Genetic and cell biological characterization of the vaccinia virus A30 and G7 phosphoproteins

The vaccinia virus proteins A30 and G7 are known to play essential roles in early morphogenesis, acting prior to the formation of immature virions. Their repression or inactivation results in the accumulation of large virosomes, detached membrane crescents, and empty immature virions. We have undertaken further study of these proteins to place them within the context of the F10 kinase, the A14 membrane protein, and the H5 phosphoprotein, which have been the focus of previous studies within our laboratory. Here we confirm that both A30 and G7 undergo F10 kinase-dependent phosphorylation in vivo and recapitulate that modification of A30 in vitro. Although the detached crescents observed upon loss of A30 or G7 echo those seen upon repression of A14, no interaction between A30/G7 and A14 could be detected. We did, however, determine that the A30 and G7 proteins are unstable during nonpermissive tsH5 infections, suggesting that the loss of A30/G7 is the underlying cause for the formation of lacy or curdled virosomes. We also determined that the temperature-sensitive phenotype of the Cts11 virus is due to mutations in two codons of the G7L gene. Phenotypic analysis of nonpermissive Cts11 infections indicated that these amino acid substitutions compromise G7 function without impairing the stability of either G7 or A30. Utilizing Cts11 in conjunction with a rifampin release assay, we determined that G7 acts at multiple stages of virion morphogenesis that can be distinguished both by ultrastructural analysis and by monitoring the phosphorylation status of several viral proteins that undergo F10-mediated phosphorylation.     

Fusion of reconstituted influenza virus envelopes with liposomes mediated by streptavidin/biotin interactions

Reconstituted influenza virus envelopes (virosomes) containing the viral hemagglutinin (HA) represent an efficient fusogenic cellular delivery system. By interaction of HA with its natural receptors, sialylated lipids (gangliosides) or proteins, virosomes bind to cells and, following endocytic uptake, deliver their contents to the cytosol through fusion from within acidic endosomes. Here, we show that binding to sialic acid is not necessary for fusion. In the presence of streptavidin, virosomes containing a biotinylated lipid fused with liposomes lacking sialic acid if these liposomes also had a biotinylated lipid in their membranes. Moreover, fusion characteristics corresponded well with fusion of virosomes with ganglioside-containing liposomes.     

Selection for Neutralization Resistance of the Simian/Human Immunodeficiency Virus SHIVSF33A Variant In Vivo by Virtue of Sequence Changes in the Extracellular Envelope Glycoprotein That Modify N-Linked Glycosylation

We previously reported on the in vivo adaptation of an infectious molecular simian/human immunodeficiency virus (SHIV) clone, SHIVSF33, into a pathogenic biologic viral variant, designated SHIVSF33A. In the present study, we show that SHIVSF33A is resistant to neutralization by human immunodeficiency virus (HIV) and SHIV antisera. Multiple amino acid substitutions accumulated over time throughout the env gene of SHIVSF33A; some of them coincided with the acquisition of the neutralization resistance of the virus. Of interest are changes that resulted in the removal, repositioning, and addition of potential glycosylation sites within the V1, V2, and V3 regions of envelope gp120. To determine whether potential glycosylation changes within these principal neutralization domains of HIV type 1 formed the basis for the resistance to serum neutralization of SHIVSF33A, mutant viruses were generated on the backbone of parental SHIVSF33 and tested for their neutralization sensitivity. The mutations generated did not alter the in vitro replication kinetics or cytopathicity of the mutant viruses in T-cell lines. However, the removal of a potential glycosylation site in the V1 domain or the creation of such a site in the V3 domain did allow the virus to escape serum neutralization antibodies that recognized parental SHIVSF33. The combination of the V1 and V3 mutations conferred an additive effect on neutralization resistance over that of the single mutations. Taken together, these data suggest that (i) SHIV variants with changes in the Env SU can be selected in vivo primarily by virtue of their ability to escape neutralizing antibody recognition and (ii) carbohydrates play an important role in conferring neutralization escape, possibly by altering the structure of envelope gp120 or by shielding principal neutralization sites.     

Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly

Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drug-resistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase IIbeta. One can prevent the interaction by introducing a C(11)-to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase IIalpha/beta in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase IIalpha and IIbeta antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases IIalpha/beta in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.     

Reassessing the role of region A in Pit1-mediated viral entry

The mammalian gammaretroviruses gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B) can use the same receptor, Pit1, to infect human cells. A highly polymorphic nine-residue sequence within Pit1, designated region A, has been proposed as the virus binding site, because mutations in this region abolish Pit1-mediated cellular infection by GALV and FeLV-B. However, a direct correlation between region A mutations deleterious for infection and loss of virus binding has not been established. We report that cells expressing a Pit1 protein harboring mutations in region A that abolish receptor function retain the ability to bind virus, indicating that Pit1 region A is not the virus binding site. Furthermore, we have now identified a second region in Pit1, comprising residues 232 to 260 (region B), that is required for both viral entry and virus binding. Epitope-tagged Pit1 proteins were used to demonstrate that mutations in region B result in improper orientation of Pit1 in the cell membrane. Compensatory mutations in region A can restore proper orientation and full receptor function to these region B mutants. Based on these results, we propose that region A of Pit1 confers competence for viral entry by influencing the topology of the authentic binding site in the membrane and hence its accessibility to a viral envelope protein. Based on glycosylation studies and results obtained by using N- and C-terminal epitope-tagged Pit1, region A and region B mutants, and the transmembrane helices predicted with the PHD PredictProtein algorithm, we propose a new Pit1 topology model.     

Determination of the multimerization state of the hepatitis delta virus antigens in vivo

Hepatitis delta virus expresses two essential proteins, the small and large delta antigens, and both are required for viral propagation. Proper function of each protein depends on the presence of a common amino-terminal multimerization domain. A crystal structure, solved using a peptide fragment that contained residues 12 to 60, depicts the formation of an octameric ring composed of antiparallel coiled-coil dimers. Because this crystal structure was solved for only a fragment of the delta antigens, it is unknown whether octamers actually form in vivo at physiological protein concentrations and in the context of either intact delta antigen. To test the relevance of the octameric structure, we developed a new method to probe coiled-coil structures in vivo. We generated a panel of mutants containing cysteine substitutions at strategic locations within the predicted monomer-monomer interface and the dimer-dimer interface. Since the small delta antigen contains no cysteine residues, treatment of cell extracts with a mild oxidizing reagent was expected to induce disulfide bond formation only when the appropriate pairs of cysteine substitution mutants were coexpressed. We indeed found that, in vivo, both the small and large delta antigens assembled as antiparallel coiled-coil dimers. Likewise, we found that both proteins could assume an octameric quaternary structure in vivo. Finally, during the course of these experiments, we found that unprenylated large delta antigen molecules could be disulfide cross-linked via the sole cysteine residue located within the carboxy terminus. Therefore, in vivo, the C terminus likely provides an additional site of protein-protein interaction for the large delta antigen.     

Introduction of cationic virosome derived from vesicular stomatitis virus as a novel gene delivery system for sf9 cells

Insect-derived cell lines are used extensively to produce recombinant proteins because they are capable of performing a range of post-translational modifications. Due to their significance in biotechnological applications, various methods have been developed to transfect them. In this study, we introduce a virosome constructed from vesicular stomatitis virus (VSV) as a new delivery system for sf9 cells. We labeled these VSV virosomes by fluorescent probe Rhodamine B chloride (R18). By fluorescence microscope observation and conducting a fusion assay, we confirmed the uptake of VSV virosomes via endocytosis by sf9 cells and their fusion with the endosomal membrane. Moreover, we incubated cationic VSV virosomes with a GFP-expressing bacmid and transfected sf9 cells, after 24?h some cells expressed GFP indicating the ability of VSV virosomes to deliver heterologous DNA to these cells. This is the first report of a virosome-based delivery system introduced for an insect cell line.     

Virosomes in vaccine development: induction of cytotoxic T lymphocyte activity with virosome-encapsulated protein antigens

Virosomes are reconstituted viral envelopes which lack the genetic material but retain the cell entry and membrane fusion characteristics of the virus they are derived from. Thus, influenza virosomes are taken up by cells via receptor-mediated endocytosis, which directs the particles to the endosomal cell compartment. Subsequently, the virosomal membrane fuses with the endosomal membrane induced by the mildly acidic pH within the endosomes. This fusion process establishes continuity between the lumen of the virosome and the cell cytosol. Upon interaction of virosomes with antigen-presenting cells (APCs), protein antigens encapsulated within virosomes will be delivered to the cell cytosol, and thus, into the MHC class I presentation pathway. Indeed, virosome-mediated delivery of antigens in vivo results in efficient priming of a class I MHC-restricted cytotoxic T lymphocyte (CTL) response.     

Competitive selection in vivo by a cell for one variant over another: implications for RNA virus quasispecies in vivo.

Infidelity of genome applications of RNA viruses leads to the generation of viral quasispecies both in vitro and in vivo. However, the biological significance of such generated variants in vivo is largely unknown and controversial. To study this issue, we continued our evaluation of the tropism of a lymphocytic choriomeningitis virus (LCMV) variant termed clone 13 with its parental virus clonal pool ARM 53b (wild-type parent) for neuronal cells in vivo. Earlier in vivo and in vitro studies noted that the wild-type virus contained a Phe at glycoprotein (GP) residue 260 which correlated with neuron tropism compared with LCMV variants containing a Leu at residue 260 which showed selected tropism for cells of the immune system (C.F. Evans, P. Borrow, J. C. de la Torre, and M. B. A. Oldstone J. Virol. 68:7367-7373, 1994; L. Villarete, T. Somasundaram, and R. Ahmed, J. Virol 68:7490-7496, 1994). Here we (i) evaluated the ability of the viral variants with either a Phe or Leu at GP residue 260 to replicate in vivo in the spleen, liver, or brain, (ii) analyzed the ability of these viruses to compete against each other for cell (neuron)-specific selection following a single viral inoculation of different ratios of both viruses, and (iii) utilized genetic reassortants of both viruses to test their ability to replicate in neurons in vivo. We found that viral variants containing either a Phe or Leu at GP residue 260 were equally capable of replicating in neurons, but when inoculated together, neurons selected for the viral population containing Phe at GP residue 260 over viruses containing a Leu at this position. This was in contrast to selection in the liver and spleen that favored viruses with Leu and not Phe at GP residue 260. Analysis of inoculations with viral reassortants indicated that genes encoded on the short RNA (the GP and nucleoprotein, not the L [polymerase] and Z proteins that are encoded by the large RNA) were associated with neurotropism. Since the nucleoprotein sequences of wild-type Armstrong and clone 13 are identical, it is likely that specific cytoplasmic factors of the neurons play a fundamental role in the selection of virus with Phe at GP residue 260.     

Hepatitis C virus NS2 coordinates virus particle assembly through physical interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes

The hepatitis C virus (HCV) NS2 protein is essential for particle assembly, but its function in this process is unknown. We previously identified critical genetic interactions between NS2 and the viral E1-E2 glycoprotein and NS3-NS4A enzyme complexes. Based on these data, we hypothesized that interactions between these viral proteins are essential for HCV particle assembly. To identify interaction partners of NS2, we developed methods to site-specifically biotinylate NS2 in vivo and affinity capture NS2-containing protein complexes from virus-producing cells with streptavidin magnetic beads. By using these methods, we confirmed that NS2 physically interacts with E1, E2, and NS3 but did not stably interact with viral core or NS5A proteins. We further characterized these protein complexes by blue native polyacrylamide gel electrophoresis and identified ≈ 520-kDa and ≈ 680-kDa complexes containing E2, NS2, and NS3. The formation of NS2 protein complexes was dependent on coexpression of the viral p7 protein and enhanced by cotranslation of viral proteins as a polyprotein. Further characterization indicated that the glycoprotein complex interacts with NS2 via E2, and the pattern of N-linked glycosylation on E1 and E2 suggested that these interactions occur in the early secretory pathway. Importantly, several mutations that inhibited virus assembly were shown to inhibit NS2 protein complex formation, and NS2 was essential for mediating the interaction between E2 and NS3. These studies demonstrate that NS2 plays a central organizing role in HCV particle assembly by bringing together viral structural and nonstructural proteins.     

Redistribution of cyclophilin A to viral factories during vaccinia virus infection and its incorporation into mature particles

Cyclophilins are peptidyl-prolyl cis-trans isomerases involved in catalyzing conformational changes and accelerating the rate of protein folding and refolding in several cellular systems. In the present study, we analyzed the expression pattern and intracellular distribution of the cellular isomerase cyclophilin A (CypA) during vaccinia virus (VV) infection. An impressive increase in CypA stability was observed, leading to a practically unchanged accumulation of CypA during infection, although its synthesis was completely inhibited at late times. By confocal microscopy, we observed that CypA went through an intense reorganization in the cell cytoplasm and colocalized with the virosomes late in infection. CypA relocation to viral factories required the synthesis of viral postreplicative proteins, and treatment of infected cells with cyclosporine (CsA) prevented CypA relocation, clearly excluding the virosomes from CypA staining. Immunoelectron microscopy of VV-infected cells showed that CypA was incorporated into VV particles during morphogenesis. Biochemical and electron microscopic assays with purified virions confirmed that CypA was encapsidated within the virus particle and localized specifically in the core. This work suggests that CypA may develop an important role in VV replication.     

Use of a dialyzable short-chain phospholipid for efficient solubilization and reconstitution of influenza virus envelopes

Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for cellular delivery of various macromolecules. To further advance the use of virosomes, we developed a novel dialysis procedure for the reconstitution of influenza virus membranes that is easily applicable to industrial production and compatible with encapsulation of a variety of compounds. This procedure relies on the use of 1,2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) as a solubilizing agent. DCPC is a short-chain lecithin with detergent-like properties and with a critical micelle concentration of 14 mM. DCPC effectively dissolved the influenza virus membranes after which the nucleocapsids could be removed by ultracentrifugation. The solubilized membrane components were reconstituted either by removal of DCPC by dialysis or by a procedure involving initial dilution of the solubilized membrane components followed by dialysis. Both protocols resulted in removal of 99.9% of DCPC and simultaneous formation of virosomes. Analysis of the virosome preparations by equilibrium sucrose density gradient centrifugation revealed co-migration of phospholipid and protein for virosomes produced by either method. Moreover, both virosome preparations showed morphological and fusogenic characteristics similar to native influenza virus. Size, homogeneity and spike density of the virosomes varied with the two different reconstitution procedures employed. The recovery of viral membrane proteins and phospholipids in the virosomes was found to be higher for the dilution/dialysis procedure than for the simple dialysis protocol. This novel procedure for the production of virosomes is straightforward and robust and allows further exploitation of virosomes as vaccines or as drug delivery vehicles not only in academia, but also in industrial settings.     

Use of a dialyzable short-chain phospholipid for efficient solubilization and reconstitution of influenza virus envelopes

Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for cellular delivery of various macromolecules. To further advance the use of virosomes, we developed a novel dialysis procedure for the reconstitution of influenza virus membranes that is easily applicable to industrial production and compatible with encapsulation of a variety of compounds. This procedure relies on the use of 1,2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) as a solubilizing agent. DCPC is a short-chain lecithin with detergent-like properties and with a critical micelle concentration of 14 mM. DCPC effectively dissolved the influenza virus membranes after which the nucleocapsids could be removed by ultracentrifugation. The solubilized membrane components were reconstituted either by removal of DCPC by dialysis or by a procedure involving initial dilution of the solubilized membrane components followed by dialysis. Both protocols resulted in removal of 99.9% of DCPC and simultaneous formation of virosomes. Analysis of the virosome preparations by equilibrium sucrose density gradient centrifugation revealed co-migration of phospholipid and protein for virosomes produced by either method. Moreover, both virosome preparations showed morphological and fusogenic characteristics similar to native influenza virus. Size, homogeneity and spike density of the virosomes varied with the two different reconstitution procedures employed. The recovery of viral membrane proteins and phospholipids in the virosomes was found to be higher for the dilution/dialysis procedure than for the simple dialysis protocol. This novel procedure for the production of virosomes is straightforward and robust and allows further exploitation of virosomes as vaccines or as drug delivery vehicles not only in academia, but also in industrial settings.     

Functional reconstitution of influenza virus envelopes.

We have examined several procedures for the reconstitution of influenza virus envelopes, based on detergent removal from solubilized viral membranes. With octylglucoside, no functionally active virosomes are formed, irrespective of the rate of detergent removal: in the final preparation the viral spike proteins appear predominantly as rosettes. Protein incorporation in reconstituted vesicles is improved when a method based on reverse-phase evaporation of octylglucoside-solubilized viral membranes in an ether/water system is employed. However, the resulting vesicles do not fuse with biological membranes, but exhibit only a non-physiological fusion reaction with negatively charged liposomes. Functional reconstitution of viral envelopes is achieved after solubilization with octaethyleneglycol mono(n-dodecyl)ether (C12E8), and subsequent detergent removal with Bio-Beads SM-2. The spike protein molecules are quantitatively incorporated in a single population of virosomes of uniform buoyant density and appear on both sides of the membrane. The virosomes display hemagglutination activity and a strictly pH-dependent hemolytic activity. The virosomes fuse with erythrocyte ghosts, as revealed by a fluorescence resonance energy transfer assay. The rate and the pH dependence of fusion are essentially the same as those of the intact virus. The virosomes also fuse with cultured cells, either at the level of the endosomal membrane or directly with the cellular plasma membrane upon a brief exposure to low pH.     

Coronavirus proteins: structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins.

The recent finding that the E1 glycoproteins of murine coronaviruses contain only O-linked oligosaccharides suggested that this unusual modification might be a distinguishing feature of coronaviruses and might play an essential role in the life cycle of this family of viruses. To examine these possibilities, we analyzed the oligosaccharide moieties of the membrane proteins of the avian coronavirus infectious bronchitis virus. In addition, we determined the effect of inhibiting the glycosylation of these proteins on viral maturation and infectivity. Infectious bronchitis virus virions contain nine proteins. Four of these proteins, GP36, GP31, GP28, and P23, are closely related structurally and appear to be homologous to the E1 proteins of murine coronaviruses. We found that the oligosaccharides of GP31 and GP28 could be removed with endoglycosidase H and that neither of these glycoproteins was detectable in tunicamycin-treated cells. These two results indicated that GP31 and GP28 contain N-linked oligosaccharides. Therefore, O-linked oligosaccharides are not a universal feature of the small coronavirus membrane glycoproteins. Tunicamycin inhibited glycosylation of all of the viral glycoproteins but did not inhibit production of virions by infectious bronchitis virus-infected cells. The virions released by these cells contained only the three non-glycosylated viral proteins P51, P23, and P14. These particles were not infectious. Therefore, it appears that glycosylated infectious bronchitis virus polypeptides are not required for particle formation. However, the viral glycoproteins are apparently indispensible for viral infectivity.     

Gating Movements of Colicin A and Colicin Ia Are Different

The effect of temperature on fusion of Sendai virus with target membranes and mobility of the viral glycoproteins was studied with fluorescence methods. When intact virus was used, the fusion threshold temperature (20–22°C) was not altered regardless of the different types of target membranes. Viral glycoprotein mobility in the intact virus increased with temperature, particularly sharply at the fusion threshold temperature. This effect was suppressed by the presence of erythrocyte ghosts and/or dextran sulfate in the virus suspension. In these cases also, no change in the fusion threshold temperature was observed. On the other hand, reconstituted viral envelopes (virosomes) bearing viral glycoproteins but lacking matrix proteins were capable of fusing with erythrocyte ghosts even at temperatures lower than the fusion threshold temperature and no fusion threshold temperature was observed over the range of 10–40°C. The mobility of viral glycoproteins on virosomes was much greater and virtually temperature-independent. The intact virus treated with an actin-affector, jasplakinolide, reduced the extent of fusion with erythrocyte ghosts and the mobility of viral glycoproteins, while the treatment of virosomes with the same drug did not affect the extent of fusion of virosomes with erythrocyte ghosts and the mobility of the glycoproteins. These results suggest that viral matrix proteins including actins affect viral glycoprotein mobility and may be responsible for the temperature threshold phenomenon observed in Sendai virus fusion.This revised version was published online in August 2005 with a corrected cover date.     

Genetic Requirement for Hemagglutinin Glycosylation and Its Implications for Influenza A H1N1 Virus Evolution

Influenza A virus has evolved and thrived in human populations. Since the 1918 influenza A pandemic, human H1N1 viruses had acquired additional N-linked glycosylation (NLG) sites within the globular head region of hemagglutinin (HA) until the NLG-free HA head pattern of the 1918 H1N1 virus was renewed with the swine-derived 2009 pandemic H1N1 virus. Moreover, the HA of the 2009 H1N1 virus appeared to be antigenically related to that of the 1918 H1N1 virus. Hence, it is possible that descendants of the 2009 H1N1 virus might recapitulate the acquisition of HA head glycosylation sites through their evolutionary drift as a means to evade preexisting immunity. We evaluate here the evolution signature of glycosylations found in the globular head region of H1 HA in order to determine their impact in the virulence and transmission of H1N1 viruses. We identified a polymorphism at HA residue 147 associated with the acquisition of glycosylation at residues 144 and 172. By in vitro and in vivo analyses using mutant viruses, we also found that the polymorphism at HA residue 147 compensated for the loss of replication, virulence, and transmissibility associated with the presence of the N-linked glycans. Our findings suggest that the polymorphism in H1 HA at position 147 modulates viral fitness by buffering the constraints caused by N-linked glycans and provide insights into the evolution dynamics of influenza viruses with implications in vaccine immunogenicity.     

Targeting influenza virosomes to ovarian carcinoma cells.

Reconstituted influenza virus envelopes (virosomes) containing the viral hemagglutinin (HA) have attracted attention as delivery vesicles for cytosolic drug delivery as they possess membrane fusion activity. Here, we show that influenza virosomes can be targeted towards ovarian carcinoma cells (OVCAR-3) with preservation of fusion activity. This was achieved by incorporating poly(ethylene glycol) (PEG)-derivatized lipids into the virosome membrane. This PEG layer serves as shield to prevent interaction of HA with ubiquitous sialic acid residues and as spatial anchor for antibody attachment. Coupling of Fab' fragments of mAb 323/A3 (anti-epithelial glycoprotein-2) to the distal ends of PEG lipids resulted in specific binding of virosomes to OVCAR-3 cells. These antibody-redirected virosomes fused with membranes of OVCAR-3 cells in a pH-dependent fashion.