Recombinant Vaccinia Virus Expression System Research Articles

Epitopes Derived by Incidental Translational Frameshifting Give Rise to a Protective CTL Response

Aberrant gene expression can be caused by several different mechanisms at the transcriptional, RNA processing, and translational level. Although most of the resulting proteins may have no significant biological function, they can be meaningful for the immune system, which is sensitive to extremely low levels of Ag. We have tested this possibility by investigating the ability of CD8+ T cells (TCD8+) to respond to an epitope whose expression results from incidental ribosomal frameshifting at a sequence element within the HSV thymidine kinase gene. This element, with no apparent functional significance, has been identified due to its ability to facilitate escape from the antiviral compound acyclovir. Using a recombinant vaccinia virus expression system, we find that in vitro and in vivo TCD8+ responses to the frameshift-dependent epitope are easily discernible. Furthermore, the in vivo response is at a sufficient level to mediate protection from a tumor challenge. Thus, the targets of immune responses to infectious agents can extend beyond the products of conventional open reading frames. On a per-cell basis, responses to such minimally expressed epitopes may be exceedingly effective due to the selective expansion of high avidity TCD8+.

Decorin Evokes Protracted Internalization and Degradation of the Epidermal Growth Factor Receptor via Caveolar Endocytosis

Decorin inhibits the epidermal growth factor receptor (EGFR) by down-regulating its tyrosine kinase activity, thereby blocking the growth of a variety of transformed cells and tumor xenografts. In this study we provide evidence that decorin directly binds to the EGFR causing its dimerization, internalization, and ultimately its degradation. Using various pharmacological agents to disrupt clathrin-dependent and -independent endocytosis, we demonstrate that decorin evokes a protracted internalization of the EGFR primarily via caveolar-mediated endocytosis. In contrast to EGF, decorin targets the EGFR to caveolae, but not to early or recycling endosomes. Ultimately, however, both EGF- and decorin-induced pathways converge into late endosomes/lysosomes for final degradation. Thus, we have discovered a novel biological mechanism for decorin that could explain its anti-proliferative and anti-oncogenic mode of action.

18] Expression and functional analyses of Rab8 and Rab11a in exocytic transport from trans-golgi network

This chapter describes the morphologic, biochemical assays used to study the role of Rab8, and Rab11a in the exit from the trans-Golgi network (TGN) of cargo proteins destined for the plasma membrane. The functional analysis of Rab proteins is facilitated by the possibility of producing mutant forms that behave as dominant negative inhibitors of membrane transport. Mutants are generated by site-directed mutagenesis of conserved domains critical for nucleotide binding or hydrolysis. A recombinant vaccinia virus expression system is used to establish a biochemical assay useful for studying the function of Rab proteins in exocytic transport. This system was utilized on account of several important advantages. First, the high transfection efficiencies obtained with this system makes it particularly well suited for the development of biochemical assays that rely on having a uniform population of cells. Second, this system enables the coexpression of multiple proteins with relative ease. Thus, multiple Rab proteins could be overexpressed in combination together with relevant transport markers.

Role of NH 2 - and COOH-Terminal Domains of the P Protein of Human Parainfluenza Virus Type 3 in Transcription and Replication

The phosphoproteins (P proteins) of paramyxoviruses play a central role in transcription and replication of the viruses by forming the RNA polymerase complex L-P and encapsidation complex (N-P) with nucleocapsid protein (N) and binding to N protein-encapsidated genome RNA template (N-RNA template). We have analyzed the human parainfluenza virus type 3 (HPIV3) P protein and deletion mutants thereof in an in vitro transcription and in vivo replication system. The in vitro system utilizes purified N-RNA template and cell extract containing L and P proteins coexpressed via plasmids using a recombinant vaccinia virus expression system. The in vivo system takes advantage of minigenome replication, which measures luciferase reporter gene expression from HPIV3 minigenomes by viral proteins in a recombinant vaccinia virus expression system. These studies revealed that the C-terminal 20-amino-acid region of P is absolutely required for transcription in vitro and luciferase expression in vivo, suggesting its critical role in viral RNA synthesis. The N-terminal 40-amino-acid region, on the other hand, is essential for luciferase expression but dispensable for transcription in vitro. Consistent with these findings, the C-terminal domain is required for binding of P protein to the N-RNA template involved in both transcription and replication, whereas the N-terminal domain is required for the formation of soluble N-P complex involved in encapsidation of nascent RNA chains during replication. Coimmunoprecipitation analysis showed that the P protein forms a stable homooligomer (perhaps a trimer) that is present in L-P and N-P complexes in the higher oligomeric forms (at least a pentamer). Interestingly, coexpression of a large excess of N- or C-terminally deleted P with wild-type P had no effect on minigenome replication in vivo, notwithstanding the formation of heterooligomeric complexes. These data indicate that P protein with a deleted terminal domain can function normally within the P heterooligomeric complex to carry out transcription and replication in vivo.

Intracellular degradation of the HIV-1 envelope glycoprotein. Evidence for, and some characteristics of, an endoplasmic reticulum degradation pathway.

Analysis of the fate of HIV-1 envelope protein gp160 (Env) has shown that newly synthesized proteins may be degraded within the biosynthetic pathway and that this degradation may take place in compartments other than the lysosomes. The fate of newly synthesized Env was studied in living BHK-21 cells with the recombinant vaccinia virus expression system. We found that gp160 not only undergoes physiological endoproteolytic cleavage, producing gp120, but is also degraded, producing proteolytic fragments of 120 kDa to 26 kDa in size, as determined by SDS/PAGE in non reducing conditions. Analysis of the 120-kDa proteolytic fragment, and comparison with gp120, showed that it is composed of peptides linked by disulfides bonds and lacks the V3-loop epitope and the C-terminal domain of gp120 (amino acids 506-516). A permeabilized cell system, with impaired transport of labeled Env from the endoplasmic reticulum (ER) to Golgi compartments, was developed to determine the site of degradation and to define some biochemical characteristics of the intracellular degradation process. In the semipermeable BHK-21 cells, there was: (a) no gp120 production (b), a progressive decrease in the amount of newly synthesized gp160 and a concomitant increase in the amount of a 120-kDa proteolytic fragment. This fragment had the same biochemical characteristics as the 120-kDa proteolytic fragment found in living nonpermeabilized cells, and (c) susceptibility of the V3 loop. This degradation process occurred in the ER, as shown by both biochemical and indirect immunofluorescence analysis. Furthermore, there was evidence that changes in redox state are involved in the ER-dependent envelope degradation pathway because adding reducing agents to permeabilized cells caused dose-dependent degradation of the 120-kDa proteolytic fragment and of the remaining gp160 glycoprotein. Thus our results provide direct evidence that regulated degradation of the HIV-1 envelope glycoprotein may take place in the ER of infected cells.

A simplified method of screening for isolation of recombination vaccinia virus.

AbstractThe utility of a recombinant vaccinia virus expression system for transient expression of genes was demonstrated in 1982 (1,2). Among a number of useful characteristics of this expression system are the capacity of the vaccinia virus genome to accommodate large genes (>20 kb pairs), availability of insertional sites on the viral genome yielding viable recombinant viruses, ability of the vaccinia virus to infect a wide range of mammalian hosts, cytoplasmic transcription and replication of the viral genome unlike many other expression systems, a relatively high level of recombinant proteins produced in infected cells, and the primary sequence-directed posttranslational modifications of recombinant proteins aim to native proteins (for a review, see ref. 3). As a first step in the construction of a recombinant vaccinia virus, the gene encoding a protein in the form of a cDNA is cloned into a plasmid vector under the control of an early or late vaccinia virus promoter (4–6), or a bacteriophage T7 promoter/the encephalomyocarditis virus 5′-untranslated leader sequence which would allow a cap independent mechanism of translation of the transcripts (7). Flanking the gene chosen for expression are DNA sequences representing portions of a nonessential viral gene such as that encoding the thymidine kinase in order to allow homologous recombination at the locus of the viral gene in vivo (6). The frequency with which the gene is integrated into the viral genome has been estimated to be about 0.1% (for a review, see ref. 3). In the original protocol for the isolation of a recombinant vaccinia virus (6), cell monolayers are infected with the wild-type virus (WR strain) and transfected with the plasmid DNA encoding the gene of interest. Since insertion of the gene of interest at the viral thymidine kinase (TK) gene locus results in recombinant viruses with the TK− phenotype, plaque isolates of recombinant viruses are selected in the presence of 5-bromodeoxyuridine (5-BrdU). The conditions chosen for selection in the presence of this analog of thymidine are lethal to replication of the wild-type virus owing to incorporation of the phosphorylated form into the viral genome, but not to the recombinant virus deficient in TK. The cell lysates containing the wild-type and recombinant viruses at appropriate dilutions are used to infect cell monolayers of human TK− cells, and then overlaid with growth medium containing agarose and 5-BrdU to prevent spreading of recombinant virus plaques and inhibit replication of the wild-type virus, respectively. A second agarose overlay containing the 5-BrdU and the neutral red stain is used to enable one to visualize recombinant virus plaques (6). A single plaque results from a productively infected cell and the cell-to-cell spread of the virus, and an agarose overlay localizes each of these plaques on a mono-layer. Since a plaque could consist of either a recombinant virus or a spontaneous TK− mutant virus often present as a contaminant in the cell lysates, a suitable hybridization technique is employed in the screening protocol for isolation of recombinants (6).KeywordsVaccinia VirusThymidine KinaseRecombinant VirusRecombinant Vaccinia VirusSingle PlaqueThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Receptor interference mediated by the envelope glycoproteins of various HIV-1 and HIV-2 isolates

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) plays a major role in the down-regulation of its receptor, CD4. This down-regulation, at least in part, is caused by the formation of gp160-CD4 intracellular complexes which fail to transport out of the endoplasmic reticulum (ER). In this report, we have evaluated the ability of envelope glycoproteins from various isolates to block CD4 transport within the endoplasmic reticulum. Using a recombinant vaccinia virus expression system in HeLa cells, we expressed different HIV-1 and HIV-2 envelope glycoproteins with CD4. Pulse-chase labeling followed by immunoprecipitation demonstrated that envelope glycoproteins from primary and lab-adapted isolates were capable of forming intracellular complexes with CD4, resulting in the partial inhibition of CD4 transport to the Golgi. Although the efficiency of CD4 modulation was variable, these differences did not correlate with the type of isolate from which the HIV-1 glycoprotein was derived. However, we did find that the HIV-2 ST envelope glycoprotein (gp150) was not as efficient at blocking CD4 as the glycoprotein (gp140) derived from HIV-2 ROD. The decreased ability of ST gp150 to block CD4 within the ER was associated with an increased efficiency of ST gp150 transport and cleavage. Thus, differences in the ability of HIV envelope glycoproteins to block CD4 transport do exist, and these differences may be determined by envelope glycoprotein transport kinetics.

Domains required for assembly of adenovirus type 2 fiber trimers.

Entry of human adenovirus into cells is a two-step process, mediated in the first step by a specific interaction between the trimeric fiber protein and a specific receptor on the surface of susceptible cells. Because of the interest in human adenovirus as a vector for gene therapy, we have mapped domains in the fiber protein that are important for proper assembly of this trimeric structure and for proper addition of O-linked N-acetylglucosamine (0-GlcNAc). Mutants of adenovirus type 2 fiber in this study were expressed in human cells by use of a recombinant vaccinia virus expression system that yielded protein indistinguishable from the fiber produced during adenovirus infection. The N-terminal half of the protein did not appear to influence fiber trimer formation, since deletions up to 260 amino acids (aa) from the N-terminal end as well as in-frame deletions within the shaft of the molecule still allowed trimerization; internal deletions in the shaft between aa 61 and 260 appeared to alter addition of 0-GlcNAc, as judged by loss of reactivity to a monoclonal antibody specific for this carbohydrate addition. Deletions from the C terminus of the molecule (as small as 2 aa) appeared to prevent trimer formation. Additions of amino acids to the C-terminal end of the fiber showed variable results: a 6-aa addition allowed trimer formation, while a 27-aa addition did not. These trimer-defective mutants were also relatively less stable, as judged kV pulse-chase experiments. Taken together, our results indicate that trimerization of the fiber requires at least two domains, the entire head (aa 400 to 582), and at least the C-terminal-most 15 aa of the shaft.

Biochemical Characterization of a Temperature-Sensitive Adenovirus DNA Polymerase

Involvement of adenovirus DNA polymerase (AdPol) in viral DNA replication was initially demonstrated using two temperature-sensitive (ts) adenoviruses, AdSts36 and Ad5ts149. Ad5ts36 is particulary interesting because a single mutation mapping to Leu391 → Phe in the AdPol gene confers ts phenotype not only for DNA replication in human cells but also for transformation in rat cells. In order to elucidate the mechanism(s) involved in the ts phenotype, we introduced the ts36 mutation in AdPol of adenovirus type 2 and overexpressed the protein using recombinant vaccinia virus expression system in order to study its biochemical properties. The results show that the ts36 AdPol is defective in the in vitro DNA replication initiation and DNA polymerase elongation assays it the protein is expressed at the nonpermissive temperature (37°) independent of whether the assays are performed at 32 or 37°. In contrast, the ts36 AdPol expressed at 32° had activities similar to that of the wild-type protein when asseyed at either temperature. Furthermore, unlike the wild-type protein, ts36 AdPol expressed at 37° failed to recognize the viral DNA replication origin, but bound to a single-stranded DNA cellulose column with greater affinity, suggesting that the defect in the ts36 AdPol for DNA replication can be attributed to its altered DNA-binding properties.

Correlation of serum antibody titers against hepatitis C virus core protein with clinical features by western blot (immunoblot) analysis using a recombinant vaccinia virus expression system.

In order to study the relationships among the clinical features of hepatitis C patients, the presence of hepatitis C virus (HCV) RNA in their blood, and their serum antibody titers against the core protein of virus and to study the antibody levels in asymptomatic HCV carriers, a recombinant vaccinia virus containing a core protein gene was constructed. The recombinant virus expressed a protein with a molecular mass of 22 kDa in RK-13 cells as determined by Western blot (immunoblot) analysis. By using the cell lysate of virus-infected cells and serially diluted serum samples, core antibody titers in the groups of patients in the chronic hepatitis phase and in the convalescent phase as well as in asymptomatic carriers were determined by enhanced chemiluminescence Western blot analysis. Almost all patients in the chronic phase were shown to have high antibody titers of more than 1:500,000 and with no exception had of HCV RNA in their sera. On the other hand, patients who had recovered naturally and were in the convalescent phase were shown to have significantly lower antibody titers, and the antibody was not detected in the lowest serum dilution of 1:500 in 43% of these patients (three of seven total patients). Antibody levels of patients who showed a good response to interferon treatment decreased to intermediate levels between those of patients in the chronic phase and those of patients in convalescent phase. The antibody titers in asymptomatic carriers varied considerably from 1:500,000 to 1:500, and 41% (11 of 27 total individuals) of these carriers showed a high titer equivalent to that of those in the chronic phase. Core antibody was detected consistently in the individuals in whom HCV RNA was detected. This system for core antibody might be useful for identifying the stage of an apparent HCV infection.

Myristylation of poliovirus capsid precursor P1 is required for assembly of subviral particles.

The poliovirus capsid precursor polyprotein, P1, is cotranslationally modified by the addition of myristic acid. We have examined the importance of myristylation of the P1 capsid precursor during the poliovirus assembly process by using a recently described recombinant vaccinia virus expression system which allows the independent production of the poliovirus P1 protein and the poliovirus 3CD proteinase (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991). We constructed a site-directed mutation in the poliovirus cDNA encoding an alanine at the second amino acid position of P1 in place of the glycine residue required for the myristic acid addition and isolated a recombinant vaccinia virus (VVP1myr-) that expressed a nonmyristylated form of the P1 capsid precursor. The 3CD proteinase expressed by a coinfecting vaccinia virus, VVP3, proteolytically processed the nonmyristylated precursor P1 expressed by VVP1myr-. However, the processed capsid proteins, VP0, VP3, and VP1, did not assemble into 14S or 75S subviral particles, in contrast to the VP0, VP3, and VP1 proteins derived from the myristylated P1 precursor. When cells were coinfected with VVP1myr- and poliovirus type 1, the nonmyristylated P1 precursor expressed by VVP1myr- was processed by 3CD expressed by poliovirus, and the nonmyristylated VP0-VP3-VP1 (VP0-3-1) protomers were incorporated into capsid particles and virions which sedimented through a 30% sucrose cushion. Thus, the nonmyristylated P1 precursor and VP0-3-1 protomers were not excluded from sites of virion assembly, and the assembly defects observed for the nonmyristylated protomers were overcome in the presence of myristylated capsid protomers expressed by poliovirus. We conclude that myristylation of the poliovirus P1 capsid precursor plays an important role during poliovirus assembly by facilitating the appropriate interactions required between 5S protomer subunits to form stable 14S pentamers. The results of these studies demonstrate that the independent expression of the poliovirus P1 and 3CD proteins by using recombinant vaccinia viruses provides a unique experimental tool for analyzing the dynamics of the poliovirus assembly process.

Gp160, the envelope glycoprotein of human immunodeficiency virus type 1, is a dimer of 125-kilodalton subunits stabilized through interactions between their gp41 domains

The molecular masses, carbohydrate contents, oligomeric status, and overall molecular structure of the env glycoproteins of human immunodeficiency virus type 1--gp120, gp160, and gp41--have been determined by quantitative electron microscopy. Using purified gp160s, a water-soluble form of env purified from a recombinant vaccinia virus expression system, we have measured the masses of several hundred individual molecules by dark-field scanning transmission electron microscopy. When combined with sequence-based information, these mass measurements establish that gp160s is a dimer of subunits with an average monomer mass of 123 kDa, of which approximately 32 kDa is carbohydrate and 91 kDa is protein. Similarly, gp120 was found to be a monomer of 89 kDa and to contain virtually all of env's glycosylation. gp41 is glycosylated only slightly, if at all, and is responsible for the interactions that stabilize the gp160s dimer. A molecular mass map of gp160s derived by image processing depicts an asymmetric dumbbell whose two domains have masses of approximately 173 and approximately 73 kDa, corresponding to a gp120 dimer and a gp41 dimer, respectively. We infer that the average monomer mass of native gp160 is 125 kDa and that in situ, env is either a dimer or a tetramer but is most unlikely to be a trimer.

Recombinant peripheral myelin protein Po confers both adhesion and neurite outgrowth‐promoting properties

To probe into the functional properties of the major peripheral myelin cell surface glycoprotein P0, its ability to confer adhesion and neurite outgrowth-promoting properties was studied in cell culture. To this aim, P0 was expressed as integral membrane glycoprotein at the surface of CV-1 cells with the help of a recombinant vaccinia virus expression system. Furthermore, the immunoglobulin-like extracellular domain of P0 (P0-ED) was expressed as soluble protein in a bacterial expression system and used as substrate coated to plastic dishes or as competitor in cell adhesion and neurite outgrowth-promoting assays. The adhesion of P0-expressing CV-1 cells to P0-ED substrate was specifically inhibitable by polyclonal P0 antibodies (54% +/- 6%). In addition, the specific interaction between P0 molecules could be reduced (49% +/- 8%) by adding soluble P0-ED to the culture medium, demonstrating that the homophilic interaction between recombinant P0 molecules can be mediated, at least on one partner of interacting molecules, by the unglycosylated Ig-like domain. Substrate-coated P0-ED also conferred adhesion and neurite outgrowth ability to dorsal root ganglion neurons with neurites of a mean length of about 150 microns. This neurite outgrowth was specifically inhibitable by soluble P0 (74% +/- 14%) and P0 antibodies (65% +/- 9%). These observations indicate that P0 is capable of displaying two different types of functional roles in the myelination process of peripheral nerves: The heterophilic interaction with neurons may be responsible for the recognition between axon and myelinating Schwann cell at the onset of myelination, whereas the homophilic interaction may indicate its role in the self-recognition of the apposing loops of Schwann cell surface membranes during the myelination process and in the mature compact myelin sheath.

Human immunodeficiency virus-like, nonreplicating, gag-env particles assemble in a recombinant vaccinia virus expression system

We report the assembly of human immunodeficiency virus (HIV)-like particles in African green monkey kidney cells coinfected with two recombinant vaccinia viruses, one carrying the HIV-1 gag and protease genes and the other the env gene. Biochemical analysis of particles sedimented from culture supernatants of doubly infected cells revealed that they were composed of gag proteins, primarily p24, as well as the env proteins gp120 and gp41. Thin-section immunoelectron microscopy showed that these particles were 100 to 120 nm in diameter, were characterized by the presence of cylindrical core structures, and displayed the mature gp120-gp41 complexes on their surfaces. Furthermore, thin-section immunoelectron microscopy analysis of infected cells showed that particle assembly and budding occurred at the plasma membrane. Nucleic acid hybridization suggested that the particles packaged only the gag mRNA but not the env mRNA. Therefore, the system we present is well suited for studies of HIV virion maturation. In addition, the HIV-like particles provide a novel and attractive approach for vaccine development.

Intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly

Coronavirus spike protein genes were expressed in vitro by using the recombinant vaccinia virus expression system. Recombinant spike proteins were expressed at the cell surface and induced cell fusion in a host-cell-dependent fashion. The intracellular transport of recombinant spike proteins was studied. The half time of acquisition of resistance to endo-beta-N-acetylglucosaminidase H was approximately 3 h for the recombinant feline infectious peritonitis virus S protein. The S protein in feline infectious peritonitis virus-infected cells was found to have a half time of acquisition of resistance to endo-beta-N-acetylglucosaminidase H of approximately 1 h. This difference can be explained by the fact that coronavirus budding takes place at intracellular membranes and that the oligosaccharides of the spike protein are modified after budding. Apparently, spike protein incorporated into budded virions is transported faster through the Golgi apparatus than is spike protein alone. These findings provide new insights into the mechanism of coronavirus budding and are discussed in relation to current models of intracellular transport and sorting of proteins.

Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes.

A novel expression system based on coinfection of cells with two recombinant vaccinia viruses has been developed. One recombinant vaccinia virus contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second recombinant vaccinia virus contained a target gene of choice flanked by bacteriophage T7 promoter and termination sequences. Maximum expression of the target gene occurred when cells were infected with 10 PFU of each recombinant virus. Although T7 RNA polymerase synthesis began shortly after infection, the target gene was not expressed until late times and was largely inhibited when DNA replication was blocked. Target gene transcripts were analyzed by agarose gel electrophoresis and had the predicted size. With this system, Escherichia coli beta-galactosidase, hepatitis B virus surface antigen, and human immunodeficiency virus envelope proteins were made. In each case, the level of synthesis was greater than had previously been obtained with the more conventional recombinant vaccinia virus expression system.

Use of a Hybrid Vaccinia Virus-T7 RNA Polymerase System for Expression of Target Genes

A novel expression system based on coinfection of cells with two recombinant vaccinia viruses has been developed. One recombinant vaccinia virus contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second recombinant vaccinia virus contained a target gene of choice flanked by bacteriophage T7 promoter and termination sequences. Maximum expression of the target gene occurred when cells were infected with 10 PFU of each recombinant virus. Although T7 RNA polymerase synthesis began shortly after infection, the target gene was not expressed until late times and was largely inhibited when DNA replication was blocked. Target gene transcripts were analyzed by agarose gel electrophoresis and had the predicted size. With this system, Escherichia coli beta-galactosidase, hepatitis B virus surface antigen, and human immunodeficiency virus envelope proteins were made. In each case, the level of synthesis was greater than had previously been obtained with the more conventional recombinant vaccinia virus expression system.