bluuebunny/crossref_metadata_2025 huggingface dataset:
Organization and expression of mouse mammary tumor virus sequences in normal and neoplastic C3Hf/HeSed mouse tissues
Authors: Popko B J , Pauley R J
Published: January 2020

The organization and expression of germinally transmitted mouse mammary tumor virus (MMTV) proviruses in C3Hf/HeSed mouse tissues were examined. Digestion with the restriction enzymes EcoRI, BamHI, and HindIII and hybridization with cloned probes specific for the long terminal repeat and the 5' and 3' regions of the MMTV genome revealed three full-length (units Ib, II, and V) and two subgenomic (units I and IX) MMTV proviruses in C3Hf/HeSed mouse germ line DNA. The EcoRI fragments (15.0 and 5.7 kilobase pairs [kbp]) that contained unit Ib were previously described as separate, subgenomic MMTV proviruses. The methylated state of each full-length MMTV provirus was examined in DNA from C3Hf/HeSed mouse livers, spleens, mammary glands, and mammary tumors by digestion with EcoRI or BamHI in combination with the methyl-sensitive restriction enzymes HhaI or HpaII. Unit Ib contained HhaI- and HpaII-sensitive sites in spleen, mammary gland, and mammary tumor DNA but was completely methylated in liver DNA. Units II and V contained HhaI- and HpaII-sensitive sites in mammary gland and mammary tumor DNA, but the sites were extensively methylated in spleen and liver DNA. The HhaI-sensitive sites were mapped to the 5' end of the 5' and 3' long terminal repeats of each full-length MMTV provirus. C3Hf/HeSed mouse tissue RNA was examined for MMTV transcripts. Mammary glands contained MMTV RNA species of 9.0, 3.8, and 1.7 kb. Mammary tumors contained high levels of the 9.0- and 3.8-kb transcripts but lacked the 1.7-kb species. A very low level of the 3.8-kb MMTV transcript was present in spleens. Livers lacked detectable MMTV RNA. These results implicate mammary tissue as the site of unit V activation in the formation of MMTV virions.
Neutralizing monoclonal antibodies to hepatitis A virus: partial localization of a neutralizing antigenic site
Authors: Hughes J V , Stanton L W , Tomassini J E , Long W J , Scolnick E M
Published: January 2020

BALB/c mice were immunized with purified preparations of hepatitis A virus (HAV) isolated after 21 days of growth in LLC-MK2 cells. The HAV antigen was isolated from CsCl gradients and consisted primarily of the following three proteins as analyzed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining: VP-1 at 33,000 daltons, VP-2 at 29,000 to 30,000 daltons, and VP-3 at 27,000 daltons. The spleen cells isolated from two BALB/c mice, immunized with two inoculations of HAV, were fused with SP 2/0 myeloma cells and grown in hypoxanthine-aminopterin-thymidine medium. Of 270 hybridomas initially screened, 72 were positive for binding HAV by a noncompetitive radioimmunoassay. All 72 were tested for the ability to neutralize the infectivity of HAV in an in vitro cell culture assay that was adapted for microtiter plates and that used detergent-treated virus for improved neutralization sensitivity and newborn cynomolgus monkey kidney cells for rapid growth. Eighteen hybridomas were positive for neutralization; 16 remained stable. Of the 16, 9 were able to compete with labeled polyclonal serum for binding to HAV. The nine competing hybridomas could be separated into two groups which appear to be directed towards two different sites on HAV and could complement each other in the competitive radioimmunoassay against polyclonal sera. Of the original 16 neutralizing hybridomas, 4 were subcloned through two cycles of limit dilutions. All four monoclonal antibodies retained their original neutralizing and competitive properties; three were immunoglobulin G2a, and one was immunoglobulin G1. All four monoclonal antibodies readily precipitate whole 125I-labeled HAV but are not able to recognize the disrupted proteins of the virus (as tested by immune precipitations of heat- and detergent-disrupted virions or Western blot analyses). However, the heterobifunctional cross-linking reagent toluene-2,4-diisocyanate was used to cross-link purified Fab fragments of two different monoclonal antibodies (2D2 and 6A5) to HAV before disruption. This reagent demonstrated a specific reaction of the monoclonal antibodies to the VP-1 of HAV, suggesting this major surface protein contains at least one of the major neutralization sites for HAV.
Vaccinia virus induces ribonucleotide reductase in primate cells
Authors: Slabaugh M B , Johnson T L , Mathews C K
Published: January 2020

Infection of monkey kidney (BSC-40) cells with vaccinia virus strain WR resulted in a marked increase in ribonucleoside diphosphate reductase (EC 1.17.4.1) activity as measured by CDP reduction in cell-free extracts. After a synchronous infection, increased activity was detected at 2 h, peaked at 4 to 5 h, and then declined between 6 and 8 h to the endogenous cellular level. The induction, detectable at 0.5 PFU/cell, correlated strongly with multiplicity of infection to 10 PFU/cell and continued to increase to 50 PFU/cell. It paralleled the previously described induction of viral DNA polymerase and thymidine kinase, suggesting that the reductase may also be a product of early transcription of the viral genome. The inhibition of DNA synthesis throughout infection resulted in prolonged accumulation of reductase activity and delayed and incomplete down-regulation at 8 h, suggesting that repression involves late functions. Rescue of fluorodeoxyuridine-inhibited DNA synthesis with exogenous thymidine restored the normal pattern. Preferential association of the induced reductase with the cytoplasmic sites of vaccinia virus DNA replication (virosomes) was not detected. The induced enzyme is similar in several respects to other eucaryotic ribonucleotide reductases, but is distinct from host cell reductase in response to certain modulators of reductase activity (M. B. Slabaugh and Christopher K. Mathews, J. Virol. 52:501-506, 1984). Full activity required an activator, exogenous reducing equivalents, and iron. Hydroxyurea, EDTA, dATP, and dTTP inhibited CDP reduction, setting this reductase apart from T4 reductase, which is not inhibited by dATP, and from herpesvirus reductase, which requires no activation and is insensitive to deoxyribonucleoside triphosphate inhibition.
Identification of distinct antigenic determinants on Semliki Forest virus by using monoclonal antibodies with different antiviral activities
Authors: Boere W A , Harmsen T , Vinjé J , Benaissa-Trouw B J , Kraaijeveld C A , Snippe H
Published: January 2020

Fifteen monoclonal antibodies (MAs) directed against either the E1 or E2 glycoprotein of Semliki Forest virus (SFV) were characterized by immunoglobulin subclass, pI traject, hemagglutination inhibition, neutralization of infectious virus, and protection against virulent infection in mice. All MAs except UM8.4 (immunoglobulin M [IgM]) belonged to various subclasses of IgG and predominantly to IgG2a, but all were unique as indicated by their banding patterns in isoelectric focusing. Competitive binding assays with these MAs revealed the presence of at least six distinct antigenic determinants (epitopes) on the E1 glycoprotein and five epitopes on the E2 glycoprotein. Two of the epitopes on E1, as defined by the properties of the MAs, were associated with hemagglutination inhibition (E1c and E1d), three were associated with neutralization (E1a, E1b, and E1f), and five were associated in various degrees with protection (E1a, E1b, E1c, E1e, and E1f) of mice against virulent SFV infection. With the MAs against E2, the epitopes on E2 were similarly defined. Epitopes E2b and E2e were associated with hemagglutination inhibition, E2c and E2d were associated with neutralization, and three epitopes were associated with in vivo protection (E2a, E2c, and E2d). Furthermore, for each MA the relative avidity to purified SFV was determined with an enzyme-linked immunosorbent assay. The binding of some MAs to purified SFV was enhanced by a second MA. The relative avidities of individual MAs did not correlate with their neutralizing capacities. From the results, we suggest that the amino acid sequence which makes up determinant E2d and is recognized by the highly protective MA UM5.1 is an excellent candidate for the production of a synthetic vaccine.
Transduction of the Chinese hamster ovary aprt gene by herpes simplex virus
Authors: Tackney C , Cachianes G , Silverstein S
Published: January 2020

The Chinese hamster ovary adenine phosphoribosyl transferase gene (aprt) was reengineered to be flanked by sequences from the thymidine kinase (tk) gene of herpes simplex virus. This construct was cotransfected with DNA from herpes simplex virus type 1, and after 3 days, virus was harvested and Tk- plaques were selected after the virus was plated on Tk- cells in the presence of bromodeoxycytosine. Recombinant viruses were identified by dot-blot hybridization, and the arrangement of aprt and tk sequences were determined by Southern blot hybridization. Analysis of the recombinants revealed that acquisition of aprt sequences resulted from insertional inactivation of the tk locus as a consequence of homology-based recombination. Recombination was precise, as evidenced by the failure to detect plasmid sequences or the synthetic restriction endonuclease sites that bounded the mutant tk gene in the aprt-tk construct. Infection of Aprt- mouse or Chinese hamster ovary cells with UV-irradiated virus and selection in medium containing azaserine and adenine resulted in the survival of numerous colonies that stably express the aprt gene. Transformed cells synthesized an aprt mRNA that is identical to wild-type mRNA as determined by Northern blot and S1 nuclease analyses. Cells lytically infected with the recombinant virus do not appear to transcribe the aprt gene. Thus, infected cells differentiate between virus and foreign promoters even when a cellular gene is cis to the virus chromosome.
Herpes simplex virus types 1 and 2 homology in the region between 0.58 and 0.68 map units
Authors: Draper K G , Frink R J , Devi G B , Swain M , Galloway D , Wagner E K
Published: January 2020

The homology between herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2, respectively) DNA between 0.58 and 0.674 map units was compared by Southern and dot blot analysis with DNA of one type of virus as a hybridization probe against the other type. Regions of high homology were interspersed with regions of detectably lower homology. However, only one region (between 0.647 and 0.653 map units) contained few or no homologous sequences. In situ RNA blot hybridization demonstrated that the mRNA species transcribed in the right-hand portion of the region are homologous between HSV-1 and HSV-2, as was previously found for the left-hand portion. A 2.7-kilobase HSV-2 transcript in the right-hand portion of the studied region was clearly that encoding HSV-2 glycoprotein C. Comparative nucleotide sequence analysis of specific regions demonstrated that homologous translational reading frames could be identified in the virus types. This analysis also demonstrated that homology could be abruptly lost outside such reading frames. Comparison of regions of homology with published HSV-1 transcription maps suggests that there can also be large divergence within translational reading frames. Some, but not complete, sequence homology was seen in the putative promoter sequence for the 730-base HSV-1 mRNA mapping to the right of glycoprotein C and the corresponding HSV-2 DNA. This suggests that the rather strict conservation of promoter sequences between homologous HSV-1 and HSV-2 transcripts seen in other regions of the genome may not be a necessary feature between these virus types.
Role of immunity in age-related resistance to paralysis after murine leukemia virus infection
Authors: Hoffman P M , Robbins D S , Morse H C
Published: January 2020

Resistance to the paralytic effects of a wild mouse (Cas-Br-M) murine leukemia virus infection develops with age and is complete by 10 days of age in susceptible NFS mice. The possibility that cell-mediated immunity plays a significant role in this resistance was suggested by the observation that treatment of 10-day-old mice with antithymocyte serum rendered them susceptible to paralysis. By comparison, mice rendered incapable of generating a humoral immune response by treatment from birth to 1 month of age with anti-immunoglobulin M serum did not develop paralysis after challenge with virus at day 10. Transfer of unseparated and T-cell-enriched populations of Cas-Br-M murine leukemia virus-immune spleen cells protected neonatally infected NFS recipients from paralysis; transfer of Cas-Br-M murine leukemia virus-immune populations enriched for B cells delayed the onset but did not ultimately protect neonatally infected NFS mice from paralysis. Transfer of naive adult spleen cells had no protective effect in neonatally infected NFS mice. High-level virus replication occurred in the spleens and brains of all mice that developed paralysis regardless of treatment; low-level virus replication in spleen and barely detectable replication in brain occurred in mice that remained clinically normal. These studies suggest that the age-acquired resistance to the paralytic effect of Cas-Br-M murine leukemia virus infection is immunologically mediated and that T cells may play a major role.
Construction of bacteriophage luminal diameterX174 mutants with maximum genome sizes
Authors: Russell P W , Müller U R
Published: January 2020

The bacteriophage phi X174 strain ins6 constructed previously was used to investigate the maximum genome size that could be packaged into the icosahedral phage without concomitant loss of phage viability. The J-F intercistronic region of ins6, which already contains an insert of 117 base pairs with a unique PvuII site, was enlarged further by insertion of HaeIII restriction fragments of the plasmid pBR322 into that PvuII site. By using a biochemical approach for the site-specific mutagenesis as well as selection of mutant genomes, a series of mutants was isolated with genomes of up to 5,730 nucleotides, 6.4% larger than that of the wild-type DNA. Phages with genomes larger than 5,550 nucleotides were highly unstable and were rapidly outgrown by spontaneously occurring deletion mutants. The data predict that genomes of at least 6,090 nucleotides could be constructed and, most likely, packaged, but the resulting phages would not grow well. We speculate that the volume of the phage capsid is not the limiting factor of genome size or is not the only limiting factor.
Two classes of transformation-deficient, immortalization-positive simian virus 40 mutants constructed by making three-base insertions in the T antigen gene
Authors: Sugano S , Yamaguchi N
Published: January 2020

We constructed two mutants of simian virus 40 (SV40) by introducing a three-base duplication at AvaII cutting sites within the large T antigen coding region, and we examined these mutants for their abilities to replicate in monkey GC7 cells, to transform rat cell line 3Y1 cells, and to transform and immortalize primary cells from newborn rats. Neither of the mutants could replicate in GC7 cells. One mutant with the duplication at 0.335 SV40 map units (m.u.) (inA942) could transform 3Y1 cells, but the other mutant with the duplication at 0.636 m.u. (inA941) could not. The two mutants could not transform primary rat cells but retained immortalization activity. The results suggest that transformation of primary cells by SV40 requires at least two distinct activities of the large T antigen, one of which can be replaced by a cellular function(s) expressed in immortalized 3Y1 cells.
Cloned duck hepatitis B virus DNA is infectious in Pekin ducks
Authors: Sprengel R , Kuhn C , Manso C , Will H
Published: January 2020

Approximately 10% of German-bred Pekin ducks were found to be chronically infected with duck hepatitis B virus (DHBV). The genomes of three German DHBV isolates analyzed were closely related but showed substantial restriction site polymorphism compared with U.S. isolates. We tested the infectivity of three sequence variants of cloned DHBV DNA by injecting them into the liver of virus-free ducklings. Most of these animals injected with double-stranded closed-circular or plasmid-integrated dimer DHBV DNA developed viremia, demonstrating the infectivity of all three cloned DHBV DNA variants. The cloned viruses produced were indistinguishable from those from naturally infected animals, implying that our experimental approach can be used to perform a functional analysis of the DHBV genome.
Classification of herpesvirus saimiri into three groups based on extreme variation in a DNA region required for oncogenicity
Authors: Medveczky P , Szomolanyi E , Desrosiers R C , Mulder C
Published: January 2020

The leftmost 7 kilobase pairs of unique sequence L-DNA of herpesvirus saimiri was found to be highly variable among different strains as determined by restriction endonuclease analysis and blot hybridization. This region in one group of viruses (group A) showed only very weak hybridization with the DNA of two other groups. Similarly, a fragment of group B hybridized to DNA of its own group much more strongly than to group A. No homology was detected within a 1.2-kilobase-pair region between strain 11 (group A virus) and strain SMHI (group B) even under reduced stringency, and the adjacent 5.5-kilobase-pair segment of the region showed only a very weak intergroup hybridization. DNA of a third group of viruses (non-A, non-B) did not hybridize significantly with cloned fragments representing the leftmost 7-kilobase-pair region of either group A or group B. Since sequences in the highly variable region are required for the oncogenicity of the virus, these results raise interesting questions regarding the origin and function of this region of the genome.
Sequence analysis of the respiratory syncytial virus phosphoprotein gene
Authors: Satake M , Elango N , Venkatesan S
Published: January 2020

A recombinant cDNA plasmid (pRSA3) containing an almost full-length copy of the mRNA encoding respiratory syncytial virus phosphoprotein was identified in a cDNA library prepared with mRNA from respiratory syncytial virus-infected cells. The cDNA insert was sequenced, and a protein of 27,150 daltons was deduced from the DNA sequence. The protein is relatively acidic, containing two clusters of acidic amino acids, one in the middle of the molecule and the other at the C-terminus. It is devoid of both cysteine and tryptophan. There was no other potential reading frame within the phosphoprotein gene of respiratory syncytial virus. This situation is unlike that with Sendai virus, a paramyxovirus, which has a nonstructural C protein encoded by a second overlapping reading frame near the 5' end of the mRNA for phosphoprotein.
Demonstration of three major species of pseudorabies virus glycoproteins and identification of a disulfide-linked glycoprotein complex
Authors: Lukàcs N , Thiel H J , Mettenleiter T C , Rziha H J
Published: January 2020

The glycoproteins of pseudorabies virus (PRV) Phylaxia were characterized with monoclonal antibodies as specific reagents. Three major structural glycoproteins with molecular weights of 155,000 (155K) (gC), 122K (gA), and 90K (gB) could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. We investigated the processing of glycoproteins gA, gB, and gC by in vitro translation, pulse-chase experiments, and in the presence of the ionophore monensin which inhibits glycosylation. gA and gB were found to compose a single polypeptide, whereas gC was found to be a disulfide-linked glycoprotein complex. Immunoprecipitates formed with the aid of anti-gC monoclonal antibodies gave rise to three glycoprotein bands (gC0 [120K], gC1 [67K], and gC2 [58K]) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Limited proteolysis of gC0, gC1, and gC2 resulted in peptide maps of gC0 related to those of both gC1 and gC2. No common peptide bands between gC1 and gC2, however, were seen. We suggest that (i) gC1 and gC2 arise by proteolytic cleavage from the same precursor molecule and stay joined via disulfide bridges and (ii) gC0 is an uncleaved precursor.
Spontaneous expression of C-type virus in DBA/2 mice is associated with an increased rate of mortality, independent of neoplastic disease
Authors: Ulrich K , Nexø B A
Published: January 2020

Ecotropic C-type retroviruses isolated from both normal and dimethylbenzanthracene-treated DBA/2 mice could be classified into three major groups, Ea, Eb, and Ec, that differed in structure and biology. Weanling DBA/2 mice were generally free of viruses, but a fraction of adult individuals became virus positive and were apparently selectively associated with a high expression of the Eb viruses. Some of the ecotropic viruses from DBA/2 mice acted as exogenous pathogens. They caused viremia and a moderate incidence of leukemia when injected into C3H and ST/a mice. In addition, they caused an appreciable number of early deaths without signs of malignancy. To evaluate the natural role of the viruses, we studied the survival of spontaneously viremic and nonviremic DBA/2 mice. The viremic animals as a group were characterized by a significantly reduced life-span that was not related to neoplasia. These observations indicated that endogenous C-type retroviruses can be pathogenic without preselection of the host for disease. They also emphasize that endogenous viruses, like their exogenous counterparts, can have a broader pathogenic spectrum than normally appreciated.
Biologically active peptides of the vesicular stomatitis virus glycoprotein
Authors: Schlegel R , Wade M
Published: January 2020

A peptide corresponding to the amino-terminal 25 amino acids of the mature vesicular stomatitis virus glycoprotein has recently been shown to be a pH-dependent hemolysin. In the present study, we analyzed smaller constituent peptides and found that the hemolytic domain resides within the six amino-terminal amino acids. Synthesis of variant peptides indicates that the amino-terminal lysine can be replaced by another positively charged amino acid (arginine) but that substitution with glutamic acid results in the total loss of the hemolytic function. Peptide-induced hemolysis was dependent upon buffer conditions and was inhibited when isotonicity was maintained with mannitol, sucrose, or raffinose. In sucrose, all hemolytic peptides were also observed to mediate hemagglutination. The large 25-amino acid peptide is also a pH-dependent cytotoxin for mammalian cells and appears to effect gross changes in cell permeability. Conservation of the amino terminus of vesicular stomatitis virus and rabies virus suggests that the membrane-destabilizing properties of this domain may be important for glycoprotein function.
Preliminary characterization of an epitope involved in neutralization and cell attachment that is located on the major bovine rotavirus glycoprotein
Authors: Sabara M , Gilchrist J E , Hudson G R , Babiuk L A
Published: January 2020

The 38,200-molecular weight (unreduced)/41,900-molecular-weight (reduced) glycoprotein of bovine rotavirus, isolate C486, was identified as the major neutralizing antigen. This glycoprotein as well as the corresponding glycoprotein of another bovine rotavirus serotype also specifically attached to cell monolayers under normal conditions for virus adsorption in vitro. Further support for this glycoprotein being directly responsible for virus attachment to cells was that (i) infectious virus of both serotypes could compete with the C486 glycoprotein for cell surface receptors, and (ii) neutralizing monospecific antiserum and neutralizing monoclonal antibodies directed toward the glycoprotein could block this virus-cell interaction. Preliminary epitope mapping of the glycoprotein with monoclonal antibodies further localized the neutralization-adsorption domain to a peptide with an approximate molecular weight of 14,000. The effect of two protein modifications, glycosylation and disulfide bridging, on the reactivity of this peptide with antibodies and cell surface receptors was investigated. It was demonstrated that, whereas glycosylation did not appear to affect these reactivities, disulfide bridging seemed to be essential.
Hemagglutinin-neuraminidase glycoprotein as a determinant of pathogenicity in mumps virus hamster encephalitis: analysis of mutants selected with monoclonal antibodies
Authors: Löve A , Rydbeck R , Kristensson K , Orvell C , Norrby E
Published: January 2020

With the aid of monoclonal antibodies directed against a specific site on the hemagglutinin-neuraminidase surface glycoprotein, four mutants of the Kilham neurotropic strain of mumps virus were isolated. All four mutants had increased neuraminidase activity. Two mutants (M10 and M12) lost their hemagglutination capacity with human O erythrocytes but retained their ability to agglutinate guinea pig erythrocytes at 4 degrees C. A third mutant (M11) showed a change in the molecular weight of the hemagglutinin-neuraminidase glycoprotein. These three mutants (M10, M11, and M12) showed unaltered capacity to infect tissue cultures and to cause encephalitis in newborn hamsters. A fourth mutant (M13) retained its hemagglutination activity and capacity to infect Vero cell cultures but showed significantly lower neurovirulence in the suckling hamster brain than did the parental Kilham strain and the other three mutants. Both the number of infected neurons and the amount of infectious virus in the brain was reduced. On the other hand, there were no apparent differences in the occurrence of viral antigen in ependymal cells, indicating a selective change in affinity for neurons in the brain. These results suggest that certain changes in the hemagglutinin-neuraminidase glycoprotein may lead to an alteration of the neuropathogenicity of the Kilham strain of mumps virus.
Feline panleukopenia virus replicates in cells in which cellular DNA synthesis is blocked
Authors: Lenghaus C , Mun T K , Studdert M J
Published: January 2020

The components of the cell cycle for a feline embryo cell line were defined. Thymidine (6mM)-supplemented medium reversibly arrested cells 1 h into the S phase of the cell cycle and was used in a double blocking procedure to synchronize cells to the early S phase. The kinetics of feline panleukopenia virus replication in synchronized cells was studied by using (i) inclusion body formation, (ii) a plaque assay for cell-associated and cell-free virus under one-step growth conditions, (iii) an enzyme immunoassay for viral protein, (iv) electron microscopy of infected cells, and (v) the detection and identification of viral replicative form DNA by restriction endonuclease analysis. Parallel studies by each of these procedures of the replication of feline panleukopenia virus in cells in which a 6 mM thymidine block was maintained indicated that parvovirus replicated with essentially similar kinetics in both unblocked, synchronized cells and in cells in which the block was maintained. Accordingly, a 6 mM thymidine-supplemented medium, although it effectively blocks cellular DNA synthesis, does not block the replication of parvovirus.
Reovirus serotype 1 intestinal infection: a novel replicative cycle with ileal disease
Authors: Rubin D H , Kornstein M J , Anderson A O
Published: January 2020

After oral inoculation, reovirus serotype 1 strain Lang was shown to specifically infect the epithelial cells of the ileum, while sparing the epithelial cells in the duodenum, jejunum, and colon. The initial site of replication was localized in cells of the crypts of Lieberkühn adjacent to Peyer's patches. Virus was subsequently found by immunoperoxidase staining in cells migrating up the crypt-villus complex throughout the ileum. The severity of the pathological changes in the ileum was proportional to the concentration of the viral inoculum. This site-specific infection of the ileum by reovirus may provide a model for diseases that are restricted to specific sites in the intestine.
Polymer-level synthesis of oxopyrimidine deoxynucleotides by Bacillus subtilis phage SP10: characterization of modification-defective mutants
Authors: Witmer H , Wiatr C
Published: January 2020

Bacillus subtilis phage SP10 DNA has two oxopyrimidines, thymidine 5'-monophosphate (dTMP) and its hypermodified analog (YdTMP). Published data suggest that both are synthesized by postreplicational modification of 5-hydroxymethyldeoxyuridylate (HOMedUMP) in nascent DNA by the following pathway: HOMedUMP----PPOMedUMP----dTMP (85%) or YdTMP (15%); PPOMedUMP is 5-(hydroxymethyl-O-pyrophosphoryl)deoxyuridylate, the pyrophosphoric acid ester of the C5CH2OH function of HOMedUMP. This paper describes aberrant DNAs synthesized at nonpermissive temperatures by a complementary series of heat-sensitive, modification-defective (mod) mutants. Collectively, these mutants encompass the major steps in the complete modification of nascent SP10 DNA. DNA produced by modA phage retains HOMedUMP as its sole oxopyrimidine, implying that (i) this mutant is defective in the pyrophosphorylation step and (ii) formation of PPOMedUMP is required for any further modification. Furthermore, studies with double mutants indicated that modA is epistatic for all other mod mutants, which supports the hypothesis that modA controls the earliest step in the modification pathway. Since their DNAs contain no YdTMP, modC and modD are defective in hypermodification (i.e., PPOMedUMP----YdTMP). However, dTMP occupies the entire oxopyrimidine fraction of modC DNA, whereas modD DNA has a normal dTMP content, but the now-missing YdTMP is replaced by either PPOMedUMP or a byproduct of abortive hypermodification. It is proposed that the modD mutants are defective in the catalytic aspects of hypermodification and that modC are defective in some regulatory function that promotes hypermodification at the expense of reductive modification (i.e., PPOMedUMP----dTMP). Reductive modification is defective in modB phage, as evidenced by the absence of dTMP. In contrast to the others, modB DNA has a complex oxopyrimidine content: HOMedUMP, ca. 30%; PPOMedUMP, ca. 40%; and YdTMP, ca. 30%. The expanded level of YdTMP suggests that at certain sites, reductive modification and hypermodification are competing reactions. Interestingly, the PPOMedUMP content of modB DNA seemingly reflects the maximum degree to which phage DNA can be pyrophosphorylated, since the loss of YdTMP from modBmodC and modBmodD DNAs results in a unilateral increase in HOMedUMP content.
A noninverting genome of a viable herpes simplex virus 1: presence of head-to-tail linkages in packaged genomes and requirements for circularization after infection
Authors: Poffenberger K L , Roizman B
Published: January 2020

The wild-type herpes simplex virus 1 genome consists of two components, L and S, which invert relative to each other, giving rise to four isomers. Previously we reported the construction of a herpes simplex virus 1 genome, HSV-1(F)I358, from which 15 kilobase pairs of DNA spanning the junction between L and S components were deleted and which no longer inverted (Poffenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2690-2694, 1983). Further studies on the structure of HSV-1(F)I358 revealed the presence of two submolar populations among packaged DNA. The first, comprising no more than 10% of total packaged DNA, consisted of defective genomes with a subunit size of 36 kilobase pairs. The results suggest that this population arose by recombination through a directly repeated sequence inserted in place of the deleted L-S junction. The second minor population consisted of HSV-1(F)I358 DNA linked head-to-tail. Analyses of the structure of HSV-1(F)I358 DNA after infection indicated that the fraction of total DNA linked head-to-tail increased to approximately 40 to 50% within 30 min after exposure of cells to virus. The formation of head-to-tail linkages did not require de novo protein synthesis. Our interpretation of the results is that the termini of full-length DNA molecules are held together during packaging, that a small fraction of the termini is covalently linked during or after packaging, and that the remainder is covalently joined after the release of viral DNA from the infecting virus by either host or viral factors introduced into the cell during infection.
Scrapie PrP 27-30 is a sialoglycoprotein
Authors: Bolton D C , Meyer R K , Prusiner S B
Published: January 2020

The major scrapie prion protein, designated PrP 27-30, exhibited both charge and size heterogeneity after purification from infected hamster brains. Eight or more discrete charge isomers of PrP 27-30 with isoelectric points ranging from approximately pH 4.6 to 7.9 were found by using non-equilibrium pH gradient electrophoresis in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The charge isomers were detected by silver staining as well as by radioiodination. The procedures used to disaggregate PrP 27-30 before electrophoresis in the first dimension do not appear to be responsible for the charge heterogeneity. However, heating PrP 27-30 to 100 degrees C for 15 min in 0.1 N NaOH or 0.1 N HCl resulted in modification of the protein and alteration of its electrophoretic pattern. A PrP 27-30 fragment (molecular weight, 17,100 to 21,900) obtained by cyanogen bromide cleavage also exhibited charge and size heterogeneity. Periodic acid-Schiff staining of PrP 27-30 electrophoresed into sodium dodecyl sulfate-polyacrylamide gels demonstrated that carbohydrate residues are attached to the protein. Digestion of PrP 27-30 with neuraminidase and endo-beta-N-acetylglucosaminidase H resulted in significant changes in the isoelectric pH of PrP 27-30 isomers, whereas digestion with alkaline phosphatase had no effect. Our results demonstrate that PrP 27-30 is a sialoglycoprotein; this is consistent with several properties of this protein and of the scrapie prion.
Localization of epitopes of herpes simplex virus type 1 glycoprotein D
Authors: Eisenberg R J , Long D , Ponce de Leon M , Matthews J T , Spear P G , Gibson M G , Lasky L A , Berman P , Golub E , Cohen G H
Published: January 2020

We previously defined eight groups of monoclonal antibodies which react with distinct epitopes of herpes simplex virus glycoprotein D (gD). One of these, group VII antibody, was shown to react with a type-common continuous epitope within residues 11 to 19 of the mature glycoprotein (residues 36 to 44 of the predicted sequence of gD). In the current investigation, we have localized the sites of binding of two additional antibody groups which recognize continuous epitopes of gD. The use of truncated forms of gD as well as computer predictions of secondary structure and hydrophilicity were instrumental in locating these epitopes and choosing synthetic peptides to mimic their reactivity. Group II antibodies, which are type common, react with an epitope within residues 268 to 287 of the mature glycoprotein (residues 293 to 312 of the predicted sequence). Group V antibodies, which are gD-1 specific, react with an epitope within residues 340 to 356 of the mature protein (residues 365 to 381 of the predicted sequence). Four additional groups of monoclonal antibodies appear to react with discontinuous epitopes of gD-1, since the reactivity of these antibodies was lost when the glycoprotein was denatured by reduction and alkylation. Truncated forms of gD were used to localize these four epitopes to the first 260 amino acids of the mature protein. Competition experiments were used to assess the relative positions of binding of various pairs of monoclonal antibodies. In several cases, when one antibody was bound, there was no interference with the binding of an antibody from another group, indicating that the epitopes were distinct. However, in other cases, there was competition, indicating that these epitopes might share some common amino acids.
Human glioblastoma cells persistently infected with simian virus 40 carry nondefective episomal viral DNA and acquire the transformed phenotype and numerous chromosomal abnormalities
Authors: Norkin L C , Steinberg V I , Kosz-Vnenchak M
Published: January 2020

A stable, persistent infection of A172 human glioblastoma cells with simian virus 40 (SV40) was readily established after infection at an input of 450 PFU per cell. Only 11% of the cells were initially susceptible to SV40, as shown by indirect immunofluorescent staining for the SV40 T antigen at 48 h. However, all cells produced T antigen by week 11. In contrast, viral capsid proteins were made in only about 1% of the cells in the established carrier system. Weekly viral yields ranged between 10(4) and 10(6) PFU/ml. Most of the capsid protein-producing cells contained enormous aberrant (lobulated or multiple) nuclei. Persistent viral DNA appeared in an episomal or "free" state exclusively in Southern blots and was indistinguishable from standard SV40 DNA by restriction analysis. Viral autointerference activity was not detected, and yield reduction assays did not indicate defective interfering particle activity, further implying that variant viruses were not a factor in this carrier system. Interferon was also not a factor in the system, as shown by direct challenge with vesicular stomatitis virus. Persistent infection resulted in cellular growth changes (enhanced saturation density and plating efficiency) characteristic of SV40 transformation. Persistent infection also led to an increased frequency of cytogenetic effects. These included sister chromatid exchanges, a variety of chromosomal abnormalities (ring chromosomes, acentric fragments, breaks, and gaps), and an increase in the chromosome number. Nevertheless, the persistently infected cells continued to display a bipolar glial cell-like morphology with extensive process extension and intercellular contacts.
Evidence for a direct role for both the 175,000- and 110,000-molecular-weight immediate-early proteins of herpes simplex virus in the transactivation of delayed-early promoters
Authors: O'Hare P , Hayward G S
Published: January 2020

We reconstructed the regulated induction of delayed-early (DE) transcription that occurs during herpes simplex virus (HSV) infection by using a transient expression system in which recombinant target genes were cotransfected into Vero cells together with intact activating genes. Plasmids containing cloned HSV-1 or HSV-2 immediate-early (IE) genes stimulated by up to 100-fold the expression from recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the DE promoter/regulatory region from the genes for an HSV-2 38,000-molecular-weight (38K) protein and the HSV-1 thymidine kinase. This activation was specific to hybrid genes containing DE regulatory regions since no significant increases in expression were observed in cotransfection experiments with the CAT gene without any promoter region or under the control of a number of other regulatory regions, including an HSV-1 IE regulatory region, the complete or enhancerless early regulatory region of simian virus 40, and an inducible cellular promoter/regulatory region. By using a variety of cotransfected plasmids containing individual or different combinations of HSV-1 or HSV-2 IE genes, we show that of the five known IE genes, two, those coding for the 175K and 110K polypeptides, each possessed the ability to stimulate expression from both DE promoters. Cleavage of the input plasmids within the known coding regions for the 175K and 110K proteins abolished stimulation of DE/CAT gene expression, whereas cleavage outside the coding regions had no effect on stimulation. We conclude that stimulation of CAT expression occurred exclusively by a transactivation mechanism in which the products encoded by these IE genes acted on the DE hybrid constructs at the transcription level. No transcriptional stimulatory function was demonstrated for the IE 68K and 63K proteins, although our results indicate that the IE 12K protein may augment the DE stimulatory activity of the 175K and 110K proteins.
In vitro phenotypic markers of a poliovirus recombinant constructed from infectious cDNA clones of the neurovirulent Mahoney strain and the attenuated Sabin 1 strain
Authors: Kohara M , Omata T , Kameda A , Semler B L , Itoh H , Wimmer E , Nomoto A
Published: January 2020

Infectious cDNA corresponding to the entire genome of the attenuated Sabin strain of type 1 poliovirus has been inserted into EcoRI site of bacterial plasmid pBR325. Two consecutive PstI fragments (nucleotide positions 1814 to 3421) of the infectious cDNA of the Sabin 1 strain were replaced by the corresponding DNA fragments prepared from an infectious DNA clone of the genome of the virulent Mahoney strain of poliovirus type 1. The exchanged segment encodes capsid protein VP1 and part of capsid protein VP3, a region in which a large number of amino acid differences between the attenuated Sabin and the parental, neurovirulent Mahoney strain cluster. The recombinant virus was obtained by DNA transfection of HeLa S3 cells, and several in vitro phenotypes of the virus were compared with those of the parental viruses. The recombinant virus was recognized by a neutralizing monoclonal antibody specific to the Mahoney strain. Growth of the Sabin strain of poliovirus has been shown to be quite dependent upon the bicarbonate concentration (d marker). The growth of the recombinant virus, however, was not highly dependent upon the concentration of bicarbonate in cell culture media, and thus resembled that of the Mahoney strain. On the other hand, the temperature-sensitive multiplication (rct marker) and the small-plaque morphology of the recombinant virus corresponded to the phenotype of the Sabin 1 strain. The in vitro recombination of infectious cDNA clones of genomic RNA and subsequent analysis of the growth properties of the recombinant virus have allowed us to correlate specific mutations in the genome of an RNA virus with certain biological characteristics of that virus.
Properties of the deoxycholate-solubilized HeLa cell plasma membrane receptor for binding group B coxsackieviruses
Authors: Krah D L , Crowell R L
Published: January 2020

Physical and chemical properties of deoxycholate-solubilized HeLa cell plasma membrane receptors for binding group B coxsackieviruses were determined. Receptors eluted from Sepharose 4B with an apparent molecular weight of 275,000 and sedimented with an S value of between 14.7 and 4.9 and a buoyant density of 1.06 to 1.10 g/cm3. Virus-binding activity was destroyed after treatment with proteases, glycosidases, and periodate but was unaffected by lipases or reducing or alkylating agents. Additionally, lectins, including concanavalin A, adsorbed receptors and inhibited virus attachment. The composite data suggested that glycoprotein is an integral part of the receptors for binding virus.
Reassortant rotaviruses as potential live rotavirus vaccine candidates
Authors: Midthun K , Greenberg H B , Hoshino Y , Kapikian A Z , Wyatt R G , Chanock R M
Published: January 2020

A series of reassortants was isolated from coinfection of cell cultures with a wild-type animal rotavirus and a "noncultivatable" human rotavirus. Wild-type bovine rotavirus (UK strain) was reassorted with human rotavirus strains D, DS-1, and P; wild-type rhesus rotavirus was reassorted with human rotavirus strains D and DS-1. The D, DS-1, and P strains represent human rotavirus serotypes 1, 2, and 3, respectively. Monospecific antiserum (to bovine rotavirus, NCDV strain) or a set of monoclonal antibodies to the major outer capsid neutralization glycoprotein, VP7 (of the rhesus rotavirus), was used to select for reassortants with human rotavirus neutralization specificity. This selection technique yielded many reassortants which received only the gene segment coding for the major neutralization protein from the human rotavirus parent, whereas the remaining genes were derived from the animal rotavirus parent. Single human rotavirus gene substitution reassortants of this sort represent potential live vaccine strains.
Primer binding sites corresponding to several tRNA species are present in DNAs of different members of the same retrovirus-like gene family (VL30)
Authors: Itin A , Keshet E
Published: January 2020

We analyzed the putative tRNA primer binding site (PBS) present in several cloned copies of the murine retrovirus-like VL30 family. In the five VL30 DNA clones analyzed, we identified PBS sequences corresponding to three different tRNA species: tRNAPro, tRNAGly, and tRNAGln. The latter two PBS sequences have not been previously encountered in other retroviral or retrovirus-like systems. A unique situation was observed in which PBS sequences complementary to two different tRNA species were flanked by otherwise identical VL30 sequences. In addition, we demonstrated the use of PBS-specific synthetic oligonucleotides for the identification of the tRNA primer and their potential utility in the direct cloning of PBS-containing DNA elements.
Cytotoxic T-lymphocyte reactivity with individual Sendai virus glycoproteins
Authors: Al-Ahdal M N , Nakamura I , Flanagan T D
Published: January 2020

Liposomes were constituted with affinity-purified Sendai virus glycoproteins HN and F and phosphatidylcholine (PC) or phosphatidylethanolamine: phosphatidylserine (PEPS). The glycoprotein-bearing recombinant vesicles (RV) were used to modify the surface of P815 mastocytoma cells (H-2d) or EL4 lymphoma cells (H-2b). The cells treated with HN-F-PCRV, HN-PEPSRV, or F-PEPSRV were shown by surface immunofluorescence to retain antigen for at least 2 h at 37 degrees C after treatment. The modified cells were used in cytotoxicity assays with effector spleen cells from either DBA/2 (H-2d) or C57BL/6 (H-2b) immunized by inoculation of active Sendai virus. Cells modified by treatment with HN/F-PCRV showed susceptibility to cytolysis similar to that in actively infected cells. Cells modified with HN-PEPSRV or with F-PEPSRV were also susceptible. The sum of reactivities of the anti-HN component and the anti-F components was close to that seen with HN- and F-bearing targets. Syngeneic but not allogeneic target cells expressing Sendai virus glycoproteins were bound and lysed by the effector cells, which was expected if the interactions were major histocompatibility complex restricted. The activity was attributed to cytotoxic T lymphocytes, since it was depleted by treatment with anti-Thy 1.2 antibody and complement.
An unusual spliced herpes simplex virus type 1 transcript with sequence homology to Epstein-Barr virus DNA
Authors: Costa R H , Draper K G , Kelly T J , Wagner E K
Published: January 2020

High-resolution transcription mapping localized a spliced 2.7-kilobase herpes simplex virus type 1 mRNA. The 4-kilobase intron of this transcript encodes a nested set of transcripts on the opposite DNA strand. The nucleotide sequence of the DNA encoding the left-hand and right-hand exons of the spliced transcript was determined, and the salient features are presented here. Of major interest is that both exons contained regions within several hundred bases of the splice donor and acceptor sites which showed homology to two regions of the Epstein-Barr virus genome, which are themselves 3 kilobases apart. The spliced herpes simplex virus transcript encoded a translational reading frame which could encode a protein with an approximate size of 75,000 daltons. This value is in agreement with in vitro translation data. The predicted amino acid sequence of the herpes simplex virus protein had significant homology with putative amino acid sequences encoded by the homologous Epstein-Barr virus DNA sequences.