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PubMed1995

Characterization of a highly attenuated Japanese encephalitis virus generated from molecularly cloned cDNA.

Six recombinant Japanese encephalitis virus (JEV) isolates were recovered from infectious RNAs transcribed by T7 RNA polymerase from molecularly cloned cDNA templates. Three of the recombinant viruses had characteristics similar to the wild-type parent virus, JaOArS982. The other 3 recombinant viruses exhibited an attenuated phenotype in mice. An avirulent recombinant virus, IC47, was characterized and compared with the wild-type parent virus and a virulent recombinant virus, IC37. IC47 produced smaller plaques than parent or IC37 viruses and exhibited no viremia or neuroinvasion in young adult mice inoculated subcutaneously and no mortality when inoculated intracerebrally. IC47 was also immunogenic and protective in the murine model. The probable basis for attenuation, revealed by nucleotide sequence analysis, was a single amino acid substitution at position 138 (Glu to Lys) in the E protein.

Animals↗
PubMed1989

Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus.

Full-length cDNA copies of tobacco vein mottling virus (TVMV) RNA were constructed downstream from bacteriophage T7 or T3 RNA polymerase promoters. The plasmids were designed to produce in vitro transcripts containing, respectively, one or two guanosine residues at the 5' terminus not derived from the TVMV sequence and a single cytidine residue at the 3' terminus following the poly(A) tail. Introduction of transcripts from either plasmid into tobacco mesophyll protoplasts resulted in the accumulation of TVMV coat protein and RNA. Neither coat protein nor viral RNA accumulated in protoplasts inoculated with linearized cDNA or with in vitro transcripts synthesized in the absence of 7-methylguanosine(5')triphospho(5')guanosine (m7GpppG). Tobacco seedlings inoculated with native TVMV RNA developed symptoms a few days before those inoculated with in vitro transcripts; however, 3 weeks after inoculation, the symptoms produced by the two inocula were indistinguishable.

Blotting, Western↗
PubMed1991

Cells that express all five proteins of vesicular stomatitis virus from cloned cDNAs support replication, assembly, and budding of defective interfering particles.

An alternative approach to structure-function analysis of vesicular stomatitis virus (VSV) gene products and their interactions with one another during each phase of the viral life cycle is described. We showed previously by using the vaccinia virus-T7 RNA polymerase expression system that when cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with defective interfering (DI) particles, rapid and efficient replication and amplification of (DI) particle RNA occurred. Here, we demonstrate that all five VSV proteins can be expressed simultaneously when cells are contransfected with plasmids containing the matrix protein (M) gene and the glycoprotein (G) gene of VSV in addition to plasmids containing the genes for the N, NS, and L proteins. When cells coexpressing all five VSV proteins were superinfected with DI particles, which because of their defectiveness are unable to express any viral proteins or to replicate, DI particle replication, assembly, and budding were observed and infectious DI particles were released into the culture fluids. Omission of either the M or G protein expression resulted in no DI particle budding. The vector-supported DI particles were similar in size and morphology to the authentic DI particles generated from cells coinfected with DI particles and helper VSV and their infectivity could be blocked by anti-VSV or anti-G antiserum. The successful replication, assembly, and budding of DI particles from cells expressing all five VSV proteins from cloned cDNAs provide a powerful approach for detailed structure-function analysis of the VSV gene products in each step of the replicative cycle of the virus.

Animals↗
PubMed1996

Synthetic transcripts of double-stranded Birnavirus genome are infectious.

We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented double-stranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines.

Animals↗
PubMed2002

Flexibility of the rings: structural asymmetry in the DnaB hexameric helicase.

DnaB is the primary replicative helicase in Escherichia coli and the hexameric DnaB ring has previously been shown to exist in two states in the presence of nucleotides. In one, all subunits are equivalent, while in the other, there are two different subunit conformations resulting in a trimer of dimers. Under all conditions that we have used for electron microscopy, including the absence of nucleotide, some rings exist as trimers of dimers, showing that the symmetry of the DnaB hexamer can be broken prior to nucleotide binding. Three-dimensional reconstructions reveal that the N-terminal domain of DnaB makes two very different contacts with neighboring subunits in the trimer of dimers, but does not form a predicted dimer with a neighboring N-terminal domain. Within the trimer of dimers, the helicase domain exists in two alternate conformations, each of which can form symmetrical hexamers depending upon the nucleotide cofactor used. These results provide new information about the modular architecture and domain dynamics of helicases, and suggest, by comparison with the hexameric bacteriophage T7 gp4 and SV40 large T-antigen helicases, that a great structural and mechanistic diversity may exist among the hexameric helicases.

Bacterial Proteins↗
PubMed1988

A unique deoxyguanosine triphosphatase is responsible for the optA1 phenotype of Escherichia coli.

Escherichia coli optA1, a mutant unable to support the growth of T7 phage containing mutations in gene 1.2, contains reduced amounts of dGTP. Extracts of E. coli optA1 catalyze the hydrolysis of dGTP at a rate 50-fold greater than do extracts of E. coli optA+. The dGTPase responsible for the increased hydrolysis has been purified to apparent homogeneity. Purification of the protein is facilitated by its high affinity for single-stranded DNA. By using this purification scheme an identical dGTPase has been purified from E. coli optA+. The purified proteins catalyze the hydrolysis of dGTP to yield deoxyguanosine and tripolyphosphate. The products of hydrolysis, chromatographic properties, denatured molecular mass of 56 kDa, N-terminal amino acid sequence, substrate specificity, and heat inactivation indicate that the proteins purified from optA1 and from optA+ cells are identical and identify the enzyme as the deoxyguanosine 5'-triphosphate triphosphohydrolase purified to homogeneity from wild-type E. coli [Seto, D., Bhatnagar, S. K. & Bessman, M. J. (1988) J. Biol. Chem. 263, 1494-1499]. OptA1 cells contain approximately equal to 50-fold more active molecules of the 56-kDa dGTPase than do E. coli optA+ cells.

Deoxyguanine Nucleotides↗
PubMed1999

Cis-acting functions of alfalfa mosaic virus proteins involved in replication and encapsidation of viral RNA.

cDNA clones of RNAs 1 and 2 of alfalfa mosaic virus (AMV) were slightly modified to permit transcription of infectious RNAs with T7 RNA polymerase. Together with transcripts of an available clone of AMV RNA 3, these transcripts were used to study cis- and trans-acting functions of AMV proteins in protoplasts from nontransgenic tobacco plants and from plants transformed with the P1 and P2 genes, encoded by RNAs 1 and 2, respectively. Transgenic P1 was unable to complement mutations in the P1 gene in RNA 1, pointing to a cis-acting function of P1 in RNA 1 replication. A study of the replication of RNA 3 mutants in nontransgenic protoplasts revealed that coat protein (CP) expressed from RNA 3 in the inoculum is required in trans for replication and encapsidation of RNAs 1 and 2 but is required in cis for replication and encapsidation of RNA 3. CP is required in the inoculum to initiate infection of nontransgenic plants and protoplasts. When protoplasts expressing both P1 and P2 (P12 protoplasts) were infected with RNAs 1, 2, and 3, initiation of replication of RNAs 1 and 2 required the presence of CP in the inoculum, whereas the initiation of replication of RNA 3 did not. This demonstrated that CP expressed from RNA 3 cannot substitute for the early function of CP in the inoculum. The results showed that CP in the inoculum is required to permit viral minus-strand RNA synthesis, whereas CP expressed from RNA 3 after the initiation of infection is required for plus-strand RNA synthesis.

Alfalfa mosaic virus↗
PubMed2000

Multiple-step kinetic mechanism of DNA-independent ATP binding and hydrolysis by Escherichia coli replicative helicase DnaB protein: quantitative analysis using the rapid quench-flow method.

The kinetic mechanism of DNA-independent binding and hydrolysis of ATP by the E. coli replicative helicase DnaB protein has been quantitatively examined using the rapid quench-flow technique. Single-turnover studies of ATP hydrolysis, in a non-interacting active site of the helicase, indicate that bimolecular association of ATP with the site is followed by the reversible hydrolysis of nucleotide triphosphate and subsequent conformational transition of the enzyme-product complex. The simplest mechanism, which describes the data, is a three-step sequential process defined by:¿eqalign¿¿¿rm Helicase+ATP¿&¿mathop¿¿rightleftharpoons¿ ¿k_1¿_¿k_¿-1¿¿¿¿rm (H-ATP)¿¿mathop¿¿rightleftharpoons¿ ¿k_2¿_¿k_¿-2¿¿¿¿rm (H-ADP¿cdot Pi)¿¿cr &¿mathop¿¿rightleftharpoons¿ ¿k_3¿_¿k_¿-3¿¿¿¿rm (H-ADP¿cdot Pi)¿ *¿The sequential character of the mechanism excludes conformational transitions of the DnaB helicase prior to ATP binding. Analysis of relaxation times and amplitudes of the reaction allowed us to estimate all rate and equilibrium constants of partial steps of the proposed mechanism. The intrinsic binding constant for the formation of the (H-ATP) complex is K(ATP)=(1.3+/-0.5)x10(5) M(-1). The analysis of the data indicates that a part of the ATP binding energy originates from induced structural changes of the DnaB protein-ATP complex prior to ATP hydrolysis. The equilibrium constant of the chemical interconversion is K(H)=k(2)/k(-2) approximately 2 while the subsequent conformational transition is characterized by K(3)=k(3)/k(-3) approximately 30. The low value of K(H) and the presence of the subsequent energetically favorable conformational step(s) strongly suggest that free energy is released from the enzyme-product complex in the conformational transitions following the chemical step and before the product release.The combined application of single and multiple-turnover approaches show that all six nucleotide-binding sites of the DnaB hexamer are active ATPase sites. Binding of ATP to the DnaB hexamer is characterized by the negative cooperativity parameter sigma=0.25(+/-0.1). The negative cooperative interactions predominantly affect the ground state of the enzyme-ATP complex. The significance of these results for the mechanism of the free energy transduction of the DnaB helicase is discussed.

Adenosine Triphosphatases↗
PubMed1991

Red clover necrotic mosaic virus infectious transcripts synthesized in vitro.

The red clover necrotic mosaic dianthovirus (RCNMV) genome is split between two essentially nonhomologous ssRNAs of 3.9 kb (RNA-1) and 1.45 kb (RNA-2) which are each capped at the 5' terminus with m7GpppA. cDNA clones short of full length by several nucleotides at both termini have been generated to both RNAs. Oligonucleotide-directed mutagenesis was employed to generate a series of RNA-1 and -2 transcription vectors in which the bacteriophage T7 RNA polymerase promoter was fused to full-length cDNA clones. Yields of in vitro transcripts initiating with wild-type viral 5'-terminal adenosine were extremely low. Efficient transcription was achieved only when one, or alternatively two, nonviral guanosines were engineered 5' to the authentic viral sequence at the transcription start site. m7GpppG-capped or -uncapped RCNMV RNA-1 and RNA-2 transcripts were infectious and induced symptoms identical to those of wild-type virus infection when coinoculated on the systemic hosts Nicotiana benthamiana and N. clevelandii, and on the local lesion host Chenopodium amaranticolor. Uncapped in vitro transcripts were somewhat less infectious. Progeny virus derived from infectious transcript inoculum was as infectious as wild-type virus. Primer extension analysis indicated that the 5'-terminal nonviral guanosine residues were not maintained in the progeny virus.

Base Sequence↗
PubMed1996

Double-stranded (ds) RNA binding and not dimerization correlates with the activation of the dsRNA-dependent protein kinase (PKR).

Upon binding to double-stranded (ds) RNA, the dsRNA-dependent protein kinase (PKR) sequentially undergoes autophosphorylation and activation. Activated PKR may exist as a dimer and phosphorylates the eukaryotic translation initiation factor 2 alpha subunit (cIF-2 alpha) to inhibit polypeptide chain initiation. Transfection of COS-1 cells with a plasmid cDNA expression vector encoding a marker gene, activates endogenous PKR, and selectively inhibits translation of the marker mRNA, dihydrofolate reductase (DHFR). This system was used to study the dsRNA binding and dimerization requirements for over-expressed PKR mutants and subdomains to affect DHFR translation. DHFR translation was rescued by expression of either an ATP hydrolysis defective mutant PKR K296P, the amino-terminal 1-243 fragment containing two dsRNA binding motifs, or the isolated first RNA binding motif (amino acids 1-123). Mutation of K64E within the dsRNA binding motif 1 destroyed dsRNA binding and the ability to rescue DHFR translation. Immunoprecipitation of T7 epitope-tagged PKR derivatives from cell lysates detected interaction between intact PKR and the amino-terminal 1-243 fragment as well as a 1-243 fragment harboring the K64E mutation. Expression of adenovirus VAI RNA, a potent inhibitor of PKR activity, did not disrupt this interaction. In contrast, intact PKR did not interact with fragments containing the first dsRNA binding motif (1-123), the second dsRNA binding motif (98-243), or the isolated PKR kinase catalytic domain (228-551). These results demonstrate that the translational stimulation mediated by the dominant negative PKR mutant does not require dimerization, but requires the ability to bind dsRNA and indicate these mutants act by competition for binding to activators.

Adenoviridae↗
PubMed2000

Coexpression of proteins in bacteria using T7-based expression plasmids: expression of heteromeric cell-cycle and transcriptional regulatory complexes.

This report describes the development and application of a dual vector coexpression system for the overproduction of heteromeric cell cycle and transcriptional regulatory protein complexes in bacteria. To facilitate these studies we constructed a T7-based expression plasmid, pRM1 that contains an origin of replication derived from p15A, and a gene encoding kanamycin resistance. This expression vector is compatible with ColE1-derived plasmids found in the pET family of T7 expression vectors, which encode ampicillin resistance. It also has the same multiple cloning sites as the pET- derived pRSET vector, allowing easy shuttling between the two expression vectors. Cotransformation of the pRM1 and pET-derived expression vectors into an Escherichia coli strain such as BL21(DE3) results in a significant level of coexpression of heteromeric protein complexes. We demonstrate the applicability of combining the pRM1 and pET-derived vectors for the coexpression of cell cycle regulatory components, pRB/E7 and pRB/E1a, and the transcriptional regulatory complexes, SRF/SAP-1 and SRF/Elk-1. We further use the pRB/E1a complex to demonstrate that these coexpressed complexes can be purified to homogeneity for further studies. Use of the pRM1 vector in combination with the pET-derived vectors should be generally applicable for the large-scale coexpression and purification of a wide variety of heteromeric protein complexes for biochemical, biophysical, and structural studies.

Bacteriophage T7↗
PubMed2002

Recombinant chimeric western and eastern equine encephalitis viruses as potential vaccine candidates.

Chimeric cDNA clones, pMWE1000 and pMWE2000, differing by five nucleotides at their 5' termini, were constructed of the 5' two-thirds of the western equine encephalitis (WEE) virus genome (encoding nonstructural proteins) and the 3' one-third of the eastern equine encephalitis (EEE) virus genome (encoding structural proteins). The WEE virus sequences were derived from full-length cDNA clones, pWE1000 and pWE2000, which were isogenic except for five nucleotide differences at their 5' termini and were responsible for significant differences in mouse virulence. Each cDNA clone was placed downstream from a T7 promoter to allow in vitro transcription of full-length RNA. Transfection of BHK-21 cells with the chimeric RNA by electroporation gave rise to high-titer infectious virus. The in vitro characteristics of each chimera virus were determined by electrophoretic analysis of its structural proteins, plaque morphology, neutralization characteristics, replication kinetics, and rate of viral RNA synthesis. With the exception of plaque morphology, the in vitro characteristics of MWE1000 and MWE2000 were indistinguishable from the parental EEE virus. Subcutaneous inoculation of 5-week-old C57BL/6 mice with varying doses of MWE1000 or MWE2000 virus demonstrated that both chimeric viruses were significantly attenuated compared to the parental WEE virus (Cba 87) and EEE virus (PE-6). Animals infected with 10(5) PFU or more of either MWE1000 or MWE2000 were completely protected from lethal challenge with the virulent EEE virus, FL91-4679, but were not protected from virulent WEE virus Cba 87 challenge. Construction of viable virus chimeras often results in attenuated viruses that may hold promise as genetically engineered alphavirus vaccine candidates (R. J. Kuhn, D. E. Griffin, K. E. Owen, H. G. M. Niesters, and J. H. Strauss, 1996, J. Virol. 70, 7900-7909).

Animals↗
PubMed1998

Detection and quantitation of human respiratory syncytial virus (RSV) using minigenome cDNA and a Sindbis virus replicon: a prototype assay for negative-strand RNA viruses.

We describe here a novel approach for detecting and quantitating human respiratory syncytial virus (RSV) based on expression of a reporter gene from an RSV minigenome. BHK cells were cytoplasmically transformed with a noncytopathic Sindbis virus replicon expressing T7 RNA polymerase. These cells were then cotransfected with T7 expression plasmids that contain the cDNA of an RSV minigenome and the genes for RSV nucleocapsid proteins N, P, and L. The minigenome contains a reporter gene such as lacZ or CAT flanked by cis-acting RSV transcription signals. Subsequent infection of these cells with RSV resulted in a high level of reporter gene expression which could be inhibited by ribavirin. Mock-infected cells exhibited background levels of expression. This assay can be used to quantitate RSV and titer neutralizing antibody and may be a valuable tool for screening compounds for anti-RSV activity. It serves as a prototype for other negative-strand RNA viruses.

Animals↗
PubMed1994

An infectious cDNA copy of the genome of a non-cardiovirulent coxsackievirus B3 strain: its complete sequence analysis and comparison to the genomes of cardiovirulent coxsackieviruses.

The genome of the non-cardiovirulent coxsackievirus B3 (CVB3) strain CVB3/0 was cloned and sequenced to aid in the elucidation of the viral genetic basis for the CVB3 cardiovirulent phenotype. Reverse-transcribed sub-genomic complementary DNA (cDNA) fragments were enzymatically amplified using generic oligonucleotide primers and were assembled as a complete infectious genomic copy (pCVB3-0) downstream of the T7 RNA polymerase promoter. Positive-strand viral RNA transcribed from pCVB3-0 using T7 RNA polymerase and transfected into HeLa cells produced infectious virus (CVB3/0c). No differences in phenotype were observed comparing growth of CVB3/0c to the parental CVB3/0 in HeLa single-step growth curves, virus yields, or plaque size. When inoculated into C3H/HeJ mice, CVB3/0c achieved cardiac titers equivalent to the parental CVB3/0 and like the parental virus, demonstrated a non-cardiovirulent phenotype. The nucleotide sequence of the cloned CVB3/0 genome was determined and compared to the genomes of infectious cDNA clones of cardiovirulent CVB3 strains. Two consistent differences among nucleotides in non-translated regions and 8 amino acid differences relative to two well-characterized infectious cDNA copies of genomes from cardiovirulent CVB3 strains were identified.

Animals↗
PubMed1999

Replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes.

The sequences involved in the replication and packaging of transmissible gastroenteritis virus (TGEV) RNA have been studied. The structure of a TGEV defective interfering RNA of 9.7 kb (DI-C) was described previously (A. Mendez, C. Smerdou, A. Izeta, F. Gebauer, and L. Enjuanes, Virology 217: 495-507, 1996), and a cDNA with the information to encode DI-C RNA was cloned under the control of the T7 promoter. The molecularly cloned DI-C RNA was replicated in trans upon transfection of helper virus-infected cells and inhibited 20-fold the replication of the parental genome. A collection of 14 DI-C RNA deletion mutants (TGEV minigenomes) was synthetically generated and tested for their ability to be replicated and packaged. The smallest minigenome (M33) that was replicated by the helper virus and efficiently packaged was 3.3 kb. A minigenome of 2.1 kb (M21) was also replicated, but it was packaged with much lower efficiency than the M33 minigenome, suggesting that it had lost either the sequences containing the main packaging signal or the required secondary structure in the packaging signal due to alteration of the flanking sequences. The low packaging efficiency of the M21 minigenome was not due to minimum size restrictions. The sequences essential for minigenome replication by the helper virus were reduced to 1,348 nt and 492 nt at the 5' and 3' ends, respectively. The TGEV-derived RNA minigenomes were successfully expressed following a two-step amplification system that couples pol II-driven transcription in the nucleus to replication supported by helper virus in the cytoplasm, without any obvious splicing. This system and the use of the reporter gene beta-glucuronidase (GUS) allowed minigenome detection at passage zero, making it possible to distinguish replication efficiency from packaging capability. The synthetic minigenomes have been used to design a helper-dependent expression system that produces around 1.0 microgram/10(6) cells of GUS.

Animals↗
PubMed1999

Altered growth characteristics of recombinant respiratory syncytial viruses which do not produce NS2 protein.

The second gene in the 3'-to-5' gene order in respiratory syncytial virus (RSV) encodes the nonstructural protein NS2, for which there is no assigned function. To study the function of NS2, we have used a recently developed reverse genetics system to ablate expression of NS2 in recombinant RSV. A full-length cDNA copy of the antigenome of RSV A2 strain under the control of a T7 promoter was modified by introduction of tandem termination codons within the NS2 open reading frame (NS2stop) or by deletion of the entire NS2 gene (DeltaNS2). The NS2 knockout antigenomic cDNAs were cotransfected with plasmids encoding the N, P, L, and M2-1 proteins of RSV, each controlled by the T7 promoter, into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase. Recombinant NS2stop and DeltaNS2 RSVs were recovered and characterized. Both types of NS2 knockout virus displayed pinpoint plaque morphology and grew more slowly than wild-type RSV. The expression of monocistronic mRNAs for the five genes examined (NS1, NS2, N, F, and L) was unchanged in cells infected with either type of NS2 knockout virus, except that no NS2 mRNA was detected with the DeltaNS2 virus. Synthesis of readthrough mRNAs was affected only for the DeltaNS2 virus, where the NS1-NS2, NS2-N, and NS1-NS2-N mRNAs were replaced with the predicted novel NS1-N mRNA. Upon passage, the NS2stop virus stock rapidly developed revertants which expressed NS2 protein and grew with similar plaque morphology and kinetics wild-type RSV. Sequence analysis confirmed that the termination codons had reverted to sense, albeit not the wild-type assignments, and provided evidence consistent with biased hypermutation. No revertants were recovered from recombinant DeltaNS2 RSV. These results show that the NS2 protein is not essential for RSV replication, although its presence greatly improves virus growth in cell culture. The attenuated phenotype of these mutant viruses, coupled with the expected genetic stability associated with gene deletions, suggests that the DeltaNS2 RSV is a candidate for vaccine development.

Animals↗
PubMed1996

Vaccinia virus A17L open reading frame encodes an essential component of nascent viral membranes that is required to initiate morphogenesis.

We generated an antiserum to the predicted C-terminal peptide of the A17L open reading frame (ORF), which encodes a 23-kDa polypeptide with hydrophobic regions characteristic of membrane proteins. Immuno-electron microscopy of infected cells indicated that the A17L protein is intimately associated with the earliest characteristic viral membranes, even those formed in the presence of the drug rifampin. To study the role of the A17L protein in morphogenesis, we constructed recombinant vaccinia viruses in which the endogenous A17L ORF was deleted and a copy of the ORF under the control of the bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor was inserted into an alternative site in the vaccinia virus genome. Growth of these recombinant viruses was entirely dependent on the induction of A17L expression by isopropyl-beta-D-thiogalactopyranoside. Electron microscopic examination of cells infected in the absence of inducer revealed the accumulation of large, well-demarcated electron-dense aggregates but no characteristic membrane-associated viral structures. Viral late protein synthesis occurred under these conditions, although the maturational proteolytic processing of structural proteins was inhibited. We conclude that the product of the A17L gene is an essential component of the immature viral membrane and has an early function in viral morphogenesis.

Animals↗
PubMed1993

A poxvirus-encoded uracil DNA glycosylase is essential for virus viability.

Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells. The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases. The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates. During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E. coli-expressed proteins migrate with an apparent molecular mass of 25 kDa. The insoluble protein aggregate generated by expression in E. coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites. The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity. Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability.

Animals↗