bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple transcription factor binding sites mediate adenovirus E1A transactivation
Authors: Pei R , Berk A J
Published: January 2020

We studied the response of simple synthetic promoter regions to transactivation by the adenovirus early region 1A (E1A) protein. Binding sites for one or two host cell transcription factors were substituted for the E1B promoter region in reconstructed virus mutants, and the response to E1A transactivation was assayed during the early phase of infection. We found that a single CREB/ATF binding site resulted in a surprisingly strong promoter which responded to E1A. A CREB/ATF binding site placed upstream of the E1B TATA box behaved much like the wild-type E1B promoter, which is composed of a single Sp1 binding site plus a TATA box. A single E2F binding site resulted in an extremely weak promoter which did not respond to E1A, much like a single Sp1 site. Two E2F sites in an inverted orientation with the same spacing as in the adenovirus type 2 E2 early promoter produced a strong, E1A-responsive promoter. Substitution of the E4 TATA box region for the E1B TATA box region produced a promoter about five times stronger than the wild-type E1B promoter in the absence of E1A. However, the E4 TATA box substitution did not respond significantly to E1A transactivation. These results directly demonstrate that many different transcription factor binding sites, including the E1B TATA box, a CREB/ATF binding site, and two E2F sites, can mediate E1A transactivation. Other transcription factor binding sites cannot mediate an E1A response; these other sites include the E4 TATA box, a single Sp1 binding site, and a single E2F binding site. Implications of these findings for the mechanism of E1A transactivation are discussed.
Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant
Authors: Whitt M A , Chong L , Rose J K
Published: January 2020

We have used transient expression of the wild-type vesicular stomatitis virus (VSV) glycoprotein (G protein) from cloned cDNA to rescue a temperature-sensitive G protein mutant of VSV in cells at the nonpermissive temperature. Using cDNAs encoding G proteins with deletions in the normal 29-amino-acid cytoplasmic domain, we determined that the presence of either the membrane-proximal 9 amino acids or the membrane-distal 12 amino acids was sufficient for rescue of the temperature-sensitive mutant. G proteins with cytoplasmic domains derived from other cellular or viral G proteins did not rescue the mutant, nor did G proteins with one or three amino acids of the normal cytoplasmic domain. Rescue correlated directly with the ability of the G proteins to be incorporated into virus particles. This was shown by analysis of radiolabeled particles separated on sucrose gradients as well as by electron microscopy of rescued virus after immunogold labeling. Quantitation of surface expression showed that all of the mutated G proteins were expressed less efficiently on the cell surface than was wild-type G protein. However, we were able to correct for differences in rescue efficiency resulting from differences in the level of surface expression by reducing wild-type G protein expression to levels equivalent to those observed for the mutated G proteins. Our results provide evidence that at least a portion of the cytoplasmic domain is required for efficient assembly of the VSV G protein into virions during virus budding.
DNA binding and gene regulation by the herpes simplex virus type 1 protein ICP4 and involvement of the TATA element
Authors: DiDonato J A , Muller M T
Published: January 2020

We report the results of fine mapping the sequences responsible for negative regulation of immediate-early (IE) gene 3 by its own gene product, ICP4. Affinity-purified ICP4 binds the transcriptional start site of IE gene 3 and protein-protein interactions induce a secondary mobility shift that footprints exactly as the primary complex. Since these DNA-protein complexes contain ICP4, it is likely that the two differ only in stoichiometry of protein. Additional data show that the DNA-binding domain recognized by ICP4 can be embedded as a cassette in foreign DNA and that native ICP4 will recognize and bind the resulting DNA. In two different immediate-early promoters, the ICP4 binding site can be located either 3' or 5' of the TATA box; however, the ICP4 site is rotationally displaced from the transcription factor IID (TFIID) site by a roughly one-half helical turn, suggesting that ICP4 and TFIID are on the opposite helical face when bound at their respective sites. In the IE1 and IE3 promoters, binding of ICP4 causes an alteration in the helical geometry of the minor groove of the TATA region as visualized by copper footprinting. In contrast, TATA hypersensitivity was not detected in the glycoprotein D promoter (an early gene promoter containing the ICP4 site separated from TATA by eight helical turns) or in an artificial IE3 promoter construct in which the TATA-A4 separation was increased from 2.5 to roughly 5 helical turns. Such stereospecific and distance-dependent conformational alterations in the TATA box under the influence of ICP4 binding may be important in the repression of immediate-early genes.
Depletion of the surface CD4 molecule by the envelope protein of human immunodeficiency virus expressed in a human CD4+ monocytoid cell line
Authors: Kawamura I , Koga Y , Oh-Hori N , Onodera K , Kimura G , Nomoto K
Published: January 2020

A CD4+ human monocytoid cell line, U937, was transfected with a constructed plasmid which has the envelope gene of human immunodeficiency virus under the transcriptional control of the human metallothionein IIA promoter and was cloned thereafter. These cloned cell lines (EH and EL cells) expressed the viral gp160 in the cytoplasm. The expression of surface CD4 antigen examined by Leu3a and OKT4 monoclonal antibodies, however, disappeared completely in EH cells, which produce a larger amount of gp160, while diminishing only partly in EL cells, which produce a smaller amount of gp160. These results indicate that the level of expression of surface CD4 antigen correlates inversely with the amount of intracellular gp160. Moreover, immunoprecipitation studies using lysate from EH cells showed that OKT4 monoclonal antibody precipitated a significant number of CD4 molecules even after surface CD4 disappeared. However, Leu3a monoclonal antibody, which recognizes the binding site for envelope protein, could not precipitate any CD4 molecules in the same cell lysate. Taken together, these results suggested that CD4 molecules are still synthesized normally after the augmented production of gp160 in the cells but form a complex with the envelope protein in the cytoplasm and become unable to be transported to the cell surface, resulting in the observed depletion of surface CD4 antigen. This mechanism may explain the decrease or absence of surface CD4 antigens in human lymphocytes infected with human immunodeficiency virus.
Identification of amino acids recognized by syncytium-inhibiting and neutralizing monoclonal antibodies to the human parainfluenza type 3 virus fusion protein
Authors: Coelingh K V , Tierney E L
Published: January 2020

Neutralizing monoclonal antibodies specific for the fusion (F) glycoprotein of human parainfluenza type 3 virus (PIV3) were used to select neutralization-resistant antigenic variants. Sequence analysis of the F genes of the variants indicated that their resistance to antibody binding, antibody-mediated neutralization or to both was a result of specific amino acid substitutions within the neutralization epitopes of the F1 and F2 subunits. Comparison of the locations of PIV3 neutralization epitopes with those of Newcastle disease and Sendai viruses indicated that the antigenic organization of the fusion proteins of paramyxoviruses is similar. Furthermore, some of the PIV3 epitopes recognized by syncytium-inhibiting monoclonal antibodies are located in an F1 cysteine cluster region which corresponds to an area of the measles virus F protein involved in fusion activity.
Nucleocapsid mass and capsomer protein stoichiometry in equine herpesvirus 1: scanning transmission electron microscopic study
Authors: Newcomb W W , Brown J C , Booy F P , Steven A C
Published: January 2020

The Brookhaven scanning transmission electron microscope was employed to measure the masses of two nucleocapsid species (of light and intermediate densities) of equine herpesvirus 1. These were found to be 196.7 +/- 9.2 and 229.0 +/- 9.5 megadaltons (MDa), respectively. Biochemical assays showed that neither nucleocapsid contained any significant amount of DNA (less than 0.2% [wt/wt]). Taking into account data on protein composition, we conclude that the difference between their masses is essentially contributed by viral protein 22 (46 kDa), which is an integral component of the maturable intermediate nucleocapsid but not of the abortive light nucleocapsid. In view of earlier ultrastructural information on capsomer symmetry, our mass determinations are consistent only with the 150 hexavalent capsomers being hexamers of the 148-kDa major capsid protein.
Identification and characterization of a human cytomegalovirus gene coding for a membrane protein that is conserved among human herpesviruses
Authors: Lehner R , Meyer H , Mach M
Published: January 2020

A rabbit antiserum was raised against envelope material from purified human cytomegalovirus strain AD169. The serum recognized polypeptides 200, 170, 160, 75, 58, and 45 kilodaltons in size. It was used to screen a cDNA library constructed from poly(A)+ RNA from human cytomegalovirus-infected cells in the expression vector lambda gt11. A recombinant bacteriophage expressing cytomegalovirus-specific sequences was identified, and the corresponding gene was mapped to the HindIII R fragment. The gene is transcribed into a late 1.5-kilobase RNA. The nucleotide sequence of the coding region was determined. Computer analysis of the gene product revealed a polypeptide containing multiple potential membrane-spanning domains, representing a type of protein not identified in the envelope of herpesviruses before. The protein shows homology on the amino acid level to hypothetical proteins from reading frames BBRF3 of Epstein-Barr virus, UL10 of herpes simplex virus type 1, and ORF50 of varicella-zoster virus. By using an antiserum raised against procaryote-expressed parts of the cytomegalovirus membrane protein, a 45-kilodalton structural component of the virus was identified as the gene product.
Genetic identification of endogenous polytropic proviruses by using recombinant inbred mice
Authors: Frankel W N , Stoye J P , Taylor B A , Coffin J M
Published: January 2020

Forty-seven endogenous polytropic murine viruses (Pmv) were identified by examination of proviral-cellular DNA junction fragment segregation in recombinant inbred (RI) mice. Most Pmv loci were found in more than one of the seven RI progenitor strains analyzed, but only four were present in all strains. Chromosomal assignments for 41 Pmv loci were determined by comparing their RI strain distribution patterns with those of known genetic markers. Pmv loci were found dispersed throughout the genome, with chromosomes 1, 3, 4, 5, 7, 11, 12, 15, and 16 each carrying three or more proviruses. Linkage analysis in the AKXD RI set suggested that the gene encoding mink cell focus-forming virus resistance (Rcmfr) of DBA/2J mice is probably not a Pmv provirus. It was also deduced that no single, AKR/J-specific Pmv provirus is required as an env gene donor for thymomagenic mink cell focus-forming viruses. In addition, a Pmv provirus was very closely associated with the albino mutation on chromosome 7.
High recombination rate of an Epstein-Barr virus-simian virus 40 hybrid shuttle vector in human cells
Authors: Stary A , James M R , Sarasin A
Published: January 2020

The stability of an Epstein-Barr virus (EBV)-simian virus 40 (SV40) hybrid shuttle vector, the p205-GTI plasmid, was analyzed in human cells during EBV- or SV40-type replication mode. When the p205-GTI plasmid was maintained as an episomal EBV vector in the human 293 cell line, no rearrangement was detected. To induce the SV40 replication mode, cells containing the episomal p205-GTI plasmid were either transfected with vectors carrying the T antigen gene or infected with SV40. Surprisingly, we observed both production and amplification of different classes of recombinant molecules. Particular types of modifications were found in most of the recombinants. The most striking rearrangement was a duplication of the promoter and enhancer regions of SV40 which was inserted in the thymidine kinase (TK) promoter. This recombination process involved a few bases of homology, and one of the recombination junctions implicated the GC boxes which constitute the essential components of the TK and SV40 early promoters. Our results suggest that a combination of a low level of base homology and a specific DNA sequence function (promoter and enhancer sites) leads to a very high level of recombinational activity during T-antigen-dependent plasmid replication.
The Epstein-Barr virus immediate-early gene product, BMLF1, acts in trans by a posttranscriptional mechanism which is reporter gene dependent
Authors: Kenney S , Kamine J , Holley-Guthrie E , Mar E C , Lin J C , Markovitz D , Pagano J
Published: January 2020

In DNA cotransfection experiments, the Epstein-Barr virus immediate-early gene product, BMLF1, stimulated the chloramphenicol acetyltransferase (CAT) activity of both latent and productive EBV promoters linked to CAT. This BMLF1-induced increase in CAT activity was out of proportion to the effect on CAT mRNA, suggesting a posttranscriptional mechanism. Furthermore, when growth hormone was used as a reporter gene instead of CAT, BMLF1 no longer functioned. Thus, the BMLF1 effect was reporter-gene dependent. The effect of the BMLF1 gene product does not then appear to be directed at promoter activation, but instead may function to increase the level of an as yet unidentified protein(s) required for Epstein-Barr virus infection.
Characterization of hepatitis B virus major surface antigen gene transcriptional regulatory elements in differentiated hepatoma cell lines
Authors: Raney A K , Milich D R , McLachlan A
Published: January 2020

The regulatory DNA sequence elements that control the expression of the hepatitis B virus major surface antigen gene in the hepatoblastoma cell line HepG2 were analyzed by using transient transfection assays. In this system, the hepatitis B virus enhancer increases transcription from the surface antigen promoter approximately twofold. The promoter elements regulating the expression of this gene are within a 200-nucleotide sequence located immediately upstream of the transcription initiation sites. The promoter consists of an 85-nucleotide distal element which increases transcription from the surface antigen gene by two- to fourfold and a proximal element of approximately 115 nucleotides which is essential for transcriptional activity. The proximal and distal promoter elements were shown to bind factors present in HepG2 nuclear extracts, which is consistent with the regulatory role demonstrated for these sequences. The regulatory role of these promoter sequences in the hepatocellular carcinoma cell lines PLC/PRF/5 and Hep3B was also demonstrated, indicating similar transcriptional regulation of the surface antigen gene in each of these differentiated hepatoma cell lines.
Human cytomegalovirus binding to fibroblasts is receptor mediated
Authors: Taylor H P , Cooper N R
Published: January 2020

The binding of radiolabeled human cytomegalovirus (HCMV) strain AD169 to human lymphocytes, lymphoblastoid cell lines, monocytes, and fibroblasts varied over a 20-fold range. Since maximum binding was observed with human foreskin fibroblasts (HFF), interactions of radiolabeled HCMV with this cell type were analyzed quantitatively. Binding of HCMV to HFF at 4 degrees C was specific and saturable; at low viral inputs specific binding averaged 16.4% of input and nonspecific binding was less than 1% of input. Binding curves yielded single-component linear Scatchard plots indicating an average Kd of 1.1 nM and 5,262 available virus-binding sites per cell. A two-component Scatchard curve was obtained at 37 degrees C and reflected viral internalization, since it could be converted to a single-component curve by the use of paraformaldehyde-fixed cells. HCMV strain Towne was found to bind to the receptor used by HCMV strain AD169 with similar affinity. HCMV failed to bind to protease-treated HFF or to HFF grown in the presence of inhibitors of glycosylation. Sialic acid residues, however, were not found to be important in binding. These data indicate that a single type of molecule, likely a glycoprotein, on the surface of HFF serves as a specific receptor for the virus.
Transcriptional activation of homologous and heterologous genes by the hepatitis B virus X gene product in cells permissive for viral replication
Authors: Colgrove R , Simon G , Ganem D
Published: January 2020

The potential of the hepadnavirus X gene product to activate gene expression in trans was tested through a series of cotransfections of X expression vectors with a variety of potential targets for transactivation. The X gene products from human hepatitis B virus (HBV), woodchuck hepatitis virus, and ground squirrel hepatitis virus are all equally active in augmenting the expression of a wide array of target promoters in both permissive and nonpermissive cells. Using the HBV genome itself as the source of X protein, we demonstrate that transactivation of HBV and heterologous genes occurs when X protein is expressed in its native state during productive infection of permissive cells. Run-on transcription analysis indicates that this transactivation occurs at the level of primary transcription.
Restricted growth of attenuated poliovirus strains in cultured cells of a human neuroblastoma
Authors: Agol V I , Drozdov S G , Ivannikova T A , Kolesnikova M S , Korolev M B , Tolskaya E A
Published: January 2020

Cultured cells of a human neuroblastoma, SK-N-MC, were found to be highly resistant to Sabin attenuated poliovirus types 1 and 2 strains; no appreciable cytopathic effect was observed, and the total harvest was generally in the order of 1 PFU per cell or less. On the other hand, related neurovirulent strains of these antigenic types produced a relatively good (2 orders of magnitude higher) yield in a markedly protracted infectious cycle. The limited growth of the attenuated virus in the neuroblastoma cells appeared to be confined to a minor cell subpopulation. Experiments with intratypic (type 1) poliovirus recombinants suggested that the major genetic determinants limiting reproduction of the attenuated polioviruses in the neuroblastoma cells are located in the 5' half of the viral RNA, although the 3' half also appears to contribute somewhat to this phenotype. The possibility that neuroblastoma cells may represent an in vitro model for studying poliovirus neurovirulence is briefly discussed.
Site of the base change in the vaccinia virus DNA polymerase gene which confers aphidicolin resistance
Authors: DeFilippes F M
Published: January 2020

An aphidicolin-resistant mutant of vaccinia virus has been shown to encode an altered viral DNA polymerase that is more resistant to aphidicolin. Marker transfer experiments with the DNA from the resistant virus localized the mutation site to an RsaI segment within the portion of the HindIII-E segment which has been shown to contain the viral DNA polymerase gene. Nucleotide sequence analysis of the mutant DNA showed a single GC to AT transition at position 2430, which indicates a leucine-to-methionine change at residue 645 in the protein.
Cell surface expression of several species of human immunodeficiency virus type 1 major core protein
Authors: Laurent A G , Krust B , Rey M A , Montagnier L , Hovanessian A G
Published: January 2020

The major core protein (p25) of the human immunodeficiency virus type 1 (HIV-1) was characterized by two-dimensional-gel isoelectric focusing. The p25 detectable in HIV-1-infected cells is composed of four species with related isoelectric points. This is due in part to the phosphorylated state of p25. The four species of p25 are expressed on the cell surfaces of infected cells, but only the two most basic species are incorporated into the HIV-1 virion. These findings emphasize the importance of p25 in understanding infection with HIV and might have implications for the development of vaccines.
Epstein-Barr virus latent infection membrane protein increases vimentin expression in human B-cell lines
Authors: Birkenbach M , Liebowitz D , Wang F , Sample J , Kieff E
Published: January 2020

Latent Epstein-Barr virus (EBV) infection activates B-lymphocyte proliferation through mechanisms which are partially known. One approach to further delineate these mechanisms is to identify cellular genes whose expression is augmented in cells latently infected with EBV. Since EBV-negative Burkitt's lymphoma cells can be grown in continuous culture and EBV can establish growth-altering latent infection in these cells, some effects of EBV on B-lymphocyte gene expression can be studied by using this in vitro system. Pursuing this latter approach, we have used cDNA cloning and subtractive hybridization to identify a gene whose expression is increased after EBV infection. This gene encodes the cytoskeletal protein vimentin. Latent infection of established EBV-negative Burkitt's lymphoma cell lines with the transforming EBV strain, B95-8, resulted in dramatic increases in vimentin mRNA and protein levels, while infection with the nontransforming P3HR1 strain failed to do so. Vimentin induction was reproduced by the expression of the single EBV gene which encodes the latent infection membrane protein (LMP). An amino-terminal LMP deletion mutant did not induce vimentin. These results are of particular interest in light of the transforming potential of LMP, as demonstrated in rodent fibroblasts, and the interaction between vimentin and LMP observed in immunofluorescent colocalization and cell fractionation studies.
Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions
Authors: Lever A , Gottlinger H , Haseltine W , Sodroski J
Published: January 2020

Sequences required for efficient packaging of human immunodeficiency virus type 1 (HIV-1) genome RNA into virus particles were identified. Deletion of 19 base pairs between the 5' long terminal repeat and the gag gene initiation codon of HIV-1 resulted in a virus markedly attenuated for replication in human T lymphocytes. The mutant virus was characterized by nearly wild-type ability to encode viral proteins and to produce virion particles. The mutant virions exhibited a significant reduction in the content of HIV-1-specific RNA. These results identify an important component of the HIV-1 packaging signal.
Phenotypic hiding: the carryover of mutations in RNA viruses as shown by detection of mar mutants in influenza virus
Authors: Valcárcel J , Ortín J
Published: January 2020

When influenza virus monoclonal antibody-resistant (mar) mutants are selected by incubation in vitro with excess antibody, 90 to 99% of the mutants are not detectable. This observation may be explained by encapsidation of mar mutant RNAs within phenotypically wild-type envelopes. This phenotypic hiding can be revealed by selection of mar mutants in vivo after virus uncoating. Using experimental procedures appropriate to detect all viable mar mutants in a virus population, we determined precisely the mutation rates to the mar genotype by the fluctuation test for two nonoverlapping monoclonal antibodies.
Characterization of the pneumococcal bacteriophage HB-3 amidase: cloning and expression in Escherichia coli
Authors: Romero A , Lopez R , Garcia P
Published: January 2020

HB-3, a temperate bacteriophage of Streptococcus pneumoniae, synthesizes its own murein hydrolase activity when multiplying on cultures of pneumococcus. The enzyme (HBL) was purified and biochemically characterized as an N-acetylmuramoyl-L-alanine amidase of 36,000 daltons, and a 2.1-kilobase DraI fragment containing the lysin gene (hbl) was cloned and expressed in Escherichia coli. Our results demonstrated that the primary product of the hbl gene is a form with low enzyme activity that can be converted to a more active form under conditions similar to those previously described for the major pneumococcal autolysin (E. García, J.L. García, C. Ronda, P. García, and R. López, Mol. Gen. Genet. 201:225-230, 1985). The phage-encoded amidase requires the presence of choline in the teichoic acids of the pneumococcal cell walls for in vivo and in vitro activity. Comparative biochemical and immunological tests of the phage-encoded and host amidases revealed a remarkable similarity between these enzymes, although analyses of their N-terminal amino acid sequences allowed us to conclude that the amidases are similar but not identical. This appears to be the first description of the cloning of a phage-encoded amidase in gram-positive bacteria.
Conditional poliovirus mutants made by random deletion mutagenesis of infectious cDNA
Authors: Kirkegaard K , Nelsen B
Published: January 2020

Small deletions were introduced into DNA plasmids bearing cDNA copies of Mahoney type 1 poliovirus RNA. The procedure used was similar to that of P. Hearing and T. Shenk (J. Mol. Biol. 167:809-822, 1983), with modifications designed to introduce only one lesion randomly into each DNA molecule. Methods to map small deletions in either large DNA or RNA molecules were employed. Two poliovirus mutants, VP1-101 and VP1-102, were selected from mutagenized populations on the basis of their host range phenotype, showing a large reduction in the relative numbers of plaques on CV1 and HeLa cells compared with wild-type virus. The deletions borne by the mutant genomes were mapped to the region encoding the amino terminus of VP1. That these lesions were responsible for the mutant phenotypes was substantiated by reintroduction of the sequenced lesions into a wild-type poliovirus cDNA by deoxyoligonucleotide-directed mutagenesis. The deletion of nucleotides encoding amino acids 8 and 9 of VP1 was responsible for the VP1-101 phenotype; the VP1-102 defect was caused by the deletion of the sequences encoding the first four amino acids of VP1. The peptide sequence at the VP1-VP3 proteolytic cleavage site was altered from glutamine-glycine to glutamine-methionine in VP1-102; this apparently did not alter the proteolytic cleavage pattern. The biochemical defects resulting from these mutations are discussed in the accompanying report.
An enhancer within the divergent promoter of Epstein-Barr virus responds synergistically to the R and Z transactivators
Authors: Cox M A , Leahy J , Hardwick J M
Published: January 2020

The EA-R and NotI repeat genes of Epstein-Barr virus (EBV) are oriented head to head and separated by a 1,000-base-pair (bp) divergent promoter region. We have identified functional domains within this divergent promoter which are important for regulation of the rightward EA-R gene. Both the R transactivator (Rta) and the Z transactivator (Zta) increase the abundance of correctly initiated EA-R transcripts. A 258-bp fragment (-114 to -372 from the EA-R cap site) contained the primary Rta and Zta response elements and was capable of transferring Rta and Zta activity to a heterologous promoter in an orientation- and position-independent manner. Rta activated this 258-bp enhancer region in both EBV-positive and EBV-negative cells. However, Zta activity appeared to be dependent on another EBV gene product, since Zta activated the enhancer efficiently (500- to 2,000-fold) in EBV-positive cells but had little or no activity in EBV-negative cells. The combination of Rta and Zta produced a striking synergistic effect on the enhancer in the absence of any additional EBV components, suggesting that the interaction between Zta and Rta accounts for the Zta response observed in EBV-positive cells. An Rta response element was mapped to a domain located 60 bp away from a Zta-binding site within the enhancer. Although Rta activated the enhancer and other early promoters without additional EBV- or B-cell-specific factors, it did not activate the lytic cycle of EBV, in contrast to Zta. Immunofluorescence patterns of Rta and Zta with antipeptide antisera indicated that they have overlapping but different subcellular localizations. Both transactivators were found in the nucleus, but Rta was also found in the cytoplasm.
Intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly
Authors: Vennema H , Heijnen L , Zijderveld A , Horzinek M C , Spaan W J
Published: January 2020

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.
Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene
Authors: Vialard J , Lalumière M , Vernet T , Briedis D , Alkhatib G , Henning D , Levin D , Richardson C
Published: January 2020

An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified recombinants, and synthesis of H and F proteins were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot detection and Coomassie blue staining. Glycosylation of the proteins appeared to be impaired somewhat, and the precursor to the F protein was not completely cleaved by the proteases present in insect host cells. On the other hand, both proteins appeared to be active in hemagglutination, hemolysis, and cell fusion assays. Levels of synthesis were in the order of 50 to 150 mg of protein per 10(8) cells.
Adenovirus DNA-binding protein forms a multimeric protein complex with double-stranded DNA and enhances binding of nuclear factor I
Authors: Stuiver M H , van der Vliet P C
Published: January 2020

The 72-kilodalton adenovirus DNA-binding protein (DBP) binds to single-stranded DNA as well as to RNA and double-stranded DNA and is essential for the replication of viral DNA. We investigated the binding of DBP to double-stranded DNA by gel retardation analysis. By using a 114-base-pair DNA fragment, five or six different complexes were observed by gel retardation. The mobility of these complexes is dependent on the DBP concentration, suggesting that the complexes arise by sequential binding of DBP molecules to the DNA. In contrast to binding to single-stranded DNA, the binding of DBP to double-stranded DNA appears to be noncooperative. DBP binds to linear DNA as well as to circular DNA, while linear DNA containing the adenovirus terminal protein was also recognized. No specificity for adenovirus origin sequences was observed. To study whether the binding of DBP could influence initiation of DNA replication, we analyzed the effect of DBP on the binding of nuclear factor I (NFI) and NFIII, two sequence-specific origin-recognizing proteins that enhance initiation. At subsaturating levels of NFI, DBP increases the rate of binding of NFI considerably, while no effect was seen on NFIII. This stimulation of NFI binding is specific for DBP and was not observed with another protein (NFIV), which forms a similar DNA-multimeric protein complex. In agreement with enhanced NFI binding, DBP stimulates initiation of adenovirus DNA replication in vitro especially strongly at subsaturating NFI concentrations. We explain our results by assuming that DBP forms a complex with origin DNA that promotes formation of an alternative DNA structure, thereby facilitating the binding of NFI as well as the initiation of DNA replication via NFI.
Levels of Epstein-Barr virus DNA in lymphoblastoid cell lines are correlated with frequencies of spontaneous lytic growth but not with levels of expression of EBNA-1, EBNA-2, or latent membrane protein
Authors: Metzenberg S
Published: January 2020

The process of Epstein-Barr virus (EBV)-induced transformation of human B lymphocytes results in a cell line that is a mixture of latently and lytically infected cells, with the lytic cells composing roughly 5% to less than 0.0001% of the overall population. A set of nine normal lymphoblastoid cell lines that span a 100- to 200-fold range in average EBV DNA content were studied, and the frequency with which these cells entered a lytic phase of viral growth correlated with their EBV DNA copy number (as a population average). However, neither factor correlated with the levels of expression of transcript for the viral genes EBNA-1, EBNA-2, and latent membrane protein, nor did they correlate with the levels of EBNA-2 protein and latent membrane protein. The rate at which a cell line enters into lytic growth spontaneously is therefore not dependent on the overall steady-state levels of expression of these latent-phase genes.
Expression of the herpes simplex virus type 1 glycoprotein C gene requires sequences in the 5' noncoding region of the gene
Authors: Weir J P , Narayanan P R
Published: January 2020

The role of the 5' noncoding region of the herpes simplex virus type 1 glycoprotein C (gC) gene in viral gene expression was investigated with recombinant herpesviruses that contained the bacterial beta-galactosidase gene under the control of the gC promoter-regulatory region. Each of these viruses had the same DNA sequences from the start of gC transcription upstream to -114 but had variable segments of the downstream 140-base-pair sequence that is between the start of gC transcription and translation. Analysis of beta-galactosidase expression and mRNA synthesis from these viruses demonstrated the importance of DNA sequences from the start of gC transcription downstream to +38 for optimal expression from the gC promoter.
Topoisomerase I and II cleavage of adenovirus DNA in vivo: both topoisomerase activities appear to be required for adenovirus DNA replication
Authors: Schaack J , Schedl P , Shenk T
Published: January 2020

Sites of topoisomerase I and II cleavage across large portions of the adenovirus type 5 genome were mapped by using the drugs camptothecin and VM26, respectively. These drugs prolong the half-lives of the covalent DNA-protein intermediates in which the DNA is transiently cleaved, and so treatment with protein denaturants after exposure to the drugs leads to DNA strand scission at the site of topoisomerase cleavage. Strong topoisomerase II cleavage sites occurred in clusters throughout the regions examined, including both transcribed regions and transcriptional control regions. The efficiency of topoisomerase II cleavage increased as the rate of adenovirus DNA replication increased and then decreased with the decreasing rate of replication late in the infection cycle. The increase was not dependent on expression of the E1A gene, whose products activate transcription of the early viral genes. Positions of topoisomerase II cleavage sites did not vary during the infection. Topoisomerase I cleavage sites were also found throughout the examined regions, with the strongest sites occurring near the ends of the transcription units. Topoisomerase I cleavage in the E1 region occurred much more frequently than topoisomerase II cleavage, was not dependent on E1A gene expression, and remained at a similar level from the early viral phase into the late viral phase. Treatment of infected cells with either drug prevented efficient replication of adenovirus DNA. Inhibition of topoisomerase I activity led to an immediate cessation of adenovirus DNA replication, while inhibition of topoisomerase II blocked replication only after completion of approximately one additional round.
Biosynthesis, maturation, and acid activation of the Semliki Forest virus fusion protein
Authors: Kielian M , Jungerwirth S , Sayad K U , DeCandido S
Published: January 2020

The Semliki Forest virus spike protein has a potent membrane fusion activity which is activated in vivo by the low pH of endocytic vacuoles. The spike protein is composed of two transmembrane subunits, E1 and E2, plus E3, a peripheral polypeptide. Acid-induced conformational changes in the E1 or E2 subunits were analyzed by using monoclonal antibodies specific for the acid-treated spike protein. E1 and E2 reacted with the antibodies after treatment of wild-type or mutant virus at the pH of fusion. The E1 conformational change resembled fusion in its requirement for both low pH and cholesterol. Pulse-chase analysis and intracellular pH treatment were then used to determine the ability of the newly synthesized spike to undergo acid-induced conformational changes. p62, the precursor to E2 and E3, was shown to undergo a pH-dependent conformational change similar to that of E2 and was sensitive to acid very soon after biosynthesis. In contrast, a posttranslational maturation event was required for the conversion of E1 to the pH-sensitive form. E1 maturation occurred fairly late in the exocytic pathway, after the virus spike had passed the medial Golgi but before incorporation of the spike into a new virus particle.
Linker insertion mutagenesis of herpesviruses: inactivation of single genes within the Us region of pseudorabies virus
Authors: de Wind N , Zijderveld A , Glazenburg K , Gielkens A , Berns A
Published: January 2020

We describe a technique for the systematic inactivation of nonessential genes within the genome of a herpesvirus without the requirement for phenotypic selection. This technique is based on the insertion of an oligonucleotide containing translational stop codons at a random site within a large cloned viral DNA fragment. Mutant virus is then reconstituted by cotransfection with overlapping viral clones, together comprising the entire viral genome, as described previously (M. van Zijl, W. Quint, J. Briaire, T. de Rover, A. Gielkens, and A. Berns, J. Virol. 62:2191-2195, 1988). This technique was used to construct, in a single experiment, a set of 13 viable pseudorabies virus strains with oligonucleotide insertions within all known genes of the Us region except for the gp50 gene, which proved essential for virus growth in cell culture. The growth rate in porcine kidney cells of mutants of all nonessential Us genes was similar to that of the parental virus, with the exception of a mutant of the recently identified protein kinase gene.
The immunogenicity of VP7, a rotavirus antigen resident in the endoplasmic reticulum, is enhanced by cell surface expression
Authors: Andrew M E , Boyle D B , Whitfeld P L , Lockett L J , Anthony I D , Bellamy A R , Both G W
Published: January 2020

The glycoprotein VP7, the major serotype antigen of rotaviruses, is localized to the endoplasmic reticulum (ER) of the cell, where it is retained as a membrane-associated protein before assembly into mature virus particles. Wild-type VP7 expressed by a recombinant vaccinia virus was also located internally and was poorly antigenic. Using recombinant techniques, a correctly processed, secreted form of VP7 (S.C. Stirzaker and G.W. Both, Cell 56:741-747, 1989) was modified by addition to its C terminus of the membrane anchor and cytoplasmic domains from the influenza virus hemagglutinin. The hybrid protein was directed to the surface of cells, where it was anchored in the plasma membrane. When expressed in mice and rabbits by a recombinant vaccinia virus, the surface-anchored antigen stimulated a level of rotavirus-specific antibodies that was greater than 100-fold above the level induced by wild-type VP7. T-cell responses to the novel antigen were also elevated in comparison with the wild-type, intracellular protein. Cell surface anchoring may provide a strategy to increase the immunogenicity of intracellular antigens from other parasites and viruses.
A fusion-defective mutant of the vesicular stomatitis virus glycoprotein
Authors: Whitt M A , Zagouras P , Crise B , Rose J K
Published: January 2020

We have recently described an assay in which a temperature-sensitive mutant of vesicular stomatitis virus (VSV; mutant tsO45), encoding a glycoprotein that is not transported to the cell surface, can be rescued by expression of wild-type VSV glycoproteins from cDNA (M. Whitt, L. Chong, and J. Rose, J. Virol. 63:3569-3578, 1989). Here we examined the ability of mutant G proteins to rescue tsO45. We found that one mutant protein (QN-1) having an additional N-linked oligosaccharide at amino acid 117 in the extracellular domain was incorporated into VSV virions but that the virions containing this glycoprotein were not infectious. Further analysis showed that virus particles containing the mutant protein would bind to cells and were endocytosed with kinetics identical to those of virions rescued with wild-type G protein. We also found that QN-1 lacked the normal membrane fusion activity characteristic of wild-type G protein. The absence of fusion activity appears to explain lack of particle infectivity. The proximity of the new glycosylation site to a sequence of 19 uncharged amino acids (residues 118 to 136) that is conserved in the glycoproteins of the two VSV serotypes suggests that this region may be involved in membrane fusion. The mutant glycoprotein also interferes strongly with rescue of virus by wild-type G protein. The strong interference may result from formation of heterotrimers that lack fusion activity.
Recombinant feline herpesviruses expressing feline leukemia virus envelope and gag proteins
Authors: Cole G E , Stacy-Phipps S , Nunberg J H
Published: January 2020

We constructed recombinant feline herpesviruses (FHVs) expressing the envelope (env) and gag genes of feline leukemia virus (FeLV). Expression cassettes, utilizing the human cytomegalovirus immediate-early promoter, were inserted within the thymidine kinase gene of FHV. The FeLV env glycoprotein expressed by recombinant FHV was processed and transported to the cell surface much as in FeLV infection, with the exception that proteolytic processing to yield the mature gp70 and p15E proteins was less efficient in the context of herpesvirus infection. Glycosylation of the env protein was not affected; modification continued in the absence of efficient proteolytic processing to generate terminally glycosylated gp85 and gp70 proteins. A recombinant FHV containing the FeLV gag and protease genes expressed both gag and gag-protease precursor proteins. Functional protease was produced which mediated the proteolytic maturation of the FeLV gag proteins as in authentic FeLV infection. Use of these recombinant FHVs as live-virus vaccines may provide insight as to the role of specific retroviral proteins in protective immunity. The current use of conventional attenuated FHV vaccines speaks to the wider potential of recombinant FHVs for vaccination in cats.
Herpes simplex virus DNA synthesis at a preformed replication fork in vitro
Authors: Rabkin S D , Hanlon B
Published: January 2020

Proteins from herpes simplex virus (HSV)-infected cells were used to reconstitute DNA synthesis in vitro on a preformed replication fork. The preformed replication fork consisted of a nicked, double-stranded, circular DNA molecule with a 5' single-strand tail that was noncomplementary to the template. The products of DNA synthesis on this substrate were rolling-circle molecules, as demonstrated by electron microscopy and alkaline agarose gel electrophoresis. The tails contained double-stranded regions, indicating that both leading- and lagging-strand DNA syntheses occurred. Rolling-circle DNA replication was dependent upon HSV DNA polymerase and ATP and was stimulated by a crude fraction containing ICP8 (HSV DNA-binding protein). Similar protein fractions from mock-infected cells were unable to support rolling-circle DNA replication. This in vitro DNA replication system should prove useful in the identification and characterization of the enzymatic activities required at the HSV replication fork.
Point mutations in the proximal Cys-His box of Rous sarcoma virus nucleocapsid protein
Authors: Dupraz P , Oertle S , Meric C , Damay P , Spahr P F
Published: January 2020

To extend our previous studies of the function of the Cys-His box of Rous sarcoma virus NC protein, we have constructed a series of point mutations of the conserved or nonconserved amino acids of the proximal Cys-His box and a one-amino-acid deletion. All mutants were characterized for production of viral proteins and particles, for packaging and maturation of viral RNA, for reverse transcriptase activity, and for infectivity. Our results indicated the following. (i) Mutations affecting the strictly conserved amino acids cysteine 21, cysteine 24, and histidine 29 were lethal; only the mutant His-29----Pro was still able to package viral RNA, most of it in an immature form. (ii) Mutation of the highly conserved glycine 28 to valine reduced viral RNA packaging by 90% and infectivity 30-fold, whereas mutant Gly-28----Ala was fully infectious. This suggests a steric hindrance limit at this position. (iii) Shortening the distance between cysteine 24 and histidine 29 by deleting one amino acid abolished the maturation of viral RNA and yielded noninfectious particles. (iv) Substitution of tyrosine 22 by serine lowered viral RNA packaging efficiency and yielded particles that were 400-fold less infectious; double mutant Tyr-22Thr-23----SerSer had the same infectivity as Tyr-22----Ser, whereas mutant Thr-23----Ser was fully infectious. (v) Changing glutamine 33 to a charged glutamate residue did not affect virus infectivity. Similarities and differences between our avian mutants and those in murine retroviruses are discussed.