VGZ3) in which the vesicular stomatitis virus glycoprotein G (VSV G) is present in the viral envelope. The gene encoding VSV G was inserted into the baculovirus genome under the control of the polyhedrin promoter such that it was expressed at very high levels in infected insect cells but not in mammalian cells. Expression of the lacZ reporter gene was driven by a promoter that is functional in mammalian cells (the Rous sarcoma virus long terminal repeat). We show by Western analysis that VSV G protein was present in purified baculovirus preparations. A VSV G monoclonal antibody blocked transduction of mammalian cells by VGZ3. This virus was morphologically distinct from baculovirus lacking VSV G, with virions adopting an oval rather than rod-shaped morphology. VGZ3 transduced human hepatoma cells in vitro at an efficiency roughly 10-fold greater than baculovirus lacking VSV G (the virus Z4). VGZ3 was also capable of transducing cell lines that could not be transduced efficiently by Z4. We provide evidence that VSV G protein may enhance transduction by increasing the efficiency of escape of baculovirus from intracellular vesicles rather than by increasing cell binding or uptake of the virus. The possible use of this and related baculoviruses in gene therapy is discussed.