Intracellular Enveloped Virus Research Articles

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

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

Vaccinia virus infection disrupts microtubule organization and centrosome function

We examined the role of the microtubule cytoskeleton during vaccinia virus infection. We found that newly assembled virus particles accumulate in the vicinity of the microtubule-organizing centre in a microtubule- and dynein–dynactin complex-dependent fashion. Microtubules are required for efficient intracellular mature virus (IMV) formation and are essential for intracellular enveloped virus (IEV) assembly. As infection proceeds, the microtubule cytoskeleton becomes dramatically reorganized in a fashion reminiscent of overexpression of microtubule-associated proteins (MAPs). Consistent with this, we report that the vaccinia proteins A10L and L4R have MAP-like properties and mediate direct binding of viral cores to microtubules in vitro. In addition, vaccinia infection also results in severe reduction of proteins at the centrosome and loss of centrosomal microtubule nucleation efficiency. This represents the first example of viral-induced disruption of centrosome function. Further studies with vaccinia will provide insights into the role of microtubules during viral pathogenesis and regulation of centrosome function.

The Vaccinia Virus A36R Protein Is a Type Ib Membrane Protein Present on Intracellular but Not Extracellular Enveloped Virus Particles

Vaccinia virus gene A36R encodes a 45-kDa protein that is conserved in orthopoxviruses. A virus lacking the A36R protein formed a small plaque, was unable to induce the polymerization of actin tails, and was avirulent in vivo. Here we present a further characterization of the A36R protein by in vitro transcription and translation and analysis of infected cells by confocal microscopy and immunoelectron microscopy of cryosections using a monoclonal antibody raised against the C-terminal domain of the A36R protein. Translation of the A36R mRNA in vitro produced a protein of the same size whether or not the translation reaction was performed in the presence of canine pancreatic microsomes. However, the polypeptide synthesized in the presence of microsomes was associated integrally with the membrane and was sensitive to digestion by exogenous protease without permeabilization of the membrane with detergent, indicating that the majority of the protein is exposed on the outside of the vesicle. Consistent with this, immunofluorescent analysis of virus-infected cells demonstrated that the C-terminal domain of A36R was not exposed on the cell surface but was detected once the cell membrane was permeabilized. Immunoelectron microscopy of cryosections of infected cells showed that the protein was absent from IMV particles but present on intracellular enveloped virus (IEV) particles, predominantly on the cytosolic face of the IEV outer membrane. Where cell-associated enveloped virus (CEV) particles were attached to the cell surface, the A36R protein was detected only on the cytosolic surface of the plasma membrane where the virus particle remained attached to the cell and not elsewhere on the plasma membrane or on the CEV particle. A36R and actin copurified with EEV particles due to the association of fragments of cellular membranes with the EEV particles. Therefore, A36R represents the first example of a virus-encoded protein that is present on IEV but not CEV particles.

The vaccinia virus A27L protein is needed for the microtubule-dependent transport of intracellular mature virus particles.

The vaccinia virus (VV) A27L gene encodes a 14 kDa protein that is required for the formation of intracellular enveloped virus (IEV) and, consequently, normal sized plaques. Data presented here show that A27L plays an additional role in VV assembly. When cells were infected with the VV WR32-7/Ind 14K, under conditions that repress A27L expression, transport of intracellular mature virus (IMV) from virus factories was inhibited and some IMV was found in aberrant association with virus crescents. In contrast, other VV mutants (vDeltaB5R and vDeltaF13L) that are defective in IEV formation produce IMV particles that are transported out of virus factories. This indicated a specific role for A27L in IMV transport. Induction of A27L expression at 10 h post-infection promoted the dispersal of clustered IMV particles, but only when microtubules were intact. Formation of IEV particles was also impaired when cells were infected with WR32-7/14K, a VV strain expressing a mutated form of the A27L protein; however, this mutation did not inhibit intracellular transport of IMV particles. Collectively, these data define two novel aspects of VV morphogenesis. Firstly, A27L is required for both IMV transport and the process of envelopment that leads to IEV formation. Secondly, movement of IMV particles between the virus factory and the site of IEV formation is microtubule-dependent.

Palate shape in first onset psychoses

Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor.

The Effects of Targeting the Vaccinia Virus B5R Protein to the Endoplasmic Reticulum on Virus Morphogenesis and Dissemination

The consequence of redirecting the vaccinia virus (VV) B5R protein to the endoplasmic reticulum (ER) has been investigated by the addition of an ER retrieval signal KKSL (K2X2) to the B5R C-terminus. This mutant B5R gene and a version of the gene with the inactive ER retrieval sequence KKSLAL (K2X4) were inserted into the thymidine kinase locus of a VV mutant lacking the B5R gene, vΔB5R. Similar levels of B5R protein were made by each virus, but the B5R-K2X2 protein remained sensitive to endoglycosidase H and colocalised with protein disulphide isomerase in the ER. In contrast, the B5R-K2X4 protein colocalised with 1,4-galactosyltransferase in the trans-Golgi network. Electron microscopy revealed that even when the B5R protein was redirected to the ER, intracellular mature virus particles were wrapped by cellular membranes to form intracellular enveloped virus particles, although more incompletely wrapped particles were evident compared with wild type. These intracellular enveloped virus particles were, however, unable to efficiently induce the polymerisation of actin and the plaque size formed by vB5R-K2X2 was small. Nevertheless, the amount and specific infectivity of EEV produced by vB5R-K2X2 were similar to those of wild type, despite the dramatic reduction in the amount of B5R protein present in vB5R-K2X2 EEV.

Vaccinia virus induces Ca2+-independent cell-matrix adhesion during the motile phase of infection.

Vaccinia virus (VV) induces two forms of cell motility: cell migration, which is dependent on the expression of early genes, and the formation of cellular projections, which requires the expression of late genes. The need for viral gene expression prior to cell motility suggests that VV proteins may affect how infected cells interact with the extracellular matrix. To address this, we have analyzed changes in cell-matrix adhesion after infection of BS-C-1 cells with VV. Whereas uninfected cells round up and detach from the culture flask in the presence of EGTA, infected cells remain attached to the culture flask with a stellate morphology. Ca2+-independent cell-matrix adhesion was evident by 10 h postinfection, after the onset of cell motility but before the formation of virus-induced cellular projections. Progression to Ca2+-independent adhesion required the expression of late viral genes but not the formation of intracellular enveloped virus particles or intracellular actin tails. Analyses of specific matrix proteins identified vitronectin and fibronectin as optimal ligands for Ca2+-independent adhesion and the formation of cellular projections. Adhesion to fibronectin was mediated via RGD motifs alone and was not inhibited by 500 micrograms of heparin/ml. Kistrin, a disintegrin which binds preferentially to the alphav beta3 (vitronectin/fibronectin) receptor inhibited the formation of cellular projections without disrupting preformed matrix interactions. Finally, we show that Ca2+-independent cell-matrix adhesion is a dynamic process which mediates changes in the morphology of VV-infected cells and uninfected cells which exhibit a transformed phenotype.

DNA Packaging Mutant: Repression of the Vaccinia Virus A32 Gene Results in Noninfectious, DNA-Deficient, Spherical, Enveloped Particles

The vaccinia virus A32 open reading frame was predicted to encode a protein with a nucleoside triphosphate-binding motif and a mass of 34 kDa. To investigate the role of this protein, we constructed a mutant in which the original A32 gene was replaced by an inducible copy. The recombinant virus, vA32i, has a conditional lethal phenotype: infectious virus formation was dependent on isopropyl-beta-D-thiogalactopyranoside (IPTG). Under nonpermissive conditions, the mutant synthesized early- and late-stage viral proteins, as well as viral DNA that was processed into unit-length genomes. Electron microscopy of cells infected in the absence of IPTG revealed normal-appearing crescents and immature virus particles but very few with nucleoids. Instead of brick-shaped mature particles with defined core structures, there were numerous electron-dense, spherical particles. Some of these spherical particles were wrapped with cisternal membranes, analogous to intracellular and extracellular enveloped virions. Mutant viral particles, purified by sucrose density gradient centrifugation, had low infectivity and transcriptional activity, and the majority were spherical and lacked DNA. Nevertheless, the particle preparation contained representative membrane proteins, cleaved and uncleaved core proteins, the viral RNA polymerase, the early transcription factor and several enzymes, suggesting that incorporation of these components is not strictly coupled to DNA packaging.

Roles of vaccinia virus EEV-specific proteins in intracellular actin tail formation and low pH-induced cell-cell fusion.

During vaccinia virus (VV) morphogenesis intracellular mature virus (IMV) is wrapped by two additional membranes to form intracellular enveloped virus (IEV). IEV particles can nucleate the formation of actin tails which aid movement of IEVs to the cell surface where the outer IEV membrane fuses with the plasma membrane forming cell-associated enveloped virus (CEV) which remains attached to the cell, or extracellular enveloped virus (EEV) which is shed from the cell. In this report, we have used a collection of VV mutants lacking individual EEV-specific proteins to compare the roles of these proteins in the formation of IEV and IEV-associated actin tails and fusion of infected cells after a low pH shock. Data presented here show that p45-50 (A36R) is not required for IEV formation or for acid-induced cell-cell fusion, but is required for formation of IEV-associated actin tails. In contrast, gp86 (A56R), the virus haemagglutinin, is not required for formation of either IEV or IEV-associated actin tails. Data presented also confirm that p37 (gene F13L), gp42 (B5R) and gp22-24 (A34R) are needed for formation of IEV-associated actin tails and for cell-cell fusion after low pH shock. The phenotypes of these mutants were not affected by the host cell type as similar results were obtained in a range of different cells. Lastly, comparisons of the phenotypes of VV strains Western Reserve, deltaA34R and deltaA36R demonstrate that actin tails are not required for low pH-induced cell-cell fusion.

The envelope of vaccinia virus reveals an unusual phospholipid in Golgi complex membranes.

We isolated forms of enveloped vaccinia virus from infected HeLa cells to obtain membranes for the analysis of lipids of the cis-Golgi network and trans-Golgi network. The intracellular mature virus obtains its envelope by wrapping itself in the membranes of the cis-Golgi network. A fraction of these virions then acquires a second envelope by enwrapping trans-Golgi network membranes to form the intracellular enveloped virus. Lipids were analyzed by high performance thin layer chromatography and digital densitometry to establish a steady-state lipid profile of viral membranes, which should reflect the compositions of the cis-Golgi network and trans-Golgi network. Phosphatidyl-inositol was slightly enriched in the cis-Golgi network of HeLa cells, whereas the trans-Golgi network showed a minor increase in phosphatidylserine and sphingomyelin. Similarly, cholesterol was only slightly more abundant in the trans-Golgi compared to the cis-Golgi. An unusual lipid, semilysobisphosphatidic acid, was present in significant amounts in vaccinia envelopes. Semilysobisphosphatidic acid was present in similar levels in infected and uninfected cells, and was therefore not induced by vaccinia infection. Subcellular fractionation of HeLa cells indicated that the recovery of semilysobisphosphatidic acid paralleled the recovery of a Golgi marker. Furthermore, a lipid species that comigrated with semilysobisphosphatidic acid was also present in lipids extracted from highly purified, intact Golgi complexes from rat liver. Together, these results suggest that semilysobisphosphatidic acid is a normal component of Golgi membranes.

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

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