bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular basis of the glycoprotein C-negative phenotypes of herpes simplex virus type 1 mutants selected with a virus-neutralizing monoclonal antibody
Authors: Homa F L , Purifoy D J , Glorioso J C , Levine M
Published: January 2020

Previously (Holland et al., J. Virol. 52:566-574, 1984; Kikuchi et al., J. Virol. 52:806-815, 1984) we described the isolation and partial characterization of over 100 herpes simplex virus type 1 mutants which were resistant to neutralization by a pool of glycoprotein C- (gC) specific monoclonal antibodies. The genetic basis for the inability of several of these gC- mutants to express an immunoreactive envelope form of gC is reported here. Comparative nucleotide sequence analysis of the gC gene of the six mutants gC-3, gC-8, gC-49, gC-53, gC-85, and synLD70, which secrete truncated gC polypeptides, with that of the wild-type KOS 321 gC gene revealed that these mutant phenotypes were caused by frameshift or nonsense mutations, resulting in premature termination of gC translation. Secretion of the gC polypeptide from cells infected with these mutants was due to the lack of a functional transmembrane anchor sequence. The six secretor mutants were tested for suppression of amber mutations in mixed infection with a simian virus 40 amber suppressor vector. Mutant gC-85 was suppressed and produced a wild-type-sized membrane-bound gC. Nucleotide sequence analysis of the six gC deletion mutants gC-5, gC-13, gC-21, gC-39, gC-46, and gC-98 revealed that they carried identical deletions which removed 1,702 base pairs of the gC gene. The deletion, which was internal to the gC gene, removed the entire gC coding sequence and accounted for the novel 1.1-kilobase mRNA previously seen in infections with these mutants. The mutant gC-44 was previously shown to produce a membrane-bound gC protein indistinguishable in molecular weight from wild-type gC. This mutant differed from wild-type virus in that it had reduced reactivity with virus-neutralizing monoclonal antibodies. Nucleotide sequence analysis of the gC gene of mutant gC-44 demonstrated a point mutation which changed amino acid 329 of gC from a serine to a phenylalanine.
Constitutive and retinoic acid-inducible expression of cytomegalovirus immediate-early genes in human teratocarcinoma cells
Authors: LaFemina R , Hayward G S
Published: January 2020

Human teratocarcinoma stem cells are nonpermissive for human cytomegalovirus (HCMV) but become permissive after being induced to differentiate by treatment with retinoic acid. We show that in uninduced teratocarcinoma stem cells, and also in transformed human 293 cells expressing adenovirus E1a gene products, the HCMV immediate-early (IE) 68,000-molecular-weight polypeptide (68K polypeptide) was not expressed, and consequently input viral genomes were not replicated. However, after differentiation of the teratocarcinoma cells, synthesis of the HCMV IE 68K polypeptide was induced, and viral DNA replication occurred. In contrast to our observations for HCMV, simian cytomegalovirus (SCMV) displayed constitutive expression of its analogous IE 94K polypeptide, and the input SCMV genomes were replicated in both uninduced stem cells and 293 cells. Since little, if any, HCMV IE RNA was detectable in human teratocarcinoma or 293 cells after infection under IE conditions, we suggest that a direct transcriptional block to permissivity occurs in these cells. The presence of tandemly repeated sequences which bind nuclear factor I protein in the promoter for the SCMV IE 94K polypeptide gene but not in the promoter for the HCMV IE 68K polypeptide gene may allow the expression of the simian but not of the human IE gene product in transformed cells.
Nucleotide sequences that affect replicative and transcriptional efficiencies of Sendai virus deletion mutants
Authors: Re G G , Kingsbury D W
Published: January 2020

Structural features of the genomes of virus deletion mutants (DI virions) influence their replication efficiency. Among nonsegmented negative-strand RNA viruses, substitution of the genomic 3' terminus by a complementary copy of the 5' terminus (so-called "copy-back" sequence) could enhance replication either because the new 3' end is a better promoter of RNA replication or because DI RNAs that possess this sequence are incapable of acting as templates for transcription. Here we provide evidence that both mechanisms operate in mixed infections with Sendai virus DI RNAs. RNAs incapable of transcription always outgrew RNA species that were transcribed. This was true even when the 3'-terminal sequence of the untranscribed RNA was identical to the genomic 3' terminus, as in the case of an internally deleted DI genome (RNA Ra) rendered transcriptionally inert by point mutations of bases 47 and 51 at the 5' end of the positive-strand leader RNA template. Nevertheless, Ra was outgrown by a copy-back DI RNA, indicating that the 3' genomic end of Ra is a less efficient site for replication initiation than the copy-back sequence.
Amplification of a tandem direct repeat within inverted repeats of Marek's disease virus DNA during serial in vitro passage
Authors: Maotani K , Kanamori A , Ikuta K , Ueda S , Kato S , Hirai K
Published: January 2020

DNA of the oncogenic strain BC-1 of Marek's disease virus contains three units of tandem direct repeats with 132 base pairs in the terminal repeat and internal repeat, respectively, of the long region of the Marek's disease virus genome, whereas the attenuated, nononcogenic viral DNA contains multiple units of the tandem direct repeats.
Establishment of a Vero cell line persistently infected with African swine fever virus
Authors: Salas J , Viñuela E
Published: January 2020

A Vero cell line persistently infected with African swine fever virus was established by infecting the cells in the presence of 10 mM NH4Cl (Vero-P cell line). The virus derived from the Vero-P cultures infected Vero cells, and virus titers were comparable to those obtained in Vero cells acutely infected with African swine fever virus. The structural proteins of the virus from Vero-P cells were similar to those of the virus produced in lytic infections. Virus production was low when the Vero-P cells were growing logarithmically and increased considerably in confluent cultures when lysis appeared in a fraction of the cell population.
Expression of herpes simplex virus glycoproteins in polarized epithelial cells
Authors: Srinivas R V , Balachandran N , Alonso-Caplen F V , Compans R W
Published: January 2020

Members of the herpesvirus family mature at inner nuclear membranes, although a fraction of the viral glycoproteins is expressed on the cell surface. In this study, we investigated the localization of herpes simplex virus type 2 (HSV-2) glycoproteins in virus-infected epithelial cells by using a panel of monoclonal antibodies directed against each of the major viral glycoproteins. All of the HSV-2 glycoproteins were localized exclusively on the basolateral membranes of Vero C1008, Madin-Darby bovine kidney, and mouse mammary epithelial cells. Using a monoclonal antibody to HSV-2 gD which cross-reacts with HSV-1 strains, we could also localize HSV-1 gD on the basolateral membranes of Madin-Darby bovine kidney cells. These results indicate that these molecules contain putative sorting signals that direct them to basolateral membrane domains.
Localization of temperature-sensitive transformation mutations and back mutations in the Rous sarcoma virus src gene
Authors: Fincham V J , Wyke J A
Published: January 2020

Cloning and sequencing of two temperature-sensitive transforming mutations of Rous sarcoma virus reveal that their lesions are due to distinct but close single amino acid changes near the carboxy terminus of the v-src gene product. Back mutations to wild type result from second mutations at either nearby or distant sites.
Internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA yields a second protein
Authors: Herman R C
Published: January 2020

In vitro translation of a mixture of the vesicular stomatitis virus (VSV) polyadenylated mRNAs yielded a previously undetected protein with a molecular weight of approximately 7,000 (7K protein). Hybrid-arrested translation demonstrated that both the 7K protein and the VSV phosphoprotein (P protein) were encoded by the P protein message. Immunoprecipitation of the 7K protein with monoclonal antiserum directed against the P protein indicated that the two products were encoded in the same open reading frame. A protein of approximately the same size was immunoprecipitated from cytoplasmic extracts of VSV-infected cells by both the polyclonal and monoclonal antisera, and it is likely that it was a previously unrecognized viral gene product. Translational mapping of the P protein mRNA in vitro indicated that the 7K protein was encoded in the 3' one-third of the sequence. The synthesis of the 7K protein in vitro was unaffected by hybrid arrest conditions which blocked the 5' two-thirds of the mRNA and inhibited synthesis of the P protein. These results imply that the ribosomes bind and initiate translation internally on the P protein mRNA at a site located hundreds of nucleotides downstream from the capped 5' end.
Mapping of phosphorylation sites in polyomavirus large T antigen
Authors: Hassauer M , Scheidtmann K H , Walter G
Published: January 2020

The phosphorylation sites of polyomavirus large T antigen from infected or transformed cells were investigated. Tryptic digestion of large T antigen from infected, 32Pi-labeled cells revealed seven major phosphopeptides. Five of these were phosphorylated only at serine residues, and two were phosphorylated at serine and threonine residues. The overall ratio of phosphoserine to phosphothreonine was 6:1. The transformed cell line B4 expressed two polyomavirus-specific phosphoproteins: large T antigen, which was only weakly phosphorylated, and a truncated form of large T antigen of 34,000 molecular weight which was heavily phosphorylated. Both showed phosphorylation patterns similar to that of large T antigen from infected cells. Peptide analyses of large T antigens encoded by the deletion mutants dl8 and dl23 or of specific fragments of wild-type large T antigen indicated that the phosphorylation sites are located in an amino-terminal region upstream of residue 194. The amino acid composition of the phosphopeptides as revealed by differential labeling with various amino acids indicated that several phosphopeptides contain overlapping sequences and that all phosphorylation sites are located in four tryptic peptides derived from a region between Met71 and Arg191. Two of the potential phosphorylation sites were identified as Ser81 and Thr187. The possible role of this modification of large T antigen is discussed.
Nucleoprotein complexes of minute virus of mice have a distinct structure different from that of chromatin
Authors: Doerig C , McMaster G , Sogo J , Bruggmann H , Beard P
Published: January 2020

We studied the structure of viral nucleoprotein complexes extracted from the nuclei of mouse cells infected with the immunosuppressive strain of the minute virus of mice (MVMi). Two types of complex were detected, with sedimentation coefficients of about 110 and 40S. The complexes sedimenting at 110S contained single-stranded MVMi DNA as well as a second form of viral DNA which apparently had a heat-sensitive secondary structure. The 110S peak also contained proteins which coelectrophoresed with the MVMi capsid proteins. Complexes sedimenting at 40S contained the double-stranded replicative form of MVMi DNA. These complexes sedimented faster than did the pure replicative form DNA (15S), but more slowly than cellular chromatin fragments containing DNA of the same length. They incorporated labeled deoxynucleoside triphosphate in vitro into the replicative form DNA. We investigated the structure of MVMi nucleoprotein complexes in the following ways. Nuclei of MVMi-infected cells were digested with staphylococcal nuclease, and the resulting DNA fragments were electrophoresed, transferred to nitrocellulose, and hybridized first with labeled MVMi DNA and then with cellular DNA. A nucleosomal repeat pattern was seen with the cellular DNA probe but not with the MVMi DNA probe. The DNA in MVMi nucleoprotein complexes was cross-linked with psoralen, purified, denatured, and examined with an electron microscope. Bubbles, indicating the presence of proteins, were seen in the MVMi DNA. The length of the DNA in the bubbles was 90 +/- 29 nucleotides. On the other hand, nucleosomes protected 160 base pairs from cross-linking by psoralen. The MVMi nucleoprotein complexes thus have a distinct structure which is different from that of chromatin.
Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies
Authors: Fleming J O , Trousdale M D , el-Zaatari F A , Stohlman S A , Weiner L P
Published: January 2020

To analyze the pathogenesis of the neurotropic murine coronavirus JHMV, we used monoclonal antibodies to the E2 viral glycoprotein to select antigenic variant viruses. Monoclonal antibodies J.7.2 and J.2.2 were shown to bind to topographically distinct regions of the E2 molecule, and the variants selected with the two antibodies demonstrated very different disease pictures in mice. Variants selected with J.7.2 were, like the parental virus, highly virulent and caused an acute encephalitic illness. By contrast, J.2.2-selected variants predominantly caused a subacute paralytic disease clinically and extensive demyelination histologically. Antigenic differences among the variants and parental virus were readily demonstrable with anti-E2 monoclonal antibodies. However, no differences between the viruses could be shown in binding studies with monoclonal antibodies directed against either E1 or N, the other two JHMV structural proteins. Since only J.2.2 selected demyelinating variants with reduced neurovirulence, it is likely that this monoclonal antibody recognizes a subregion of the E2 molecule that is particularly important in JHMV pathogenesis.
Characterization of murine-specific leukemia virus receptor from L cells
Authors: Johnson P A , Rosner M R
Published: January 2020

The host cell receptor for Moloney murine leukemia virus was solubilized from murine L-cell membranes and characterized. In initial studies designed to identify a receptor-rich cell line, different mouse cells were screened for binding to Moloney gp70, the viral envelope glycoprotein which determines host cell-binding specificity. gp70 binding to murine L cells was specific and saturable, with an apparent affinity constant (Ka) of 4 X 10(8) M-1, and the number of receptors per cell (6 X 10(5)) was similar to that of other mouse fibroblast cell lines. Characterization of the gp70 receptor with regard to extraction by detergents, protease sensitivity, and heat denaturation suggests that the receptor is an intrinsic membrane protein. Upon extraction of L-cell membranes with 0.2% deoxycholic acid and precipitation with acetone, specific and saturable binding of gp70 could be detected. The solubilized gp70-binding component was eluted upon gel filtration on Sephacryl S-300 into a species with an approximate molecular weight of 110,000.
Human papillomaviruses in Buschke-Löwenstein tumors: physical state of the DNA and identification of a tandem duplication in the noncoding region of a human papillomavirus 6 subtype
Authors: Boshart M , zur Hausen H
Published: January 2020

Six Buschke-Löwenstein tumors, i.e., highly differentiated squamous cell tumors of the genital region, were shown to contain human papillomavirus 6 (HPV 6) or HPV 11 genomes. The viral DNA was found in an episomal state, including a very small fraction of circular oligomers. HPV 6a and HPV 6d genomes were cloned from two of the tumors. Comparison with HPV 6b, cloned from a benign genital wart (E. -M. de Villiers, L. Gissmann, and H. zur Hausen, J. Virol. 40:932-935, 1981) by restriction mapping and partial sequence analysis, revealed a very high degree of homology with the different HPV 6 subtypes. A tandem duplication of 459 base pairs within the noncoding region of the genome was found in the new subtype HPV 6d. This structural rearrangement in a region containing the putative control elements for early gene transcription might influence the biological potential of that virus. No evidence for rearrangement of this region was found in the HPV DNA from the five other tumors.
Identification with monoclonal antibodies of virus-specific DNA-binding proteins in the nuclei of cells infected with three serotypes of Marek's disease virus-related viruses
Authors: Nakajima K , Ikuta K , Ueda S , Kato S , Hirai K
Published: January 2020

Two groups of virus-specific polypeptides were identified in the nuclei of infected cells by cross-reacting monoclonal antibodies with three serotypes of Marek's disease virus. Of these, a 135,000-molecular-weight polypeptide common to all three serotypes was found to bind to both double-stranded and single-stranded DNAs.
Sequences near the 5' long terminal repeat of avian leukosis viruses determine the ability to induce osteopetrosis
Authors: Robinson H L , Reinsch S S , Shank P R
Published: January 2020

Avian leukosis virus (ALV)-induced osteopetrosis is associated with the accumulation of unintegrated viral DNA in osteoblasts. Viruses constructed from the DNAs of an osteopetrosis-inducing ALV (Br21) and a non-osteopetrosis-inducing ALV (RAV-0) have been used to test for the role of viral genes in the induction of osteopetrosis. Our results map osteopetrotic potential to a 1,400-base-pair region near the 5' long terminal repeat. This region contains signals for the splicing, translation, and packaging of viral RNAs and coding sequences for the gag proteins p19 and p10 and the N terminus of p27.
The neurovirulent determinants of ts1, a paralytogenic mutant of Moloney murine leukemia virus TB, are localized in at least two functionally distinct regions of the genome
Authors: Yuen P H , Tzeng E , Knupp C , Wong P K
Published: January 2020

To better understand the molecular mechanism involved in retrovirus ts1-induced paralytic disease in mice, we constructed a panel of recombinant viruses between ts1 and the wild-type viruses Moloney murine leukemia virus (MoMuLV) and MoMuLV-TB, a strain of MoMuLV. These recombinant viruses were constructed in an attempt to identify the sequence(s) in the genome of ts1 which contains the critical mutation(s) responsible for the neurovirulence of ts1. Two functionally distinct sequences in the genome of ts1 were found to be responsible for its paralytogenic ability. One of these sequences, the 0.77-kilobase-pair XbaI-BamHI (nucleotides 5765 to 6537) fragment which encodes the 5' half of gp70 and 11 base pairs upstream of the env gene coding sequence, determines the inability of ts1 to process Pr80env. The other sequence, the 2.30-kilobase-pair BamHI-PstI (nucleotides 538 to 8264 and 1 to 567) fragment, which comprises nearly two-thirds of the env gene, the long terminal repeat, and the 5' noncoding sequence, determines the enhanced neurotropism of ts1. Replacement of any one of these two regions with the homologous region from either one of the two wild-type viruses resulted in recombinant viruses which either totally failed to induce paralysis or induced a greatly attenuated form of paresis in some of the infected mice.
Characterization of a progressive neurodegenerative disease induced by a temperature-sensitive Moloney murine leukemia virus infection
Authors: Bilello J A , Pitts O M , Hoffman P M
Published: January 2020

A progressive neurodegenerative disease occurred following infection of mice with a temperature-sensitive (ts) isolate of Moloney (Mo) murine leukemia virus (MuLV), ts Mo BA-1 MuLV. This NB-tropic ecotropic MuLV, which was ts for a late function, induced a syndrome of tremor, weakness of the hind limbs, and spasticity following infection of several strains of laboratory neonatal mice, including NFS, C3H/He, CBA, SJL, and BALB/c. The latent period of 8 to 16 weeks was considerably longer than that observed for the acute paralytic diseases observed following neonatal infection with other ts Mo-MuLV, rat-passaged Friend MuLV, and some wild mouse ecotropic MuLVs. Spongiform pathology without inflammation and degeneration of neurons devoid of budding virions occurred in the cerebellar grey matter, brain stem, and upper spinal cord; but lower spinal cord anterior horn cells were less obviously affected than in other MuLV-associated neuroparalytic syndromes. ts Mo BA-1 MuLV differed from other ts Mo-MuLV mutants that are capable of inducing a neuroparalytic syndrome in that while infected nervous system tissue contained high levels of MuLV p30 and gp70, no evidence of precursor accumulation or abnormal processing of MuLV p30 or gp70 could be demonstrated. The localization of virus within the nervous system suggests that direct neuronal infection may not be the etiologic mechanism in this MuLV-induced neurodegenerative disease.
Evolutionary changes of transcriptional control region in a minute-plaque viable deletion mutant of BK virus
Authors: Watanabe S , Yoshiike K
Published: January 2020

Two plaque morphology BK virus (BKV) mutants (pm526 and pm527) rescued from a hamster pineocytoma cell line Pc13 were characterized and compared with a similarly rescued and previously characterized mutant (pm522), its derivatives (tr530, tr531, and tr532), and the wild type (501) for their biological activities and for the structures of their transcriptional control regions. The two mutants grew somewhat more slowly in human embryonic kidney cells but transformed rat 3Y1 cells more efficiently than did the wild-type BKV. BKV pm526 formed minute plaques and had the shortest transcriptional control region, having only one 68-base-pair element, which is triplicated in the wild-type BKV. BKV pm526 was unstable during repeated replication in human embryonic kidney cells and yielded large-plaque viruses with longer HindIII C segments encompassing the BKV DNA replication origin. Comparison of nucleotide sequences of the transcriptional control regions among the mutants and the wild type showed that pm526 is a parent virus, from which all the other mutants had evolved, and that the evolutionary changes of the plaque size, from minute to small to large, were due to duplications of a certain segment containing the adenovirus E1A enhancer core or the simian virus 40 enhancer core found in the BKV 68-base-pair element. The activities to enhance early transcription, as measured by the ability to direct the synthesis of chloramphenicol acetyltransferase, approximately paralleled the plaque size. The duplication containing the adenovirus E1A enhancer core did not affect the transforming capacity of the parent virus, but the duplication including the simian virus 40 enhancer core significantly lowered the transforming capacity for rat cells.
In vivo transformation of human skin with human papillomavirus type 11 from condylomata acuminata
Authors: Kreider J W , Howett M K , Lill N L , Bartlett G L , Zaino R J , Sedlacek T V , Mortel R
Published: January 2020

Human papillomaviruses (HPVs) have been implicated in the development of a number of human malignancies, but direct tests of their involvement have not been possible. We describe a system in which human skin from various sites was infected with HPV type 11 (HPV-11) extracted from vulvar condylomata and was grafted beneath the renal capsule of athymic mice. Most of the skin grafts so treated underwent morphological transformation, resulting in the development of condylomata identical to those which occur spontaneously in patients. Foreskins responded with the most vigorous proliferative response to HPV-11. The lesions produced the characteristic intranuclear group-specific antigen of papillomaviruses. Both dot blot and Southern blot analysis of DNA from the lesions revealed the presence of HPV-11 DNA in the transformed grafts. These results demonstrate the first laboratory system for the study of the interaction of human skin with an HPV. The method may be useful in understanding the mechanisms of HPV transformation and replication and is free of the ethical restraints which have impeded study. This system will allow the direct study of factors which permit neoplastic progression of HPV-induced cutaneous lesions in human tissues.
Measurement of the mutation rates of animal viruses: influenza A virus and poliovirus type 1
Authors: Parvin J D , Moscona A , Pan W T , Leider J M , Palese P
Published: January 2020

Epidemiologic and genetic evidence suggests that influenza A viruses evolve more rapidly than other viruses in humans. Although the high mutation rate of the virus is often cited as the cause of the extensive variation, direct measurement of this parameter has not been obtained in vivo. In this study, the rate of mutation in tissue culture for the nonstructural (NS) gene of influenza A virus and for the VP1 gene in poliovirus type 1 was assayed by direct sequence analysis. Each gene was repeatedly sequenced in over 100 viral clones which were descended from a single virion in one plaque generation. A total of 108 NS genes of influenza virus were sequenced, and in the 91,708 nucleotides analyzed, seven point changes were observed. A total of 105 VP1 genes of poliovirus were sequenced, and in the 95,688 nucleotides analyzed, no mutations were observed. We then calculated mutation rates of 1.5 X 10(-5) and less than 2.1 X 10(-6) mutations per nucleotide per infectious cycle for influenza virus and poliovirus, respectively. We suggest that the higher mutation rate of influenza A virus may promote the rapid evolution of this virus in nature.
Human parainfluenza virus type 3: messenger RNAs, polypeptide coding assignments, intergenic sequences, and genetic map
Authors: Spriggs M K , Collins P L
Published: January 2020

cDNA clones of mRNAs for the major nucleocapsid protein (NP), the nucleocapsid P protein plus the nonstructural C protein (P+C), and the matrix protein (M) of human parainfluenza virus type 3 (PF3) were identified by hybrid arrest and hybrid selection of in vitro translation. Previously, cDNA clones were identified and sequenced for the hemagglutinin-neuraminidase glycoprotein (HN) and the fusion glycoprotein (F) mRNAs (N. Elango, J. E. Coligan, R. C. Jambou, and S. Venkatesan, J. Virol. 57:481-489, 1986; M. K. Spriggs, R. A. Olmsted, S. Venkatesan, J. E. Coligan, and P. L. Collins, Virology 152:241-251, 1986). Synthetic oligonucleotides, designed from nucleotide sequences of the cDNAs, were used to direct dideoxynucleotide sequencing of gene junctions in PF3 genomic RNA (vRNA). From sequencing of vRNA, a sixth viral gene was detected and identified as the large nucleocapsid protein (L) gene by hybridization of a synthetic oligonucleotide to intracellular PF3 mRNAs separated by gel electrophoresis. The order of the six PF3 genes on vRNA was 3'-NP-P+C-M-F-HN-L-5'. The five intergenic regions consisted of the trinucleotide 3'-GAA. The PF3 genes initiated with semiconserved 10-nucleotide gene-start sequences and terminated with semiconserved 12-nucleotide gene-end sequences. The M gene terminated with an aberrant gene-end sequence; analysis of intracellular mRNA showed that this aberrant sequence correlated with a disproportionately high accumulation of readthrough mRNA. These studies showed that PF3 encodes six unique mRNAs (NP, P+C, M, F, HN, and L) that encode seven proteins (NP, P, C, M, F, HN, and L) and provided evidence of a close relationship between PF3 and Sendai (murine parainfluenza type 1) viruses.
Characteristics of Bacteriophage N1 and Its Attachment to Cells of
Micrococcus lysodeikticus
Authors: Lovett Paul S. , Shockman Gerald D.
Published: January 2020

Bacteriophage N1 was purified by differential and equilibrium gradient centrifugation and characterized with respect to bouyant density in CsCl, one-step growth properties, host range, and morphology by electron microscopy. In a tris (hydroxymethyl) aminomethane-magnesium buffer (
p
H 7.15), the irreversible adsorption of N1 to cells of
Micrococcus lysodeikticus
strain 1 (ML-1) followed first-order reaction kinetics with an adsorption-velocity constant of 1.6 × 10
−9
/min at 32 C. The rate of phage attachment was not significantly altered when adsorption mixtures contained 0.01
m
KCN or 1% casein hydrolysate, 0.01
m
CaCl
2
, and 0.001
m
tryptophan. The activation energy for the irreversible adsorption reaction was 8.6 kcal. Treatment of ML-1 cells by any of the following procedures reduced the irreversible phage receptor activity over 90%: (i) mechanical disruption, (ii) lysozyme digestion, (iii) incubation in 1% cetyltrimethylammonium bromide, or (iv) incubation of heated cells (100 C, 15 min) with trypsin, Pronase, or lysozyme. The sensitivity of the phage receptor activity of ML-1 cells to lysozyme suggests that the bacterial cell wall is involved in the receptor site for the virus. Destruction of receptor activity by the other treatments cited above implies that, in addition to the cell wall, other cellular components may participate in the irreversible attachment of N1 phage to cells.
Membrane Binding of Input Arbovirus Ribonucleic Acid: Effect of Interferon or Cycloheximide
Authors: Friedman Robert M. , Sreevalsan T.
Published: January 2020

By 1 hr after infection, 36% of input Semliki Forest virus ribonucleic acid (RNA) which was cell associated was found in a membrane structure. This structure had many similarities to the membrane-associated replication complex (MRC) which had previously been identified in arbovirus infections. Interferon treatment did not affect the association of viral RNA with the MRC structure, but cycloheximide treatment inhibited it.
Defective Virions of Reovirus
Authors: Nonoyama M. , Watanabe Y. , Graham A. F.
Published: January 2020

When purified preparations of stock reovirus, type 3, were digested with chymotrypsin, the virions were converted into two different types of particle. These new particles could be separated from each other by isopycnic centrifugation in cesium chloride gradients. One particle banded at a buoyant density of 1.43 g/cm
3
, the other at a density of 1.415 g/cm
3
. The former particle is termed the heavy (H) particle, the latter is the light (L) particle. The ratio of H/L particles varied between 0.5 and 0.25 in various purified preparations of virus. In electron micrographs, both H and L particles had the appearance and dimensions of viral cores. H particles were infectious for L cells. When plaques formed by stock virus, or by H particles, were picked and propagated in L cells, the majority of the clones gave rise only to H particles on chymotrypsin digestion. On continued serial passage of the clones, virions containing L particles again appeared in the progeny. The simplest explanation of these results was that stock virus was comprised of two populations of virions. One type of virion which contained H particles was infectious, whereas the other, which contained L particles, was not itself infectious and could replicate only in cells coinfected with an H particle virion. Added weight was given to this hypothesis by two observations. First, a small but definite separation of H and L virions could be achieved by isopycnic centrifugation in a gradient of cesium chloride. Second, L particles and virions containing L particles were both shown to lack the largest of the ten segments of double-stranded ribonucleic acid genome. Thus, L particle virions have defective genomes.
New Generalized Transducing Bacteriophage in
Echerichia coli
Authors: Mise Katsutoshi , Suzuki Kenji
Published: January 2020

A new generalized transducing bacteriophage similar to P1 was isolated from
Salmonella oranienburg.
Temperature-Sensitive Simian Virus 40 Mutant Defective in a Late Function
Authors: Kit Saul , Tokuno S. , Nakajima K. , Trkula D. , Dubbs D. R.
Published: January 2020

A temperature-sensitive simian virus 40 (SV40) mutant, tsTNG-1, has been isolated from nitrosoguanidine-treated and SV40-infected African green monkey kidney (CV-1) cultures. Replication of virus at the nonpermissive temperature (38.7 C) was 3,000-fold less than at the permissive temperature (33.5 C). Plaque formation by SV40tsTNG-1 deoxyribonucleic acid (DNA) on CV-1 monolayers occurred normally at 33.5 C but was grossly inhibited at 38.7 C. The time at which virus replication was blocked at 38.7 C was determined by temperature-shift experiments. In shift-up experiments, cultures infected for various times at 33.5 C were shifted to 38.7 C. In shift-down experiments, cultures infected for various times at 38.7 C were shifted to 33.5 C. All cultures were harvested at 96 hr postinfection (PI). No virus growth occurred when the shift-up occurred before 40 hr PI. Maximum virus yields were obtained at 96 hr PI when the shift-down occurred at 66 hr, but only about 15% of the maximum yield was obtained when the shift-down occurred at 76 hr PI. These results indicate that SV40tsTNG-1 contains a conditional lethal mutation in a late viral gene function. Mutant SV40tsTNG-1 synthesized T antigen, viral capsid antigens, and viral DNA, and induced thymidine kinase activity at either 33.5 or 38.7 C. The properties of the SV40 DNA synthesized in mutant-infected CV-1 cells at 33.5 or 38.7 C were very similar to those of SV40 DNA made in parental virus-infected cells, as determined by nitrocellulose column chromatography, cesium-chloride-ethidium bromide equilibrium centrifugation, and by velocity centrifugation in neutral sucrose gradients. Mutant SV40tsTNG-1 enhanced cellular DNA synthesis in primary cultures of mouse kidney cells at 33.5 and 38.7 C and also transformed mouse kidney cultures at 36.5 C. SV40tsTNG-1 was recovered from clonal lines of transformed cells after fusion with susceptible CV-1 cells and incubation of heterokaryons at 33.5 C, but not at 38.7 C.
Requirement for a Functional
int
Product in Temperature Inductions of Prophage P22
ts mnt
Authors: Gough Michael
Published: January 2020

Prophage P22
ts mnt
was found to be inducible by high temperatures. This property was maintained when the
ts mnt
allele was recombined into a phage that cannot synthesize phage deoxyribonucleic acid at high temperature. Prophage P22
int ts mnt
, on the other hand, was not inducible at high temperature. This result leads to the suggestion that the
int
enzyme, which is involved in excision, is regulated by the product of gene
mnt
.
Effect of Rifampicin on Poxvirus Protein Synthesis
Authors: Tan K. B. , McAuslan B. R.
Published: January 2020

Poxvirus strains differ with respect to the effect of rifampicin on viral protein synthesis. Rifampicin severely depressed vaccinia-directed protein synthesis but had little effect on the rate of cowpox-directed protein synthesis, including one late virus-induced enzyme. The spectrum of polypeptides synthesized in cowpox-infected cells was similar in the presence or absence of rifampicin except for one significant difference. After removal of rifampicin, viruslike particles assembled to some extent, even when protein synthesis was inhibited by cycloheximide. However, reversal was more extensive if protein synthesis was allowed.
Genetic Mapping of a Defective Bacteriophage on the Chromosome of
Bacillus subtilis
168
Authors: Garro Anthony J. , Leffert Hyam , Marmur Julius
Published: January 2020

A genetic marker responsible for the killing activity of PBSX, a defective phage carried by
Bacillus subtilis
168, has been located on the bacterial chromosome. Two mutant strains of
B. subtilis
168, which produced tailless phage particles upon mitomycin C induction, were shown to carry lesions, designated
xtl-1
and
xtl-2
, which were linked by transformation and PBS1-mediated transduction to
metC
. The link-age relationship between
xtl
and adjacent auxotrophic markers was determined by three-factor PBS1 transduction, the suggested order of markers being
argO 1 metA metC xtl.
Nucleic Acid Synthesis in
Bacillus subtilis
Infected with Bacteriophage β22
Authors: Hemphill H. Ernest , Whiteley H. R.
Published: January 2020

Changes in the synthesis of host and phage nucleic acid after infection of
Bacillus subtilis
with virulent bacteriophage β22 were analyzed by deoxyribonucleic acid (DNA)-DNA and ribonucleic acid (RNA)-DNA hybridization. Host DNA replication continued during the first third of the 55-min latent period and then ceased at approximately the time replication of the phage genome was initiated. Host-specific RNA was synthesized concurrently with phage RNA during the first half of the latent period but was repressed late in the infection. For much of the latent period, the population of phage-specific RNA changed continually as new species were transcribed and earlier species were repressed; detectable changes ceased coincidentally with the appearance of intracellular phage. Control over transcription of phage DNA was to some degree an intrinsic property of the interaction of
B. subtilis
DNA-dependent RNA polymerase and the phage genome, since only the early species of phage RNA were synthesized in vitro by
B. subtilis
polymerase and pure β22 DNA. In vitro transcription of late functions was demonstrated by using the endogenous RNA polymerase activity of the nucleoprotein complex (nuclear fraction) from infected cells.
Control of the Replication Complex of Bacteriophage P22
Authors: Levine Myron , Chakravorty Maharani , Bronson Morley J.
Published: January 2020

A replication complex for the vegetative synthesis of the deoxyribonucleic acid (DNA) of the temperate phage P22 previously has been described. This complex is an association of parental phage DNA, most of the newly synthesized phage DNA made during pulses with
3
H-thymidine, and other cell constituents, and has a sedimentation rate in neutral sucrose gradients of at least 1,000
S
. The complex is one of the intermediates, intermediate I, in the synthesis and maturation of phage P22 DNA after infection or induction. Evidence supporting the replicative nature of intermediate I is presented. Phage replication is repressed in lysogenic bacteria. On superinfection of P22 lysogens with nonvirulent phage, little association of the input phage DNA with a rapidly sedimenting fraction is demonstrable. However, after induction with ultraviolet light, the superinfecting parental phage DNA quickly acquires the rapid sedimentation rate characteristic of intermediate I; phage DNA synthesis follows; and progeny phages are produced. Infection with a virulent mutant of P22 produces progeny phages in lysogens. Its DNA associates with intermediate I. In mixed infection with the virulent phage, replication of nonvirulent phage P22 is still repressed, even though the virulent replicates normally. The nonvirulent input DNA does not associate with intermediate I. The repressor of the lysogenic cell prevents replication by interfering with the physical association of template material with intermediate I. A phage function is required for association of phage template with the replication machinery.
Interruption by Rifampin of an Early Stage in Vaccinia Virus Morphogenesis: Accumulation of Membranes Which Are Precursors of Virus Envelopes
Authors: Grimley Philip M. , Rosenblum Edith N. , Mims Sharon J. , Moss Bernard
Published: January 2020

Assembly of vaccinia virus envelopes and immature vaccinia particles was interrupted in HeLa cells treated with rifampin (
rifampicin
). The primary action of rifampin on vaccinia morphogenesis appeared to occur during the stage of envelope formation. When envelopes and immature particles were already present, maturation could continue, even in the presence of rifampin. It was demonstrated that the trilaminar membranes of irregular contour which accumulate in the presence of rifampin are precursors of virus envelopes. When rifampin was removed under controlled conditions, synchronous transitions were observed as the precursor membranes rapidly converted into uniformly curved envelope units with a 10- to 12-nm coat on the convex surface. These experiments provided an opportunity to examine the sequence of some early events in vaccinia morphogenesis. Initially, nascent envelopes remained in clusters around dense viroplasm. Large numbers of single immature particles appeared within 10 min. Nucleation of immature particles was the first evidence of core differentiation and began within 5 to 10 min. Development of lateral bodies and modeling of the biconcave cores was observed within 30 min, and structurally mature virions were present by 2 hr after the removal of rifampin. High resolution autoradiography showed that viral deoxyribonucleic acid, which labeled with
3
H-thymidine during rifampin treatment, was incorporated by the mature vaccinia which formed after rifampin was removed. Concentration of the viral deoxyribonucleic acid in core material evidently occurred after envelope assembly, probably coincident with nucleoid formation. Cytoplasmic crystalloid bodies accumulated during rifampin treatment; they appeared morphologically identical to vaccinia nucleoids and were heavily labeled by
3
H-thymidine.
Development of Bacteriphage α: Requirement for the Integrity of One Strand of Parental Deoxyribonucleic Acid
Authors: Strniste Gary F. , Taylor William D.
Published: January 2020

Bacteriophage α-
Bacillus megaterium
complexes are as sensitive as free phage to the decay of
32
P incorporated in parental phage deoxyribonucleic acid during the eclipse period.
Basis for Variable Response of Arboviruses to Guanidine Treatment
Authors: Friedman Robert M.
Published: January 2020

The effect of guanidine on the replication of the group A arboviruses, Sindbis virus, and Semliki Forest virus (SFV) was studied. Guanidine rapidly, but reversibly, inhibited SFV ribonucleic acid (RNA) synthesis. The synthesis of all species of viral RNA was inhibited, but that of ribonuclease-resistant forms was least affected. This inhibition occurred when the drug was added at any point during the log phase of virus growth. The growth of SFV was also markedly inhibited, but Sindbis virus growth was unimpaired. Infection of guanidine-treated cells with the viruses together resulted in a significant inhibition of the yields of both. It appears that, in the case of Sindbis virus, viral RNA is ordinarily produced in such excess that inhibition of its synthesis does not reduce virus yields. In the case of SFV, guanidine also markedly distorts the pattern of RNA synthesis by greatly decreasing the production of the 26
S
interjacent RNA form. This may account for the observed inhibition of SFV growth in the presence of guanidine.
Integration of the Deoxyribonucleic Acid of Adenovirus Type 12 into the Deoxyribonucleic Acid of Baby Hamster Kidney Cells
Authors: Doerfler Walter
Published: January 2020

In a previous report, evidence was presented that the deoxyribonucleic acid (DNA) of adenovirus type 12 (Ad12) is integrated by covalent linkage into the DNA of baby hamster kidney cells (BHK-21 cells). These studies have been extended. The DNA of Ad12 and that of BHK-21 cells grown in medium containing 5-bromodeoxyuridine could be separated by equilibrium centrifugation in alkaline CsCl density gradients. BHK-21 cells were infected with
3
H-labeled Ad12, and the total intracellular DNA was analyzed at various times after infection in alkaline CsCl density gradients. The
3
H label in the position of cellular DNA hybridized predominantly with viral DNA and to a lesser extent also with cellular DNA. Replication of viral DNA could not be detected in BHK-21 cells. The appearance of viral
3
H label in the density stratum of cellular DNA was not significantly affected when DNA synthesis in Ad12-infected BHK-21 cells was inhibited >96% by cytosine arabinoside. These findings provided additional evidence for integration of Ad12 DNA into the DNA of BHK-21 cells. It could be calculated that 5 to 55 Ad12 DNA equivalents per cell are integrated. Replication of viral or cellular DNA was not required for integration. Inhibition of protein or ribonucleic acid synthesis interfered with integration only slightly.
Simian Virus 40 Deoxyribonucleic Acid Transcription In Vitro: Binding and Transcription Patterns with a Mammalian Ribonucleic Acid Polymerase
Authors: Herzberg Max , Winocour Ernest
Published: January 2020

The in vitro transcription pattern of simian virus 40 (SV40) deoxyribonucleic acid (DNA) by a mammalian ribonucleic acid (RNA) polymerase, was studied by electron microscopy and velocity sedimentation techniques. It was found that (i) the majority of supercoiled SV40 DNA molecules displayed a single binding site for the enzyme, (ii) the supercoiled structure of SV40 DNA was frequently retained during transcription, and (iii) the majority of RNA molecules synthesized from the supercoiled SV40 DNA template showed no self-complementarity and sedimented relatively homogeneously in the 15
S
to 16
S
region of a sucrose gradient (in contrast, the RNA product synthesized from the nicked-circular SV40 DNA template showed self-complementarity and sedimented heterogeneously). RNA polymerase preparations isolated from SV40-infected monkey cells were more active than those isolated from uninfected monkey cells.
Two African Viruses Serologically and Morphologically Related to Rabies Virus
Authors: Shope Robert E. , Murphy Frederick A. , Harrison Alyne K. , Causey Ottis R. , Kemp Graham E. , Simpson D. I. H. , Moore Dorothy L.
Published: January 2020

Lagos bat virus and an isolate from shrews (IbAn 27377), both from Nigeria, were found to be bullet-shaped and to mature intracytoplasmically in association with a distinct matrix. They were related to, but readily distinguishable from, rabies virus and each other by complement fixation and neutralization tests. The three viruses, including rabies, form a subgrouping within the rhabdoviruses.
Mapping of Prophage and Mature Deoxyribonuleic Acid from Temperate
Bacillus
Bacteriophage φ105 by Marker Rescue
Authors: Armentrout Richard W. , Rutberg Lars
Published: January 2020

By using temperature-sensitive (
ts
) and suppressor-sensitive (
sus
) mutants, 11 essential genes have been identified in phage φ105. The order of the genes has been established in two- and three-factor crosses. The genes can be arranged in a linear order; this order is identical in the vegetative phage and in the prophage. One gene essential for phage deoxyribonucleic acid (DNA) synthesis has been found. Marker rescue from prophage and mature DNA, taken up by competent bacteria, was studied by superinfection with phage carrying one
sus
and one
ts
mutation. In prophage DNA, all single markers studied are rescued at similar frequencies. The frequency of co-rescue of two markers is proportional to the recombinational distance between the markers. Thus, colinearity between the genetic map and the position on the DNA molecule of those mutations used to establish the map is demonstrated. The results indicate that the recombination frequencies observed in vegetative crosses are a relative measure of the physical distance between markers. All single markers are not rescued at equal frequencies from mature DNA. The frequency of co-rescue of two markers is related to the recombinational distance only over a distance about one-fourth or less of the genetic map. Markers separated by 10% recombination, or more, are co-rescued at 5 to 10% of the frequency of rescue of single markers. Shearing of mature DNA into half-sized molecules reduces the efficiency by which single markers are rescued by a factor of 5 to 10. The results of experiments on co-rescue of two markers from half-sized mature DNA indicate a preferred break-point near the middle of the genetic map; the results are compatible with a nonpermuted sequence in mature DNA. It is pointed out and discussed that mature DNA exhibits several anomalies in marker rescue experiments.