T7 RNA Polymerase Expression Research Articles

Identification of Pseudomonas aeruginosa glpM, whose gene product is required for efficient alginate biosynthesis from various carbon sources.

In a mucB (algN) genetic background, insertion of an omega element approximately 200 bp downstream of glpD, encoding sn-glycerol-3-phosphate dehydrogenase from Pseudomonas aeruginosa, had an adverse effect on alginate biosynthesis from various carbon sources. The insertion inactivated glpM, a gene encoding a 12,040-M(r) hydrophobic protein containing 109 amino acids. This protein, which was expressed in a T7 RNA polymerase expression system, appears to be a cytoplasmic membrane protein.

Expression of the sodium ion pump methylmalonyl-coenzyme A-decarboxylase from Veillonella parvula and of mutated enzyme specimens in Escherichia coli

The structural genes of the sodium ion pump methylmalonyl-coenzyme A (CoA)-decarboxylase from Veillonella parvula have recently been cloned on three overlapping plasmids (pJH1, pJH20, and pJH40) and sequenced. To synthesize the complete decarboxylase in Escherichia coli, the genes were fused in the correct order (mmdADECB) on a single plasmid (pJH70). A DNA region upstream of mmdA apparently served as promoter in E. coli because expression of the mmd genes was not dependent on the correct orientation of the lac promoter present on the pBluescript KS(+)-derived expression plasmid. To allow controlled induction of the mmd genes, the upstream region was deleted and the mmd genes were cloned behind a T7 promoter. The derived plasmid, pT7mmd, was transformed into E. coli BL21(DE3) expressing T7 RNA polymerase under the control of the lac promoter. The synthesized proteins showed the typical properties of methylmalonyl-CoA-decarboxylase, i.e., the same migration behavior during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, stimulation of the decarboxylation activity by sodium ions, and inhibition with avidin. In methylmalonyl-CoA-decarboxylase expressed in E. coli from pT7mmd, the gamma subunit was only partially biotinylated and the alpha subunit was present in substoichiometric amounts, resulting in a low catalytic activity. This activity could be considerably increased by coexpression of biotin ligase and by incubation with separately expressed alpha subunit. After these treatments methylmalonyl-CoA-decarboxylase with a specific activity of about 5 U/mg of protein was isolated by adsorption and elution from monomeric avidin-Sepharose. To analyze the function of the delta and epsilon subunits, the corresponding genes were deleted from plasmid pT7mmd. E. coli cells transformed with pJHdelta2, which lacks mmdE and the 3' -terminal part of mmdD, showed no methylmalonyl-CoA-decarboxylase activity. In addition, a contrast, catalytically active methylmalonyl-CoA-decarboxylase was expressed in E. coli from plasmid pJHdelta1, which contained a deletion of the mmdE gene only. The mutant enzyme could be isolated, reconstituted into proteolipsomes, and shown to function in the transport of Na+ ions coupled to methylmalonyl-CoA decarboxylation. The small epsilon subunit therefore has no catalytic function within the methylmalonyl-CoA-decarboxylase complex but appears to increase the stability of this complex.

Whole-virus vaccine development by continuous culture on a complementing host.

We have evaluated an adaptive strategy for generating whole-virus vaccines using a bacteriophage model. Wildtype phage T7 was cultivated in a two-stage continuous stirred-tank reactor (CSTR) utilizing a recombinant E. coli host that constitutively expressed T7 RNA polymerase, an essential enzyme of the early viral metabolism. Over the course of 180 generations a diversity of phage variants emerged, outgrew the wildtype, and were subsequently eclipsed by yet fitter variants, based on host-ranges, restriction patterns, and one-step growth responses of isolated clones. The fittest variant, which required complementation by the recombinant host in order to grow, deleted at least 12 percent of its genome and replicated twice as fast as the wildtype. Moreover, this variant was immunogenically indistinguishable from the wildtype, based on cross-reactivities of antisera raised against both. These results suggest the feasibility of the proposed strategy for the development of safe whole-virus vaccines.

DUTPase from the Retrovirus Equine Infectious Anemia Virus: High-Level Expression in Escherichia coli and Purification

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. The dUTPase gene of the retrovirus equine infectious anemia virus (EIAV) was cloned and overexpressed in Escherichia coli using the T7 RNA polymerase expression system. The recombinant vector (pET-3a/EDU), constructed by mutagenic PCR, was transformed into E. coli BL21(DE3) pLysS cells, resulting in expression of EIAV dUTPase at about 40% of the extracted protein, This level of overproduction is very high compared to previous reports on heterologous expression of dUTPases in E. coli. A one-step purification procedure using phosphocellulose chromatography results in a homogeneous preparation of the enzyme in a yield of 45 mg liter −1 of bacterial culture. The purified EIAV dUTPase, run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows an apparent molecular mass of 15.1 kDa in accordance with the gene structure. The isoelectric point (p I) was determined to 5.6. Gel filtration under nondenaturating conditions gives a retention volume corresponding to a molecular mass of 40.8 kDa, suggesting a trimeric organization of the enzyme. The amino acid composition and amino-terminal sequence of the recombinant dUTPase are in agreement with predictions from the DNA sequence.

Role of basic residues in the proteolytic activation of Sendai virus fusion glycoprotein

Cleavage activation of the Sendai virus (Fushimi strain) fusion (F) protein was analyzed by site-directed mutagenesis of the amino acids proximal to the highly conserved fusion peptide. In addition, the functional properties of the wild-type and mutant proteins were examined to determine their ability to elicit the formation of syncytia when co-expressed with the hemagglutinin-neuraminidase (HN) glycoprotein. Viral genes were expressed from recombinant T7 transcription vectors (pT7/T3 plasmids) containing F or HN genes, after transfection into cells previously infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3). The wild-type F protein sequence (112VPQSRF) which contains a monobasic cleavage activation site was altered to include a tribasic, 112VPRKRF (mB3), or a pentabasic sequence, 112RRRKRF (mB5) adjacent to the fusion peptide. Although addition of basic residues to the normal protein sequence resulted in enhanced cleavage activation of the mB3 and mB5 proteins, only the mB5 protein was able to induce syncytia formation in CV-1 or HeLa T4 cells. Further analysis by the introduction of acidic residues upstream of the cleavage activation site was performed to determine whether increased hydrophilicity of the surrounding residues might contribute to cleavage activation. The mutants examined, mAcB1 (104NDDEENAGVPQSRF), mAcB3 (104NDDEENAGVPRKRF), and mAcB5 (104NDDEENAGRRRKRF) all contained DEE in replacement for the wild-type TTQ sequence (104NDTTQNAGVPQSRF). Analysis showed that only mAcB3 was efficiently cleaved by the endogenous cellular proteases, while mAcB1 was minimally cleaved, and mAcB5 not at all. Consequently, only the mAcB3 mutant was able to support fusion of CV-1 or HeLa T4 cells when co-expressed with HN.

A hybrid baculovirus-bacteriophage T7 transient expression system.

A hybrid recombinant baculovirus-bacteriophage T7 expression system was developed for transient expression in insect cells of plasmids with foreign genes provided with a T7 promoter. The coding sequence for T7 RNA polymerase, with or without a nuclear localization signal, was inserted into the genome of Autographa californica nuclear polyhedrosis virus. Recombinant viruses stably expressed T7 RNA polymerase in insect cells. Upon transfection of infected insect cells with plasmids containing the genes for chloramphenicol acetyltransferase (CAT), the hepatitis B virus precore-, core- or e- antigens under control of the T7 promoter, transient expression of these genes was detected by ELISA. The results obtained indicate that this baculovirus/T7 system provides a simple and widely applicable tool for transient gene expression studies.

Tight transcriptional control mechanism ensures stable high-level expression from T7 promoter-based expression plasmids.

One of the more efficient systems for high-level expression of cloned genes in Escherichia coli makes use of a phage T7 late promoter whose activity depends on a regulatable transcription unit supplying the specific T7 RNA polymerase. Using various T7 RNA polymerase/T7 promoter-based vector host systems with differential control on expression of the T7 RNA polymerase, we document that leaky expression of the latter is responsible for the frequently observed loss of the culture's ability to express genes of interest. We further show that the inability to achieve detectable expression levels can be overcome by using a tightly repressed expression system. We describe a novel and efficient control system in which basal level expression of T7 RNA polymerase is attenuated by a series of tandemly arranged transcription terminators. The plasmids also incorporate the phage lambda-derived nutL/N protein antitermination function, allowing conditional reversion of attenuation upon induction. The applicability of the system is illustrated by the strictly regulatable, high-level production of several cytokines of human and murine origin.

Thermoregulation of virB transcription in Shigella flexneri by sensing of changes in local DNA superhelicity.

Transcription of the virB gene, a transcriptional regulator of invasion genes on the large plasmid of Shigella flexneri, is strictly regulated by growth temperature; when bacteria are grown at 37 degrees C, virB transcription is highly activated, while at 30 degrees C the level of virB transcription decreases to less than 5% of that at 37 degrees C. Transcription from the virB promoter is activated by VirF, which is encoded on the same plasmid, in a DNA superhelicity-dependent manner (T. Tobe, M. Yoshikawa, T. Mizuno, and C. Sasakawa, J. Bacteriol. 175:6142-6149, 1993). Here we provide evidence supporting the involvement of negative superhelicity in the thermoregulation of virB transcription. A local negatively supercoiled domain in the virB promoter region was created by activating a divergent transcription from the T7 RNA polymerase-dependent promoter, phi 10, which was placed upstream of the virB promoter in the opposite orientation. Transcription from the virB promoter was activated even at 30 degrees C by induction of divergent transcription. Levels of virB transcription correlated with levels of expressed T7 RNA polymerase. Transcriptional activation of virB by the system depended completely upon VirF function. The level of virB transcription achieved by introducing a negatively supercoiled domain was enough to give rise to expression of invasion capacity at 30 degrees C. These results indicated that the repression of virB transcription at 30 degrees C was caused by a reduction in negative superhelicity around the virB promoter region at 30 degrees C.

Gene Therapy Using Dc-Chol Liposomes

AbstractGene therapy offers the potential to deliver genetic material inside the cells for correcting a disease phenotype. Various techniques for the transgene delivery have been developed, among which cationic liposome mediated delivery is widely acceptable. DC-Chol/DOPE cationic liposomes, synthesized in our laboratory, have several alluring features such as low toxicity, non-immunogenicity, ease of utilization and stability. These liposomes have been successfully used as a vector in clinical trials for treating melanoma and cystic fibrosis. Only a small portion of the DNA-liposome complex taken up by the cell is eventually available for gene expression in the nucleus. To overcome the problem of limited expression, we have developed a cytoplasmic expression system in which the reporter gene is placed under the control of a bacteriophage T7 promoter. To initiate the expression of the gene either purified T7 RNA polymerase or T7 autogene expression vector is transfected along with the reporter gene. Prolo...

Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein.

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.

Expression in Escherichia coli: Production and Purification of Both Subunits of the Human General Transcription Factor TFIIE

Both subunits of the human class II transcription factor TFIIE (rTFIIEα and rTFIIEβ) have been overexpressed in Escherichia coli at 26°C using a T7 RNA polymerase expression system and further purified to apparent homogeneity. As in this system rTFIIEα was poorly expressed and copurified with a truncated form, we expressed rTFIIEα as a fusion protein. These overexpressed subunits of TPILE, are similar to the endogenous subunits according to the following criteria: molecular weight, microsequencing, and transcription activity.

High-Level Expression in Escherichia coli and Purification of Yeast Transcription Factor IIIA

Saccharomyces cerevisiae transcription factor IIIA, a sequence-specific DNA binding protein that is required for transcription of 5S rRNA genes by RNA polymerase III, has been expressed in Escherichia coli in a full length, native form. High level expression was achieved through the combined use of a T7 RNA polymerase expression system and of a multicopy plasmid carrying an E.coli gene, argU, which codes for a minor Arg(AGA/AGG) tRNA species. Recombinant yeast transcription factor IIIA was purified to 95% homogeneity, at a final yield of 8 mg/liter of bacterial culture, by three chromatographic steps, and it was shown to be at least 55% active by quantitative in vitro transcription assays.

Low efficiency of the 5' nontranslated region of hepatitis A virus RNA in directing cap-independent translation in permissive monkey kidney cells.

To characterize in vivo the translational control elements present in the 5' nontranslated region (5'NTR) of hepatitis A virus (HAV) RNA, we created an HAV-permissive monkey kidney cell line (BT7-H) that stably expresses T7 RNA polymerase and carries out cytoplasmic transcription of uncapped RNA from transfected DNA containing the T7 promoter. The presence of an internal ribosomal entry site (IRES) within the 5'NTR of HAV was confirmed by using BT7-H cells transcribing bicistronic RNAs in which the 5'NTR was placed within the intercistronic space, controlling translation of a downstream reporter protein (bacterial chloramphenicol acetyltransferase). However, translation directed by the 5'NTR in these bicistronic transcripts and in monocistronic T7 transcripts in which the HAV 5'NTR was placed upstream of the chloramphenicol acetyltransferase coding sequence was very inefficient compared with the translation of monocistronic transcripts containing either the IRES of encephalomyocarditis (EMC) virus or a short nonpicornavirus 5' nontranslated leader sequence. A large deletion within the HAV IRES (delta 355-532) eliminated IRES activity in bicistronic transcripts. In contrast, larger deletions within the IRES in monocistronic transcripts (delta 1-354, delta 1-532, delta 1-633, and delta 158-633) resulted in 4- to 14-fold increases in translation. In the latter case, this was most probably due to a shift from IRES-directed translation to translation initiation by 5'-end-dependent scanning. Translation of RNAs containing either the EMC virus IRES or the nonpicornavirus leader was significantly enhanced by cotransfection of the reporter constructs with pEP2A, which directs transcription of RNA containing the EMC virus IRES fused to the poliovirus 2Apro coding region. This 2Apro enhancement of cap-independent translation suggests a greater availability of limiting cellular translation factors following 2Apro-mediated cleavage of the p220 subunit of the eukaryotic initiation factor eIF-4F and subsequent shutdown of 5' cap-dependent translation. In contrast, pEP2A cotransfection resulted in severe inhibition of translation directed by the HAV IRES in either monocistronic or bicistronic transcripts. This inhibition was due to competition from the EMC virus IRES present in pEP-2A transcripts, as well as the expression of proteolytically active 2Apro. 2Apro-mediated suppression of HAV translation was not seen with transcripts containing large deletions in the HAV IRES (delta 158-633, delta 1-532, or delta 1-633). These data suggest that the HAV IRES may have a unique requirement for intact p220 or that it may be dependent on active expression of another cellular translation factor which is normally present in severely limiting quantities.(ABSTRACT TRUNCATED AT 400 WORDS)

Self-amplifying expression from the T7 promoter in 3T3 mouse fibroblasts

To increase the levels of exogenous or foreign gene expression in mammalian cells, this study sought to develop an ‘autogene’ that will self-amplify. An autogene plasmid, pT7-G1, containing the T7 phage RNA polymerase-encoding modified gene ( G1) under control of its cognate T7 promoter, was only obtained when the plasmid contained the encephalomyocarditis (EMC) untranslated sequence. In vitro transcription and translation studies confirmed that both the T7 promoter and the G1 gene were completely functional in the pT7-G1 plasmid. Expression from pT7-G1 was initiated in vivo either by co-transfection with its in vitro transcript or by transfection into NIH3T3 cell lines that stably expressed T7 RNA polymerase enzyme. Use of the pT7-G1 autogene enabled an approx. 50-fold increase in foreign protein production. Northern analysis suggested that this increased expression resulted from the self-amplification of the autogene. By allowing greater expression in cell lines with low T7 RNA polymerase expression, the pT7-Gl plasmid increases the usefulness of the T7 gene system for expression within mammalian cells.

Efficient use of lactose for the lac promoter-controlled overexpression of the main antigenic protein of the foot and mouth disease virus in Escherichia coli under fed-batch fermentation conditions.

Derivatives of the lac promoter (tac, pac, rac) belong to the strongest bacterial promoters which are frequently used for the induced overexpression of foreign genes in Escherichia coli. However, their use in fermentation processes is strongly restricted because of the high cost of the inducer iso-propyl-beta-D-thiogalactopyranoside (IPTG). The aim of this work was to investigate the possibility of using lac-derived promoters in high cell density processes resulting in a high yield of the induced recombinant protein if glucose is the main carbon and energy source. Lactose is tested as inducer of the main antigenic coat protein (VP1) of the foot and mouth disease (FMD) virus in a T7-RNA polymerase expression system. It was shown that lactose is able to induce the expression of the recombinant gene to an amount of the VP1 protein corresponding to 20% of the total cell protein.

The sequence of a second member of the lima bean lectin gene family and the expression and characterization of recombinant lectin in Escherichia coli.

The lima bean lectin recognizes terminal alpha-D-GalNAc groups and agglutinates human type A erythrocytes. We have cloned a portion of the gene encoding the alpha subunit of the lima bean lectin. The clone was obtained using the polymerase chain reaction and verified from a genomic clone encoding the mature protein of 253 amino acids. The deduced amino acid sequence has significant overall homology with other leguminous plant lectins and contains all of the known peptide sequences isolated from lima bean lectin (LBL). Southern blot analysis reveals the presence of several genes which hybridize to the cloned gene and which we propose are genes included in the lima bean lectin gene family. We report here the sequence, expression, and characterization of LBL 2, the second member of this gene family. Milligram quantities of soluble active recombinant lima bean lectin (rLBL) were obtained from Escherichia coli, using the T7 RNA polymerase expression system. SDS-polyacrylamide gel electrophoresis and Western blot analysis indicate expression of one protein band of about 27 kDa in induced E. coli cells. This protein cross-reacts with polyclonal antibodies raised against seed lectin (sLBL) and gave a reaction of identity with seed lectin by Ouchterlony double diffusion, specifically agglutinates type A blood cells, and is specifically inhibited by D-GalNAc. The isoelectric point of rLBL is 5.86, whereas those of the seed lectin subunits were determined to be 5.86, 5.58, and 5.20 (previously designated alpha, beta, alpha', respectively). rLBL binds to hydrophobic ligands independent of sugar binding, an observation similar to results obtained with sLBL. However, despite the similar activities described, several significant differences between recombinant and native lima bean lectin were found, including mobility on gel filtration, aggregation in solution, and its CD spectrum. These differences may be due to a number of factors, which will be discussed.

Serological Responses of Patients with Nasopharyngeal Carcinoma to an N-Terminal Epstein-Barr Virus DNA Polymerase Protein Expressed in Prokaryotic Cells.

A 1.7-kb cDNA clone, pGEM-cDP, was isolated from a cDNA library of IUdR-induced p3HR1 cells. It contains the upstream nucleotide sequence of the Epstein-Barr virus (EBV) DNA polymerase gene from 156,859 to 155,088, and was subcloned into expression vector pET3cp* by the polymerase chain reaction, giving the plasmid pDP1. Using a T7 RNA polymerase expression system, a 77-kD polypeptide was produced from pDP1 in Escherichia coli and specific hyperimmune serum was generated in mice. The truncated EBV DNA polymerase was shown to possess the authentic antigenicity by an indirect immunofluorescence assay and by immunoblotting using EBV-containing cells as antigens. Serum from nasopharyngeal carcinoma (NPC) patients and healthy donors was examined for antibodies against the 77-kD polypeptide by Western blot analyses and ELISAs. About 70% NPC patients were positive, while less than 15% of healthy persons showed weak reactivities in ELISAs. Copyright 1994 S. Karger AG, Basel

Expression of the Rubella Virus Nonstructural Protein ORF and Demonstration of Proteolytic Processing

To analyze the proteins produced from the rubella virus (RUB) nonstructural protein open reading frame (NSP-ORF), a DNA containing the RUB NSP-ORF was introduced into the expression vector pTM3 in which the sequences to be expressed are downstream from a T7 RNA polymerase promoter. In cells infected with a vaccinia virus recombinant which expresses T7 RNA polymerase and transfected with this plasmid, three RUB-specific products with electrophoretic mobilities of 200, 150, and 97 kDa were clearly visible. By computer alignment, the presence of a cysteine protease was predicted within the NSP-ORF (A. E. Gorbalenya et al., FEBS Lett. 288, 201-205, 1991. When the Cys proposed as the catalytic residue of this protease (Cys 1151) was mutated to a Gly, only the 200-kDa product was produced, demonstrating that the Cys is important in the activity of the protease responsible for the processing of the RUB NSPs and that the 150- and 97-kDa species are processing products. Transfections with deletion mutants revealed that the 150-kDa processing product is derived from the amino-terminal two-thirds of the ORF and that both the protease and the cleavage site on the COOH-terminal side of the 150-kDa product are between amino acids 1005 and 1507 of the ORF.

Rescue of synthetic genomic RNA analogs of rabies virus by plasmid-encoded proteins

Proteins entirely expressed from cDNA were used to rescue synthetic RNA genome analogs into infectious defective particles of rabies virus (RV). Synthetic negative-stranded RNAs containing 3'- and 5'-terminal RV sequences and transcriptional signal sequences were transcribed from plasmids transfected into cells expressing T7 RNA polymerase from recombinant vaccinia virus. After simultaneous expression of RV N, P, and L proteins from plasmids containing a T7 RNA polymerase promoter, the synthetic genomes were encapsidated, replicated, and transcribed by the RV polymerase proteins. Insertion of the bacterial chloramphenicol acetyltransferase gene or beta-galactosidase (lacZ) gene between the 3' and 5' termini containing transcriptional signal sequences resulted in transcription of mRNAs and expression of chloramphenicol acetyltransferase and beta-galactosidase, respectively. Upon simultaneous expression of N, P, M, G, and L proteins, virions carrying the foreign genes were assembled and released into the supernatant. The possibility of rescuing cDNA into rabies virions by proteins also expressed entirely from cDNA opens the possibility of studying the functions of each RV protein and analyzing cis-acting signals of the RV genome.

Paramyxovirus mediated cell fusion requires co-expression of both the fusion and hemagglutinin-neuraminidase glycoproteins

Syncytia formation in either CV-1 or HeLa T4 + cells required recombinant expression of both fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins from the human parainfluenza virus type 3 (HPIV3), human parainfluenza virus type 2 (HPIV2), and simian virus 5 (SV5). In this system, recombinant T7 transcription vectors (pT7-5 or pGEM) containing F or HN, were transfected individually or in combination into cells previously infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3). While both proteins were processed and expressed at the cell surface, syncytia formation occurred only when both glycoproteins were co-expressed. The function of HN in the fusion process could not be replaced using lectins or by co-expression of heterologous F and HN proteins. Further, cell fusion was not observed when experiments were performed using individually expressed F and HN proteins in adjacent cells. The data presented in this report support the notion that a specific interaction between both paramyxoviral glycoproteins is required for the formation of syncytia in tissue culture monolayers.