bluuebunny/crossref_metadata_2025 huggingface dataset:
Cas spleen focus-forming virus. II. Further biological and biochemical characterization
Authors: Langdon W Y , Ruscetti S K , Silver J E , Hankins W D , Buckler C E , Morse H C
Published: January 2020

A new isolate of a murine erythroblastosis-inducing spleen focus-forming virus (Cas SFFV), derived from the wild mouse ecotropic murine leukemia virus Cas-Br-M, was further characterized after the production of a nonproducer cell line. When rescued from the nonproducer cells with a helper murine leukemia virus, the Cas SFFV induced rapid splenic enlargement and focus formation when inoculated into adult NFS/N mice. The Cas SFFV nonproducer cell line was also utilized to compare the envelope-related glycoprotein of Cas SFFV with gp52s from three strains of Friend SFFV. Cas SFFV was found to encode a 50,500-dalton glycoprotein (gp50) distinct in size to the envelope-related glycoproteins of the Friend SFFVs. The Cas SFFV was also compared in RNA blot hybridization studies. The genomic viral RNA of Cas SFFV was found to be slightly larger than two polycythemia-inducing strains of Friend SFFV and markedly larger than the anemia-inducing strain. Further comparisons between the SFFVs were made by examining their transforming capabilities in an in vitro erythroid burst assay. The erythroid bursts induced by Cas SFFV and the anemia-inducing strain of Friend SFFV showed similarities in their erythropoietin requirements. This study supports our recent observations that Cas SFFV is biologically similar to the anemia-inducing strain of Friend SFFV yet biochemically distinct from all Friend SFFVs.
Formation and possible functions of alpha-putrescinylthymine in bacteriophage phi W-14 DNA: analysis of bacteriophage mutants with decreased levels of alpha-putrescinylthymine in their DNAs
Authors: Miller P B , Scraba D G , Leyritz-Wills M , Maltman K L , Warren R A
Published: January 2020

The DNA synthesized in the nonpermissive host by the noncomplementing mutants am36 and am42 of bacteriophage phi W-14 contains about half the wild-type level of alpha-putrescinylthymine (putThy) and a correspondingly greater level of thymine. The mechanisms whereby thymine nucleotides are excluded from replicating DNA are functional in both mutants because neither of them incorporates exogenous thymidine into DNA. It is proposed that (i) in wild-type phi W-14, the conversion of hydroxymethyluracil to putThy at the polynucleotide level is sequence specific, but that to thymine is nonspecific; and (ii) in the mutants, the sequence-specific recognition is impaired so that more thymine and less putThy are formed. The thymine-rich DNA can be packaged into phage particles. In the case of am42, the phage particles are morphologically indistinguishable from and have essentially the same polypeptide composition as wild-type particles. However, the DNA molecules they contain are about 11% shorter than those in wild-type phage, am42rev4, a revertant of am42, contains DNA with about 70% of the normal level of putThy; these molecules are about 3% shorter than wild-type DNA. The properties of am42 and am42rev4 are consistent with the suggestion that putThy facilitates the very tight packing of phi W-14 DNA (Scraba et al., Virology 124:152-160, 1983). It also appears that the putThy content of phi W-14 DNA can be reduced by no more than 30% without adversely affecting the production of viable progeny; for example, the burst size of am42rev4 is about 25% of that of the wild type.
Molecular cloning and physical mapping of murine cytomegalovirus DNA
Authors: Ebeling A , Keil G M , Knust E , Koszinowski U H
Published: January 2020

Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons. Of the 16 HindIII fragments, 15 were cloned in plasmid pACYC177 in Escherichia coli HB101 (recA). The recombinant plasmid clones were characterized by cleavage with the enzymes XbaI and EcoRI. In addition, fragments generated by double digestion of cloned fragments with HindIII and XbaI were inserted into the plasmid vector pACYC184. The results obtained after hybridization of 32P-labeled cloned fragments to Southern blots of MCMV DNA cleaved with HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, or KpnI allowed us to construct complete physical maps of the viral DNA for the restriction endonucleases HindIII, XbaI, and EcoRI. On the basis of the cloning and mapping experiments, it was calculated that the MCMV genome spans about 235 kilobase pairs, corresponding to a molecular weight of 155,000,000. All fragments were found to be present in equimolar concentrations, and no cross-hybridization between any of the fragments was seen. We conclude that the MCMV DNA molecule consists of a long unique sequence without large terminal or internal repeat regions. Thus, the structural organization of the MCMV genome is fundamentally different from that of the human cytomegalovirus or herpes simplex virus genome.
Nonviable mutants of simian virus 40 with deletions near the 3' end of gene A define a function for large T antigen required after onset of viral DNA replication
Authors: Tornow J , Cole C N
Published: January 2020

Deletion mutants of simian virus 40 (SV40) with lesions at the three DdeI sites near the 3' end of the early region were constructed. Mutants with deletions at 0.203 and 0.219 map units (mu) which did not change the large T antigen reading frame were viable. This extends slightly the upstream boundary for the location of viable mutants with deletions in the 3' end of the A gene. Mutants with frameshift deletions at 0.193 and 0.219 mu were nonviable. These are the first nonviable mutants with deletions in this portion of the A gene. None of the three nonviable mutants with deletions at 0.219 mu produced progeny viral DNA. These three mutants all used the alternate reading frame located in this portion of the SV40 early region. The mutant with a deletion at 0.193 mu, dlA2459, was positive for viral DNA replication and was defective for adenovirus helper function. All of these mutations were located in the portion of the SV40 large T antigen which has no homology to the polyoma T antigens. These results indicate that this portion of large T antigen is required for some late step in the viral growth cycle and suggest that adenovirus helper function is required for productive infection by SV40.
Polyadenylic acid addition sites in the adenovirus type 2 major late transcription unit
Authors: Le Moullec J M , Akusjärvi G , Stålhandske P , Pettersson U , Chambraud B , Gilardi P , Nasri M , Perricaudet M
Published: January 2020

The cytoplasmic mRNAs which are transcribed from the major late adenovirus promoter can be arranged into five 3'-coterminal families, L1 to L5. We have defined the polyadenylation sites of the mRNAs that belong to the five families at the nucleotide level. From the results, the following conclusions can be made. (i) The hexanucleotide sequence AAUAAA is present at the 3' end of all late adenovirus type 2 mRNAs and precedes the site of polyadenylation by 12 to 30 nucleotides. (ii) Between one and three A residues are present in the genomic sequence at the polyadenylation site. (iii) A sequence with the composition (T)n (A)p (T)q (n, p, q greater than or equal to 1) is found 4 to 24 nucleotides beyond all the adenovirus-specific polyadenylation sites except the 3'-coterminal family L4. This sequence is also found beyond many cellular polyadenylation sites. (iv) The L1 and L2 polyadenylation sites are very similar in structure. The other polyadenylation sites show no apparent sequence relationship, except for the hexanucleotide sequence.
Mutations in the J-F intercistronic region of bacteriophages phi X174 and G4 affect the regulation of gene expression
Authors: Romantschuk M L , Müller U R
Published: January 2020

The J-F intercistronic region of the genomes of bacteriophages phi X174 and G4 is transcribed but not translated. It contains the potential for a perfectly base-paired hairpin structure, which has been proposed to act as a terminator of transcription or as a regulatory region for mRNA turnover (or both). We measured phage-specific protein synthesis in mutants with modifications of the hairpin sequence and found a relative decrease in the expression of the upstream gene D as compared to the downstream genes F, G, and H in all mutants. The mutations also appeared to affect the efficiency of the gene F ribosome-binding site. These data strongly support the regulatory significance of the J-F intercistronic region and the putative hairpin structure therein.
Comparison of nonphosphorylated and phosphorylated species of polyomavirus major capsid protein VP1 and identification of the major phosphorylation region
Authors: Anders D G , Consigli R A
Published: January 2020

The major virion protein of polyomavirus, VP1, consists of about six isoelectric species designated A through F. The minor species D, E, and F are phosphorylated and are thought to serve as viral receptors. We first wanted to distinguish whether all VP1 species are derived by post-translational modification from a common amino acid sequence or whether one or more of the species contain a region(s) of altered amino acid sequence resulting from alternate mRNA processing. We compared the VP1 species by detailed peptide mapping with several combinations of specific protease and radioisotopic labels. This approach enabled us to examine more than 80% of the predicted VP1 sequence, including the amino-and carboxy-termini. We found no evidence of sequence differences among any of the VP1 species. The specific incorporation of 32Pi was found to be the same for all of the phosphorylated species. Comparison of the phosphorylation sites of in vivo 32Pi-labeled D, E, and F by peptide mapping showed them to be identical. Each phosphorylated species contained a single major phosphopeptide and several minor phosphopeptides. The major phosphoamino acid, identified by acid hydrolysis, was phosphothreonine, with phosphoserine also present. By using chemical cleavage methods, we localized the major phosphorylation region to a central portion of the VP1 sequence. We discuss some features of this region and relate this information to functional implications of phosphorylation.
Quantitation of a simian virus 40 nonhomologous recombination pathway
Authors: Dorsett D L , Keshet I , Winocour E
Published: January 2020

We describe an infectious-center in situ plaque hybridization procedure which quantitates simian virus 40 (SV40) nonhomologous recombination in terms of the number of recombinant-producing cells in the DNA transfected cell population. Using this assay to measure the efficiency of recombination with SV40 DNA in permissive monkey BSC-1 cells, we found that: (i) over a range of DNA concentrations, polyomavirus DNA (which is partially homologous to SV40 DNA) cannot be distinguished from nonhomologous phi X174 RF1 DNA with respect to its ability to recombine with SV40 DNA; (ii) at defined DNA concentrations, polyomavirus and phi X174 RF1 DNA compete with each other for recombination with SV40 DNA; (iii) virtually all segments of the phi X174 genome recombine, apparently at random, with SV40 DNA; (iv) the frequency of recombinant-producing cells, among the successfully transfected (virion-producing) cells, depends upon the input SV40 DNA concentration in the transfection solution; and (v) replication-defective SV40 mutant DNAs compete with wild-type SV40 DNA for recombination with phi X174 RF1 DNA. From these observations, we conclude that the efficiency of recombination with SV40, in the system under study, is unaffected by nucleotide sequence homology and that a limiting stage in the recombination pathway occurs before SV40 DNA replication. Comparison of the dependency of recombination on initial SV40 DNA concentration with the dependency on initial phi X174 RF1 DNA concentration indicates that SV40 DNA sequences are a controlling factor in the nonhomologous recombination pathway.
Structure of simian virus 40-phiX174 recombinant genomes isolated from single cells
Authors: Winocour E , Lavie V , Keshet I
Published: January 2020

Three simian virus (SV40)-phi X174 recombinant genomes were isolated from single BSC-1 monkey cells cotransfected with SV40 and phi X174 RF1 DNAs. The individual cell progenies were amplified, cloned, and mapped by a combination of restriction endonuclease and heteroduplex analyses. In each case, the 600 to 1,000 base pairs of phi X174 DNA (derived from different regions of the phi X174 genome) were present as single inserts, located in either the early or late SV40 regions; the deletion of SV40 DNA was greater than the size of the insert; and the remaining portions of the hybrid genome were indistinguishable from wild-type SV40 DNA, as judged by both mapping and biological tests. Hence, apart from the deletion which accommodates the phi X174 DNA insert, no other rearrangements of SV40 DNA were detected. The restriction map of a SV40-phi X174 recombinant DNA isolate before molecular cloning was indistinguishable from those of two separate cloned derivatives of that isolate, indicating that the species cloned was the major amplifiable recombinant structure generated by a single recombinant-producing cell. The relative simplicity of the SV40-phi X174 recombinant DNA examined is consistent with the notion that most recombinant-producing BSC-1 cells support single recombination events generating only one amplifiable recombinant structure.
Viral proteins and RNAs in BHK cells persistently infected by lymphocytic choriomeningitis virus
Authors: van der Zeijst B A , Bleumink N , Crawford L V , Swyryd E A , Stark G R
Published: January 2020

Some Syrian hamster cell lines persistently infected with lymphocytic choriomeningitis virus (LCMV) do not produce extracellular virus particles but do contain intracytoplasmic infectious material. The proteins of these cells were labeled with [35S]methionine or with [3H]glucosamine and [3H]mannose, and immunoprecipitates were prepared with anti-LCMV sera. A substantial amount of the LCMV nucleocapsid protein (molecular weight about 58,000) was detected, along with GP-C, the precursor of the virion glycoproteins GP-1 and GP-2. GP-1 and GP-2 themselves were not detected. A new method of transferring proteins electrophoretically from sodium dodecyl sulfate-polyacrylamide gels to diazotized paper in high yield revealed several additional LCMV proteins present specifically in the persistently infected cells, at apparent molecular weights (X10(3] of 112, 107, 103, 89, 71 (probably GP-C), 58 (nucleocapsid protein), 42 to 47 (probably GP-1), and 40 (possibly GP-2). By iodinating intact cells with I3, GP-1 but not GP-2 or GP-C was revealed on the surfaces of the persistently infected cells, whereas both GP-1 and GP-C were found on the surfaces of acutely infected cells. The absence of GP-C from the plasma membrane of the persistently infected cells might be related to defective maturation of the virus in these cells. Cytoplasmic viral nucleoprotein complexes were labeled with [3H]uridine in the presence or absence of actinomycin D, purified partially by sedimentation in D2O-sucrose gradients, and adsorbed to fixed Staphylococus aureus cells in the presence of anti-LCMV immunoglobulin G. Several discrete species of viral RNA were released from the immune complexes with sodium dodecyl sulfate. Some were appreciably smaller than the 31S and 23S species of standard LCMV virions, indicating that defective interfering viral RNAs are probably present in the persistently infected cells. Ribosomal 28S and 18S RNAs, labeled only in the absence of actinomycin D, were coprecipitated with anti-LCMV serum but not with control serum, indicating their association with LCMV nucleoproteins in the cells.
Microinjected simian virus 40 cRNA is spliced, as evidenced by electron microscopy
Authors: Graessmann M , Graessmann A , Westphal H
Published: January 2020

Simian virus 40 cRNA was transcribed in vitro from the early viral DNA strand. The RNA was injected through glass capillaries into the nuclei of monkey cells. After a 2-h incubation, the RNAs were extracted and hybridized to single-stranded simian virus 40 DNA sequences contained in a bacteriophage M13 vector. Electron microscopy revealed processed cRNAs with splice loops in the region of the intron of large T antigen.
Characterization of virus-specific RNA synthesized in bovine cells infected with bovine viral diarrhea virus
Authors: Purchio A F , Larson R , Collett M S
Published: January 2020

Infection of bovine kidney cells with bovine viral diarrhea virus resulted in the synthesis of a single species of virus-specific RNA. Electrophoresis of this RNA on agarose-urea and agarose-formaldehyde gels indicated that it had a molecular weight of 2.9 X 10(6), corresponding to 8,200 bases (8.2 kilobases). This 8.2-kilobase RNA was resistant to RNase A treatment at 1 microgram/ml but was digested at higher concentrations of RNase (10 micrograms/ml). Sedimentation on neutral sucrose gradients indicated that the majority of this RNA (98%) sedimented at 21S, with a small amount sedimenting at 33S. Sedimentation on formaldehyde-containing sucrose gradients resulted in the conversion of all of the RNA to the faster-sedimenting form. At no time after infection were we able to detect virus-specific RNA species of lower molecular weight than the 8.2-kilobase RNA. The implications of these findings with respect to the means of replication of various togaviruses are discussed.
Comparative inhibition of cellular transcription by vesicular stomatitis virus serotypes New Jersey and Indiana: role of each viral leader RNA
Authors: Grinnell B W , Wagner R R
Published: January 2020

We compared the ability of the leader RNAs of the New Jersey and Indiana serotypes of vesicular stomatitis virus to inhibit transcription in infected host cells. The level of cellular RNA synthesis in cells infected with either serotype was drastically reduced by 5 h after infection. Studies with UV-inactivated virus demonstrated that shutoff of cellular RNA synthesis directly correlated with the ability of the infecting virus to transcribe its plus-stranded leader RNA. Although both serotypes inhibited cellular RNA synthesis, the Indiana serotype reduced synthesis to lower levels. In addition, an examination of the kinetics of leader RNA synthesis in vivo indicated that up to four times more leader RNA was produced in cells infected with the Indiana serotype than in those infected with the New Jersey serotype. However, in vivo studies also suggested that the leader RNA of the New Jersey serotype was a more efficient RNA inhibitor than was the Indiana serotype leader RNA. Although up to 2,900 copies of the leader RNA per cell could be detected in infected cells, only 550 copies of the Indiana and 100 copies of the New Jersey leader RNAs per cell were present in infected cells that were demonstrating 50% of the maximal inhibition of RNA synthesis. In an in vitro system, leader RNAs of both serotypes inhibited DNA-dependent transcription of the adenovirus late promoter and adenovirus-associated RNA genes, but the New Jersey serotype leader was also a better inhibitor in this reconstituted system. Data from the dose response of inhibition by each leader suggest that polymerase III transcription was more sensitive to inhibition by viral leaders than was polymerase II transcription. Polyadenylated viral mRNAs and the NS and N gene starts transcribed by both serotypes did not significantly inhibit transcription at levels at which the corresponding leader RNAs were inhibitory. Overall, our results strongly suggest a role for the plus-stranded leader RNAs of the New Jersey and Indiana serotypes of vesicular stomatitis virus in inhibiting cellular transcription in vivo. We discuss differences in the nucleotide sequences of the two leader RNAs in relation to their differences in biological activity and to potential regulatory sequences.
Initiation of simian virus 40 DNA replication in vitro
Authors: Ariga H , Sugano S
Published: January 2020

Exogenously added simian virus 40 (SV40) DNA can be replicated semiconservatively in vitro by a mixture of a soluble extract of HeLa cell nuclei and the cytoplasm from SV40-infected CosI cells. When cloned DNA was used as a template, the clone containing the SV40 origin of DNA replication was active, but a clone lacking the SV40 origin was inactive. The major products of the in vitro reaction were form I and form II SV40 DNAs and a small amount of form III. DNA synthesis in extracts began at or near the in vivo origin of SV40 DNA synthesis and proceeded bidirectionally. The reaction was inhibited by the addition of anti-large T hamster serum, aphidicolin, or RNase but not by ddNTP. Furthermore, this system was partially reconstituted between HeLa nuclear extract and the semipurified SV40 T antigen instead of the CosI cytoplasm. It is clear from these two systems that the proteins containing SV40 T antigen change the nonspecific repair reaction performed by HeLa nuclear extract alone to the specific semiconservative DNA replication reaction. These results show that these in vitro systems closely resemble SV40 DNA replication in vivo and provide an assay that should be useful for the purification and subsequent characterization of viral and cellular proteins involved in DNA replication.
Sequences locating the 5' ends of the major simian virus 40 late mRNA forms
Authors: Piatak M , Ghosh P K , Norkin L C , Weissman S M
Published: January 2020

The 5' sequences of late mRNA specified by several constructed or naturally occurring deletion or duplication mutants of simian virus 40 were examined. The mutants included viruses with various small deletions centered about 25 nucleotides upstream from the major transcription initiation site, as well as viruses containing tandem duplications of a sequence of 50 nucleotides or less embedding the major transcription initiation site. The results show that the sequences 25 to 30 nucleotides upstream from the major initiation site in the position of the TATA box of other polymerase II promoters are not essential for the precise localization of the initiation site of late mRNA. Rather, we deduce that the major late mRNA start site is determined primarily by sequences located very close to the initiation site, and that the relative abundance of the 5' ends with this initiation site is modulated by nearby downstream sequences. Modification of six nucleotides adjacent upstream to the initiation site almost completely prevents the utilization of this site. Various deletions and substitutions of sequences 21 nucleotides or more downstream from the major initiation site causes upstream shifts in the localization of the most abundantly utilized 5' ends. The sequences immediately downstream from the major simian virus 40 initiation sites contain inverted symmetries that could give rise to secondary structures in either single-stranded DNA or RNA; the possibility that these inverted symmetries function in transcription initiation at the level of DNA structure rather than in RNA stabilization is discussed. Finally, we present additional evidence that precursor species with certain 5' termini are selectively spliced to form 19S RNA, whereas other 5' termini are preferred for forming the 16S RNA splice. We discuss the possibility that this is a consequence of the influence of leader structure on downstream splicing events.
Molecular cloning of the Aleutian disease virus genome: expression of Aleutian disease virus antigens by a recombinant plasmid
Authors: Mayer L W , Aasted B , Garon C F , Bloom M E
Published: January 2020

Three nonoverlapping segments representing approximately 80% of the 4.8-kilobase pair Aleutian disease virus (ADV-G) duplex genome were molecularly cloned into either bacteriophage M13mp9 (M13bm2 = 0.07 to 0.15 map unit; M13bm1 = 0.15 to 0.54 map unit) or plasmid pUC8 (pBM1 = 0.54 to 0.88 map units). In addition the 0.54- to 0.88-map unit segment of a Danish isolate of ADV (DK ADV) was also cloned into pUC8 (pBM2). The recombinant plasmids pBM1 and pBM2 induced expression of several polypeptides in Escherichia coli JM103 that were specifically recognized by sera from mink infected with ADV. The same three proteins with approximate molecular weights of 55,000, 34,000, and 27,000 were detected both by immune blotting and by immunoprecipitation of [35S]methionine-labeled JM103 (pBM1). None of these proteins were recognized in JM103 or JM103 (pUC8), nor were they detected by sera from normal mink. Purified pBM1 and pBM2 DNA appeared identical in size by gel analysis and contour length measurement, and electron microscopic heteroduplex mapping revealed no visible areas of heterology. However, restriction endonuclease mapping showed that pBM2 was different from pBM1, indicating that this segment of the ADV genome was similar but not identical for two strains of ADV (ADV-G and DK ADV). Furthermore, when cloned DNA from ADV-G was labeled with [32P]dCTP by nick translation, DNA relatedness to several field strains of ADV (Utah I, Pullman, and DK), but not to mink enteritis virus or cellular DNA, was shown by Southern blot hybridization.
Characterization of replicative intermediate RNA of mouse hepatitis virus: presence of leader RNA sequences on nascent chains
Authors: Baric R S , Stohlman S A , Lai M M
Published: January 2020

Mouse hepatitis virus A59 codes for seven mRNAs in infected cells. These mRNAs are transcribed from a minus (-) strand template of genome length and contain a leader RNA at their 5' ends. To further elucidate the mechanism of coronavirus transcription, we examined the structure of mouse hepatitis virus replicative intermediates (RIs) isolated by 2 M NaCl precipitation and Sepharose 2-B column chromatography. Purified RIs migrated as a single species on agarose gels and sedimented between 12 and 38S on 10 to 25% sucrose gradients. The complexes were readily heat denatured into a heterogeneous population of smaller RNA molecules which probably represent nascent plus (+) strands. RNase A digestion of RIs produced a single replicative form which sedimented between 30 and 32S. These data suggest that the RI is composed of a single genome-sized (-) strand hydrogen bonded to an average of 4 to 6.5 nascent (+) strands. In contrast, a column-purified replicative form was extremely resistant to RNase A digestion and heat denaturation and migrated as a single RNA species on agarose gels and sucrose gradients. Oligonucleotide fingerprinting of an RI revealed the presence of the 5' leader RNA on the nascent (+) strands. In addition, an average of 6.2 cap structures were present in each RI, which agrees with the average number of nascent (+) strands per RI. These data suggest that the leader RNA is utilized as a primer for mouse hepatitis virus RNA transcription and is not added to mRNA post-transcriptionally.
Viral Transcription During
Autographa californica
Nuclear Polyhedrosis Virus Infection: a Novel RNA Polymerase Induced in Infected
Spodoptera frugiperda
Cells
Authors: Fuchs L. Yolanda , Woods Michael S. , Weaver Robert F.
Published: January 2020

Autographa californica
nuclear polyhedrosis virus-specific RNA synthesis in isolated nuclei of
Spodoptera frugiperda
cells in culture was monitored at different times postinfection. Up to 8 h postinfection viral RNA synthesis remained sensitive to 5 μg of α-amanitin per ml. During the course of infection this sensitivity decreased, and at 24 h postinfection RNA synthesis was completely resistant to α-amanitin. DEAE-Sephadex profiles of RNA polymerase isolated at 24 h postinfection showed a new, chromatographically distinct, α-amanitin-resistant form whose kinetics and response to divalent cations differed from those of the host RNA polymerases. The possibility that this enzyme may be responsible for viral late transcription is discussed.
Discontinuities in the DNA synthesized by an avian retrovirus
Authors: Taylor J M , Cywinski A , Smith J K
Published: January 2020

The unintegrated linear DNA synthesized in cells infected by Rous sarcoma virus is a predominantly double-stranded structure in which most of the minus-strand DNA, complementary to the viral RNA genome, is genome sized, whereas the plus-strand DNA is present as subgenomic fragments. We previously reported the application of benzoylated naphthoylated DEAE-cellulose chromatography to demonstrate that of the linear viral DNA species synthesized in quail embryo fibroblasts infected with Rous sarcoma virus greater than 99.5% contain single-stranded regions and these regions are predominantly composed of plus-strand DNA sequences (T. W. Hsu and J. M. Taylor, J. Virol. 44:47-53, 1982). We now present the following additional findings. (i) There were on the average 3.5 single-stranded regions per linear viral DNA, and these single-stranded regions could occur at many locations. (ii) With a probe to the long terminal repeat, we detected, in addition to a heterogeneous size distribution of subgenomic plus-strand DNA species, at least three prominent discrete size classes. Each of these discrete species had its own specific initiation site, but all had the same termination site. Such species were analogous to those reported by Kung et al. (J. Virol. 37: 127-138, 1981). (iii) These discrete size classes of plus-strand DNA were present not only on the major size class of linear DNA but also on a heterogeneous of slower-sedimenting species, which we have called immature linears. Our interpretation is that we have thus detected several additional sites for the initiation of plus-strand DNA. (iv) The 340-base plus-strand strong-stop DNA was only found associated with the immature linears. (v) From a size and hybridization comparison of these discrete size classes of plus-strand DNA with minus-strand DNA species, as synthesized in the endogenous reaction of melittin-disrupted virions, it was found that the putative additional initiation sites for plus-strand DNA synthesis corresponded to many of the pause sites in the synthesis of minus-strand DNA.
Two distant clusters of partially homologous small repeats of Epstein-Barr virus are transcribed upon induction of an abortive or lytic cycle of the virus
Authors: Freese U K , Laux G , Hudewentz J , Schwarz E , Bornkamm G W
Published: January 2020

The two regions of the Epstein-Barr virus genome carrying partially homologous clusters of short tandem repeats (DSL and DSR [duplicated sequences, left and right, respectively] ) are transcribed into polyadenylated RNA upon spontaneous or chemical induction of the lytic virus cycle. In Raji, an Epstein-Barr virus genome carrying a nonproducer cell line, transcription of DSL and DSR is only observed upon induction of an abortive life cycle of the virus. In the nonproducer cell line Raji, the polyadenylated transcripts of DSL and DSR are about 2,500 and 2,700 bases, respectively, in length. Four different spontaneous Epstein-Barr virus producer lines, M-ABA, CC34-5, QIMR-WIL, and B95-8, differ in the length of their DSL and/or DSR regions by different numbers of tandem repeats. The size of the RNAs corresponds in all cases to the size of the respective cluster of repeats, indicating that a large part of each RNA species is colinearly transcribed from the entire tandem repeat arrays. Both the DSL and the DSR RNAs have the same polarity proceeding from right to left on the Epstein-Barr virus genome. DNA sequence analysis of the DSR repeat revealed that translation of the RNA would be possible in three open reading frames within the repeat cluster. Short homologies to herpes simplex virus IR-TR sequences and to immunoglobulin switch region sequences (IgH-S) are discussed.
Variation in p30-related proteins in gross virus-induced tumor cell lines derived from H-2 congenic mice
Authors: Wolfe J H , Blankenhorn E P , Blank K J
Published: January 2020

p30-related proteins were examined in three in vitro--passaged Gross virus--induced cell lines derived from mice congenic at the H-2 complex: BALB.K-gv-1 (H-2k) (BkGV1), which produces infectious, oncogenic virus, and BALB.B-gv-1 (H-2b) (BbGV1) and BALB/c-gv-1 (H-2d) (BdGV1), which stopped producing infectious virus early in their passage history. The producer BkGV1 line made p30 and its expected precursors. BbGV1 and BdGV1 cells had multiple changes in p30-related protein expression. BbGV1 cells (i) synthesized a truncated gag precursor polyprotein of ca. 47 kilodaltons, (ii) expressed fewer p30-related molecules per cell on their surface than BkGV1 cells, (iii) produced functional reverse transcriptase, and (iv) budded morphologically mature virions which were neither infectious nor oncogenic. BdGV1 cells (i) produced no p30 but expressed some high-molecular-weight proteins with p30 determinants, (ii) expressed fewer p30-related determinants on their surface than BbGV1 cells, and (iii) harbored immature intracisternal viral particles (type A) but did not exhibit budding virions.
Selective cloning of a DNA single-strand initiation determinant from phi X174 replicative-form DNA
Authors: Strathearn M D , Low R L , Ray D S
Published: January 2020

An M13 phage deletion mutant, M13 delta E101, developed as a vector for selecting DNA sequences that direct DNA strand initiation on a single-stranded template, has been used for cloning restriction enzyme digests of phi X174 replicative-form DNA. Initiation determinants, detected on the basis of clear-plaque formation by the chimeric phage, were found only in restriction fragments containing the unique effector site in phi X174 DNA for the Escherichia coli protein n' dATPase (ATPase). Furthermore, these sequences were functional only when cloned in the orientation in which the phi X174 viral strand was joined to the M13 viral strand. A 181-nucleotide viral strand fragment containing this initiation determinant confers a phi X174-type complementary-strand replication mechanism on M13 chimeras. The chimeric phage is converted to the parental replicative form in vivo by a mechanism resistant to rifampin, a specific inhibitor of the normal RNA polymerase-dependent mechanism of M13. In vitro, the chimeric single-stranded DNA promotes the assembly of a functional multiprotein priming complex, or primosome, identical to that utilized by intact phi X174 viral strand DNA. Chimeric phage containing the sequence complementary to the 181-nucleotide viral strand sequence shows no initiation capability, either in vivo or in vitro.
Analysis of the herpes simplex virus genome during in vitro latency in human diploid fibroblasts and rat sensory neurons
Authors: Wigdahl B , Scheck A C , Ziegler R J , De Clercq E , Rapp F
Published: January 2020

We have previously designed in vitro model systems to characterize the herpes simplex virus type 1 (HSV-1) genome during in vitro virus latency. Latency was established by treatment of infected human embryo lung fibroblast (HEL-F) cells or rat fetal neurons with (E)-5-(2-bromovinyl)-2'-deoxyuridine and human leukocyte interferon and was maintained by increasing the incubation temperature after inhibitor removal. Virus was reactivated by reducing the incubation temperature. We have now examined the HSV-1-specific DNA content of latently infected HEL-F cells and rat fetal neurons treated with (E)-5-(2-bromovinyl)-2'-deoxyuridine and human leukocyte interferon and increased temperature. The HEL-F cell population contained, on an average, between 0.25 and 0.5 copies of most, if not all, HSV-1 HindIII and XbaI DNA fragments per haploid cell genome equivalent. In contrast, the latently infected neurons contained, on an average, 8 to 10 copies per haploid cell genome equivalent of most HSV-1 BamHI DNA fragments. There was no detectable alteration in size or molarity of the HSV-1 terminal or junction DNA fragments obtained by HindIII, XbaI, or BamHI digestion of the latently infected neuron or HEL-F cell DNA, as compared with digestion of a reconstruction mixture of purified HSV-1 virion and HEL-F cell DNAs. These data suggest that the predominant form of the HSV-1 genome in either latently infected cell population is nonintegrated, linear, and nonconcatameric.
Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA
Authors: Jones E V , Moss B
Published: January 2020

The previous demonstration that a phosphonoacetate (PAA)-resistant (PAAr) vaccinia virus mutant synthesized an altered DNA polymerase provided the key to mapping this gene. Marker rescue was performed in cells infected with wild-type PAA-sensitive (PAAs) vaccinia by transfecting with calcium phosphate-precipitated DNA from a PAAr mutant virus. Formation of PAAr recombinants was measured by plaque assay in the presence of PAA. Of the 12 HindIII fragments cloned in plasmid or cosmid vectors, only fragment E conferred the PAAr phenotype. Successive subcloning of the 15-kilobase HindIII fragment E localized the marker within a 7.5-kilobase BamHI-HindIII fragment and then within a 2.9-kilobase EcoRI fragment. When the latter was digested with ClaI, marker rescue was not detected, suggesting that the PAAr mutation mapped near a ClaI site. The sensitive ClaI site was identified by cloning partial ClaI-EcoRI fragments and testing them in the marker rescue assay. The location of the DNA polymerase gene, about 57 kilobases from the left end of the genome, was confirmed by cell-free translation of mRNA selected by hybridization to plasmids containing regions of PAAr vaccinia DNA active in marker rescue. A 100,000-dalton polypeptide that comigrated with authentic DNA polymerase was synthesized. Correspondence of the in vitro translation product with purified vaccinia DNA polymerase was established by peptide mapping.
Binding of simian virus 40 a protein to DNA with deletions at the origin of replication
Authors: Lewton B A , DeLucia A L , Tegtmeyer P
Published: January 2020

Previous studies with wild-type simian virus 40 DNA have shown that the sequence 5'-GAGGC-3' directs the binding of A protein (T antigen). The functional origin of replication contains four recognition pentanucleotides each of which is separated by a single base pair and arranged a two pairs of direct repetitions that are inverted relative to each other. Analysis of A protein binding to a series of nonviable mutants progressively deleting these contact sites leads to the following conclusions: (i) stable binding of subunits of A protein to three origin pentanucleotides is not sufficient for the initiation of DNA replication, (ii) the stability of DNA binding depends on interactions between bound protein subunits, and (iii) a single pentanucleotide is sufficient to bind and orient a subunit of A protein.
N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus
Authors: Patton J T , Davis N L , Wertz G W
Published: January 2020

Genomic replication of the negative-strand RNA viruses is dependent upon protein synthesis. To examine the requirement for protein synthesis in replication, we developed an in vitro system that supports the genome replication of defective interfering particles of the negative-strand rhabdovirus vesicular stomatitis virus (VSV), as a function of protein synthesis (Wertz, J. Virol. 46:513-522, 1983). The system consists of defective interfering nucleocapsid templates and an mRNA-dependent reticulocyte lysate to support protein synthesis. We report here an analysis of the requirement for individual viral proteins in VSV replication. Viral mRNAs purified by hybridization to cDNA clones were used to direct the synthesis of individual proteins in the in vitro system. By this method, it was demonstrated that the synthesis of the VSV nucleocapsid protein, N, alone, resulted in the replication of genome-length RNA by both defective interfering intracellular nucleocapsids and virion-derived nucleocapsids. Neither the viral phosphoprotein, NS, nor the matrix protein, M, supported RNA replication. The amount of RNA replication for a given amount of N protein was the same in reactions in which either all of the VSV proteins or only N protein were synthesized. In addition, RNA replication products synthesized in reactions containing only newly made N protein assembled with the N protein to form nucleocapsids. These results demonstrate that the major nucleocapsid protein (N) can by itself fulfill the requirement for protein synthesis in RNA replication and allow complete replication, i.e., initiation and elongation, as well as encapsidation of genome-length progeny RNA.
Phenotypic mixing of pyocin R2 and bacteriophage PS17 in Pseudomonas aeruginosa PAO
Authors: Shinomiya T
Published: January 2020

Previous results indicate that a group of bacteriocins in Pseudomonas aeruginosa, named R-type pyocins, have a structure resembling bacteriophage tails and share some serological homology with certain bacteriophages. This paper presents genetic evidence which strongly suggests that components of pyocin R2, an R-type pyocin of P. aeruginosa PAO, and tail components of bacteriophage PS17 are interchangeable. Complementation tests with pyocin R2-deficient mutants of PAO and ts mutants of PS17 revealed that various phenotypic interactions occur between the pyocin and bacteriophage in PAO cells lysogenized or infected with PS17. (i) Certain pyocin R2-deficient mutations were phenotypically suppressed in cells carrying PS17 prophage. (ii) A temperature-sensitive mutant of PS17, tsQ31, was phenotypically suppressed in PAO cells treated with mitomycin C. (iii) Phenotypically mixed phages with receptor and serological specificities of pyocin R2 were formed in PS17 lysogens of certain pyocin R2-deficient mutants.
Simian virus 40 mutant T antigens with relaxed specificity for the nucleotide sequence at the viral DNA origin of replication
Authors: Margolskee R F , Nathans D
Published: January 2020

Base substitution of the ori region of simian virus 40 leads to plaque morphology mutants with markedly decreased DNA replication. Second-site mutations within the simian virus 40 T antigen gene suppress the plaque phenotype and replication defect of base-substituted ori mutants. Two second-site mutations have been mapped to a small segment of the T antigen gene, just beyond the distal splice junction. DNA sequence analysis revealed a single missense change in this segment of the T antigen gene of each of these second-site revertants, leading to a change in codon 157 in one case and codon 166 in the other. The mutant T antigens displayed relaxed specificity for the ori signal, i.e., they can function with several variously modified ori sequences, including those with small nucleotide deletions or insertions that are inactive for replication when coupled with wild-type T antigen. Thus a region of T antigen has been identified that appears to be intimately involved in vivo in binding to the ori sequence to initiate viral DNA replication.
Two types of deletion within integrated viral sequences mediate reversion of simian virus 40-transformed mouse cells
Authors: Maruyama K , Oda K
Published: January 2020

Simian virus 40 (SV40) DNA insertions from SV40-transformed mouse cell line W-2K-11 and its revertants M18, M31, and M42 were cloned. W-2K-11 cells contain 1.5 copies of the SV40 sequences in a partially tandem duplicated form. The endpoints of the viral sequences at the virus-host junctions are located very close to those reported by others, indicating that there are some preferred sites for integration and rearrangement in SV40 sequences. One flanking cellular sequence is a long stretch of adenine and thymine with repeated AAAT, and the other is a stretch of guanine and cytosine with repeated CCG. There are patchy homologies between the flanking cellular sequences and the corresponding parental SV40 sequences. The sequences around both junctions were retained in all the revertants, whereas most of the internal SV40 sequences coding for large T antigen were deleted. The coding sequences for small T antigen are intact, and small T antigen was expressed in all the revertants. The fragments cloned from M18 and M42 were identical and 3.9 kilobases of SV40 sequences were deleted. The parental SV40 sequences around the deletion site have sequences capable of forming a secondary structure which might reduce the effective distance between the two regions. The SV40 DNA retained in M31 is colinear with SV40 virion DNA, and a unit length of SV40 DNA was deleted within the SV40 sequences present in W-2K-11 cells. These results indicated that two types of deletion occurred during the reversion, one between homologous sequences and the other between nonhomologous sequences.
DNA sequence of the Bryan high-titer strain of Rous sarcoma virus: extent of env deletion and possible genealogical relationship with other viral strains
Authors: Lerner T L , Hanafusa H
Published: January 2020

The genetic structure of the Bryan high-titer strain of Rous sarcoma virus (BH-RSV) was analyzed by using a molecular clone obtained from proviral DNA. DNA sequencing of the pol-src junction of BH-RSV revealed that the env sequence was almost entirely absent; only six base pairs following the pol termination codon remained. Beginning at nucleotide 7 (relative to the end of pol), a 91-base pair sequence identical to the 91 base pairs immediately upstream from src in other strains of RSV was found. The helper virus-related sequence of about 100 base pairs, which is present as a direct repeat in the 5' and 3' regions flanking src in other RSVs, was present only on the 3' side of src in BH-RSV. The 3' end of BH-RSV, from the last 16 base pairs of src through the U3 region, was virtually identical to a region downstream of env through U3 in the nontransforming helper virus Rous-associated virus-2, suggesting that BH-RSV may have been derived by recombination between Rous-associated virus-2 and cellular src DNA. The possibility that the original RSV may have been a defective transforming virus and a precursor of the nondefective RSV strains is discussed.
Initiation by bacteriophage T1 of DNA packaging at a site between the P and Q genes of bacteriophage lambda
Authors: Drexler H
Published: January 2020

The growth of phage T1 on cells tandemly lysogenic for heteroimmune lambdoid prophages leads to a nonrandom packaging of lambda DNA by T1. A site, called esp-lambda, is located between the P and Q genes of lambda and results in increased packaging to the left by T1. When cloned into pBR322, the esp-lambda site causes a significant increase in transduction of the plasmid by T1. The nin5 deletion inactivates esp-lambda.
Vesicular stomatitis virus mutants resistant to defective-interfering particles accumulate stable 5'-terminal and fewer 3'-terminal mutations in a stepwise manner
Authors: O'Hara P J , Horodyski F M , Nichol S T , Holland J J
Published: January 2020

We have studied the evolution of sequences which include the RNA polymerase binding sites at the 5' and 3' termini of vesicular stomatitis virus mutants (Sdi-) resistant to defective-interfering particles. We observed a striking stepwise accumulation of stable base substitutions within the area of replication initiation at the 5'-terminal 54 nucleotides of Sdi- mutants isolated at intervals from persistent infections and undiluted lytic passage series. Fewer mutations accumulated in the region of transcription initiation at the 3' end and in those portions of the N and L protein coding cistrons examined. The termini changes are not strictly required to obtain the Sdi- phenotype. However, it is possible that they represent stepwise compensatory changes to accommodate Sdi- mutations affecting viral replication or encapsidation gene products or both. These results have important implications for RNA virus genome evolution.