bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic and DNA mapping of the late regulation and lysis genes of Salmonella bacteriophage P22 and coliphage lambda
Authors: Wiggins B A , Hilliker S
Published: January 2020

Genetic and DNA heteroduplex analyses of lambda imm22 hybrid phages were used to compare the Salmonella bacteriophage P22 and coliphage lambda genes which control late gene regulation and lysis. Homologous DNA sequences were correlated with P22 gene 23 and lambda gene Q (late gene regulation) and with P22 gene 13 and lambda gene S (lysis control). Nonhomologous DNA sequences were correlated with P22 gene 19 and lambda gene R (lysozyme and endolysin) and with the region encoding the P22 alpha and lambda 6S transcripts.
Effect of human alpha A interferon on influenza virus replication in MDBK cells
Authors: Ransohoff R M , Maroney P A , Nayak D P , Chambers T M , Nilsen T W
Published: January 2020

To determine the molecular mechanism whereby interferon induces resistance to influenza virus, we began an investigation of influenza virus replication in MDBK cells treated with recombinant human alpha A interferon. Negative- and positive-strand virus-specific RNA accumulation was monitored by blot hybridization with cloned probes. Primary transcription (transcription of infecting viral negative strands by the virion-associated polymerase) was inhibited by interferon treatment of MDBK cells. At moderate levels of interferon treatment (10 U/ml), this inhibition was restricted to transcripts of polymerase genes, whereas at higher levels of interferon treatment (50 U/ml), accumulation of all primary transcripts was markedly inhibited. Secondary transcripts and viral negative strands did not accumulate to any significant extent in interferon-treated MDBK cells. These results suggest that interferon-induced mechanisms which inhibit influenza virus replication in MDBK cells act at the level of primary transcription.
Normal expression of polymorphic endogenous retroviral RNA containing segments identical to mink cell focus-forming virus
Authors: Levy D E , Lerner R A , Wilson M C
Published: January 2020

In the absence of infectious virus, strains of mice express polyadenylated RNA transcripts homologous to the genome of murine leukemia virus. In addition to transcripts consistent with full-length and spliced env retroviral RNAs, several unique RNA species which lack the env sequence accumulate in a tissue-specific manner. These RNA species are presumed to be transcribed from endogenous retroviral sequences that constitute the bulk of the murine leukemia virus-related sequences in the murine genome. To determine the relationship of these RNA transcripts to infectious murine leukemia virus and the precise structural basis of the heterogeneity observed for the env-lacking transcripts, we isolated and sequenced cDNA recombinants representing the RNAs expressed in strain 129 GIX+ mice. Comparisons of the nucleotide sequences demonstrated that the endogenous retroviral transcripts differed in pol, p15E, and R-peptide regions by single nucleotide changes. In contrast, the gp70-coding regions of two cDNA clones derived from epididymis and liver were completely homologous over a 599-nucleotide overlapping sequence. The structures of env-lacking transcripts were examined in two independent cDNA clones, and each was found to contain a different deletion that was potentially mediated by seven-base pair direct repeats in the intact sequence. The extensive sequence homology between cDNAs allowed construction of a cumulative sequence map of the 3' end of an intact endogenous retroviral transcript. A comparison of this sequence with infectious ecotropic and mink cell focus-forming viruses revealed that the endogenous transcripts are highly homologous with the substituted portions of leukemogenic mink cell focus-forming viruses and therefore further define the boundaries of recombination required to generate these viruses.
Biological and biochemical differences between variants of spleen focus-forming virus can be localized to a region containing the 3' end of the envelope gene
Authors: Ruscetti S , Wolff L
Published: January 2020

Two variants of the spleen focus-forming virus (SFFV), SFFVP and SFFVA, induce acute erythroleukemia in mice but differ in their effects on erythroid cells as well as in the posttranslational modification of the product of their envelope genes. To localize the region of the SFFV envelope gene responsible for these differences, we utilized a recombinant virus containing the 3' half of the SFFVP env gene, where the vast majority of the differences between SFFVP and SFFVA reside, and the SFFVP long terminal repeat (LTR) on an SFFVA background. Analysis of the recombinant virus indicates that it is capable of inducing all of the biological effects previously associated with SFFVP, including the ability to proliferate and differentiate without the need for erythropoietin. In addition, the env gene product of the recombinant virus can be detected on the cell surface, a property previously associated only with SFFVP. Although the recombinant virus also contains LTR sequences from SFFVP, we do not believe it is likely that the four LTR nucleotides that are unique to SFFVP are responsible for the biological or biochemical differences observed. These results strengthen the argument that the SFFVP env gene product acts at the cell surface to alter the hormonal requirements for erythroid cell growth and differentiation.
Membrane association of the transforming protein of avian sarcoma virus UR2 and mutants temperature sensitive for cellular transformation and protein kinase activity
Authors: Garber E A , Hanafusa T , Hanafusa H
Published: January 2020

The localization of the transforming protein P68gag-ros of avian sarcoma virus UR2, which has a hydrophobic region at the N terminus of its ros-specific tyrosine kinase-encoding sequence, was examined by subcellular fractionation. P68 behaved as an integral membrane protein associated with the plasma membrane of transformed cells. P68 became membrane associated very rapidly in its biogenesis. Three temperature-sensitive mutants of UR2 were isolated and characterized. Cells infected with the mutants were temperature sensitive for morphological alteration and colony formation. The mutant P68 proteins were membrane associated in mutant-infected cells regardless of the temperature but were active as protein kinases only at the permissive temperature. The results suggest that P68 is a membrane-associated protein whose kinase activity plays a crucial role in UR2-mediated cell transformation.
Epstein-Barr virus genome may encode a protein showing significant amino acid and predicted secondary structure homology with glycoprotein B of herpes simplex virus 1
Authors: Pellett P E , Biggin M D , Barrell B , Roizman B
Published: January 2020

We report significant sequence and predicted secondary structure homology between the herpes simplex virus 1 glycoprotein B (gB) and a protein predicted to be encoded by the BALF4 reading frame of Epstein-Barr virus (EBV). Homology was detectable at the DNA level and was highly significant at the protein level and when evolutionary substitution frequencies of amino acids in related proteins were taken into account. Hydropathic analyses predicted that the two proteins possess conserved N-terminal and C-terminal hydrophobic domains. The N-terminal hydrophobic domains share features in common with known cleavable membrane insertion signal sequences. The amino acid sequences of the C-terminal hydrophobic domains predict three adjacent membrane-spanning segments as had been previously predicted for gB. In an alignment of the two amino acid sequences, 247 of 903 gB residues had a matched pair in the BALF4 sequence, and 247 of 854 BALF4 residues were found to have a matched pair in the gB sequence. In addition, all 10 cysteine residues located outside the predicted signal sequence of both proteins were conserved, as were four predicted N-linked glycosylation sites. In all, 43% of the residues in the aligned sequences are predicted to possess equivalent secondary structures. gB is a virion envelope glycoprotein required for virus entry into cells. The domain of gB determining the rate of entry into cells has been mapped; the predicted structure of this domain in gB and the predicted EBV protein are almost identical. Similarly, the cytoplasmic domain of gB postulated to interact with submembrane proteins was also nearly identical in predicted structure to that of the EBV protein. These results suggest that EBV encodes a protein similar in structure and function to the herpes simplex virus 1 gB.
Analysis of reassortment of genome segments in mice mixedly infected with rotaviruses SA11 and RRV
Authors: Gombold J L , Ramig R F
Published: January 2020

Seven-day-old CD-1 mice born to seronegative dams were orally inoculated with a mixture of wild-type simian rotavirus SA11 and wild-type rhesus rotavirus RRV. At various times postinfection, progeny clones were randomly isolated from intestinal homogenates by limiting dilution. Analysis of genome RNAs by polyacrylamide gel electrophoresis was used to identify and genotype reassortant progeny. Reassortment of genome segments was observed in 252 of 662 (38%) clones analyzed from in vivo mixed infections. Kinetic studies indicated that reassortment was an early event in the in vivo infectious cycle; more than 25% of the progeny clones were reassortant by 12 h postinfection. The frequency of reassortant progeny increased to 80 to 100% by 72 to 96 h postinfection. A few reassortants with specific constellations of SA11 and RRV genome segments were repeatedly isolated from different litters or different animals within single litters, suggesting that these genotypes were independently and specifically selected in vivo. Analysis of segregation of individual genome segments among the 252 reassortant progeny revealed that, although most segments segregated randomly, segments 3 and 5 nonrandomly segregated from the SA11 parent. The possible selective pressures active during in vivo reassortment of rotavirus genome segments are discussed.
Semipermissive Replication of a Nuclear Polyhedrosis Virus of
Autographa californica
in a Gypsy Moth Cell Line
Authors: McClintock J. Thomas , Dougherty Edward M. , Weiner Ronald M.
Published: January 2020

Several gypsy moth cell lines have been previously described as nonpermissive for the multiple-embedded nuclear polyhedrosis virus of
Autographa californica
(AcMNPV). In this report, we demonstrate the semipermissive infection of a gypsy moth cell line, IPLB-LD-652Y, with AcMNPV. IPLB-LD-652Y cells infected with AcMNPV produced classic cytopathic effects but failed to yield infectious progeny virus. Results of experiments employing DNA-DNA dot hybridization suggested that AcMNPV DNA synthesis was initiated from 8 to 12 h postinfection (p.i.), continued at a maximum rate from 12 to 20 h p.i., and declined from 20 to 36 h p.i. The rate of AcMNPV DNA synthesis approximated that observed in the permissive TN-368 cell line. AcMNPV-infected IPLB-LD-652Y cells, pulse-labeled with [
35
S]methionine at various time intervals p.i. and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed four virus-induced proteins, one novel to the semipermissive system and three early α proteins, synthesized from 1 to 20 h p.i. Thereafter, both host and viral protein synthesis was completely suppressed. These results suggest that AcMNPV adsorbed, penetrated, and initiated limited macromolecular synthesis in the semipermissive gypsy moth cell line. However, the infection cycle was restricted during the early phase of AcMNPV replication.
Properties of cells transformed by the middle T-antigen-coding region of polyomavirus
Authors: Priehs C , Friderici K , Winberry L , Fluck M M
Published: January 2020

A series of 10 Fischer rat transformed clonal cell lines were independently obtained in infections with a defective polyomavirus containing a scrambled genome except for an intact middle and small T-antigen-coding region. These cells synthesize middle and small T antigens; no fragment of large T antigen can be detected in any of them. The transformed phenotype of this set of cell lines (designated LT-) has been studied with respect to serum dependence, saturation density, and anchorage independence and compared with the phenotype of a set of six transformants (designated LT+) which synthesize detectable to high levels of shortened or normal-sized large T antigen. Both the LT+ and the LT- groups of polyomavirus transformants display a range of transformed phenotypes. These ranges overlap, and the variations within each group are larger than the variations between the two groups. Thus, the results suggest that, for established Fischer rat fibroblasts, the maintenance of any of the three phenotypes tested and, in particular, of serum independence is not necessarily correlated with the levels of large T antigen or fragments thereof.
Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis
Authors: Sawicki S G , Sawicki D L
Published: January 2020

The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times after infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [3H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minus-strand RNA synthesis was three- to fourfold more sensitive to inhibition by cycloheximide than was plus-strand synthesis.
Analysis of pp60c-src protein kinase activity in hamster embryo cells transformed by simian virus 40, human adenoviruses, and bovine papillomavirus 1
Authors: Amini S , Lewis A M , Israel M A , Butel J S , Bolen J B
Published: January 2020

We have examined the effect of DNA tumor virus transformation of primary hamster embryo cells on the tyrosyl kinase activity of pp60c-src. Our present study demonstrates that some clones of hamster embryo cells transformed by simian virus 40, adenovirus type 2, adenovirus type 12, or bovine papillomavirus 1 can possess elevated pp60c-src kinase activity when compared with normal hamster embryo cells. However, other clones of hamster embryo cells transformed by these same viruses were found to have normal levels of pp60c-src kinase activity. In those clones of transformed cells where pp60c-src kinase activity was elevated, the increased levels of kinase activity were the result of an apparent increase in the specific activity of the pp60c-src phosphotransferase rather than an increase in the amount of the src gene product. Additionally, pp60c-src was not found to be physically associated with tumor antigens known to be encoded by these viruses. These results indicate that elevated levels of pp60c-src kinase activity can be found in hamster embryo cells transformed by several different DNA tumor viruses and suggest that the molecular mechanism by which pp60c-src kinase activity is elevated may differ from that previously observed in polyomavirus-transformed cells. These results also imply that elevation of pp60c-src kinase activity is not required for the transformation of hamster cells by these viruses.
Molecular cloning and characterization of mouse mammary tumor proviruses from a T-cell lymphoma
Authors: Dudley J P , Arfsten A , Hsu C L , Kozak C , Risser R
Published: January 2020

Five mouse mammary tumor virus proviruses and their flanking cellular DNA sequences have been cloned from a transplanted C57BL/6 (B6) T-cell lymphoma containing additional copies of mouse mammary tumor virus DNA. Characterization of these proviruses and their flanking DNA indicates that B6 lymphomas contain many newly integrated mouse mammary tumor virus copies synthesized by a mechanism(s) which generates polymorphism or deletions or both.
Ribosomal initiation at alternate AUGs on the Sendai virus P/C mRNA
Authors: Curran J A , Richardson C , Kolakofsky D
Published: January 2020

Peptide sera specific for the C, C/C', and P proteins of Sendai virus have been used to confirm that the viral nonstructural proteins originate from internal AUG codons and are translated in a different reading frame from that of the P protein. The C protein undergoes aberrant migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is expressed at higher levels in infected cells than are the P and C' proteins.
Characterization of a new type of human papillomavirus found in a lesion of Bowen's disease of the skin
Authors: Kawashima M , Jablonska S , Favre M , Obalek S , Croissant O , Orth G
Published: January 2020

The genome of a human papillomavirus (HPV) found in a patient with Bowen's disease of the skin was molecularly cloned. Blot hybridization experiments, performed under stringent conditions, revealed no cross-hybridization between this HPV DNA and the other known HPV DNAs, showing that this HPV represents a new type, tentatively named HPV34. In relaxed hybridization conditions, the highest cross-hybridization was observed with HPV16 DNA. The physical map of HPV34 DNA was aligned with the genetic map of HPV16 DNA by heteroduplex mapping. HPV34 was not detected in 12 additional patients with Bowen's disease of the skin, but a closely related HPV DNA was found in one patient with penile Bowenoid papulosis.
Nononcogenic deletion mutants of herpesvirus saimiri are defective for in vitro immortalization
Authors: Desrosiers R C , Silva D P , Waldron L M , Letvin N L
Published: January 2020

Herpesvirus saimiri L-DNA sequences between 0.0 and 4.0 map units (4.5 kilobase pairs) are required for oncogenicity; these sequences are not required for replication of the virus. To investigate the basis for the lack of oncogenicity of mutants with deletions in this region and to study the function of this region, we developed a reliable system for in vitro immortalization by herpesvirus saimiri. In contrast to peripheral blood lymphocytes from cotton-top tamarins (Saguinus oedipus) and owl monkeys (Aotus sp.), infection of peripheral blood lymphocytes from common marmosets (Callithrix jacchus) in vitro with herpesvirus saimiri consistently yielded continuously growing lymphoblastoid cell lines. Such cell lines were established using strains of herpesvirus saimiri from group A and group non-A, non-B; however, repeated attempts to immortalize common marmoset peripheral blood lymphocytes using strains from group B were not successful. Common marmoset cell lines immortalized by herpesvirus saimiri were T12+, T8+, T4-, and B1-, indicating that they were derived from suppressor/cytotoxic T lymphocytes. Cell lines could not be established using the nononcogenic mutants 11att and S4, both of which were derived from the group A strain 11 virus. Strain 11att has a spontaneous deletion and S4 has a constructed deletion in the 0.0 to 4.0 map unit region. Constructed strains which had these deleted sequences restored did immortalize common marmoset peripheral blood lymphocytes. Thus, the nononcogenic deletion mutants are defective for immortalization. This system should facilitate attempts to define the sequences responsible for immortalization and to determine their function.
Characterization of an Epstein-Barr virus-induced thymidine kinase
Authors: de Turenne-Tessier M , Ooka T , de The G , Daillie J
Published: January 2020

Previous work from our laboratory suggested that the selective inhibition of Epstein-Barr virus (EBV) replication by 1-beta-D-arabinofuranosylthymine in human lymphoid cell lines involved the induction of a new thymidine kinase (TK) able to phosphorylate the thymidine analog. We further characterized this enzyme induced in various EBV-positive cell lines after viral genome activation with a combination of sodium butyrate and 12-O-tetradecanoylphorbol-13-acetate. The following results confirmed the existence of an EBV-specific deoxypyrimidine kinase: induction of EBV-related TK was connected with the appearance of viral early antigens in EBV-carrying cells; unexpected behaviors of the enzyme activity upon different fractionating treatments led to the conclusion that EBV-induced TK was extracted as a complex molecular form, larger than other known cellular or viral isozymes; enzymatic properties distinguished EBV-induced TK from host lymphoid cell isozymes but made it resemble other herpesvirus-specific deoxypyrimidine kinases, i.e., by partial inhibition by dTTP or ammonium sulfate, insensitiveness to dCTP, and nonstringent specificity for normal TK substrates. Genetic evidence is required to definitively ensure that EBV-specific TK actually is virus coded in EBV-transformed human lymphoid cells.
New common nomenclature for glycoprotein genes of varicella-zoster virus and their glycosylated products
Authors: Davison A J , Edson C M , Ellis R W , Forghani B , Gilden D , Grose C , Keller P M , Vafai A , Wroblewska Z , Yamanishi K
Published: January 2020

The accumulation of recent data concerning the reactivity of monoclonal antibodies with particular varicella-zoster virus (VZV) glycoproteins and the mapping of several of their respective genes on the VZV genome has led to a unified nomenclature for the glycoprotein genes of VZV and their mature glycosylated products. Homologs to herpes simplex virus glycoprotein genes are noted.
High-frequency RNA recombination of murine coronaviruses
Authors: Makino S , Keck J G , Stohlman S A , Lai M M
Published: January 2020

The RNA genome of coronaviruses consists of a single species of nonsegmented RNA. In this communication, we demonstrate that the RNA genomes of different strains of murine coronaviruses recombine during mixed infection at a very high frequency. Susceptible cells were coinfected with a temperature-sensitive mutant of one strain of mouse hepatitis virus (MHV) and a wild-type virus of a different strain. Of 21 randomly isolated viruses released from the coinfected cells at the nonpermissive temperature, 2 were recombinants which differed in the site of recombination. After three serial passages of the original virus pool derived from the mixed infection, the majority of the progeny viruses were recombinants. These recombinant viruses represented at least five different recombination sites between the two parental MHV strains. Such a high-frequency recombination between nonsegmented RNA genomes of MHV suggests that segmented RNA intermediates might be generated during MHV replication. We propose that the RNA replication of MHV proceeds in a discontinuous and nonprocessive manner, thus generating free segmented RNA intermediates, which could be used in RNA recombination via a copy-choice mechanism.
Cell-free transcription and translation of isolated restriction fragments localize bacteriophage T5 pre-early genes
Authors: Blaisdell P W , Warner H R
Published: January 2020

In vitro transcription and translation of isolated restriction fragments containing all or part of the terminal redundancies of bacteriophage T5 localized virtually every pre-early gene to a small 6.3-kilobase BglI fragment. Among these genes were those encoding the A1 and A2 proteins, which are responsible for complete entry of the viral genome into its host, and the deoxynucleoside 5'-monophosphatase. A 3.9-kilobase BglI fragment containing the remainder of the pre-early region induced no proteins under these same conditions. Proteins induced by fragments including the right and left terminal redundancies were also compared and found to be identical. DNA immediately flanking the pre-early regions induced few proteins in vitro. Thus, this technique has allowed the overall gene organization of the pre-early region of T5 to be described.
The adenovirus type 12 early-region 1B 58,000-Mr gene product is required for viral DNA synthesis and for initiation of cell transformation
Authors: Shiroki K , Ohshima K , Fukui Y , Ariga H
Published: January 2020

An E1B 58K mutant of adenovirus type 12 (Ad12), dl207, was constructed by the deletion of 852 base pairs in the E1B 58K coding region. The mutant could grow efficiently in 293E1 cells but not in HeLa, KB, or human embryo kidney (HEK) cells. Viral DNA replication of dl207 was not detected in HeLa and KB cells and was seldom detected in HEK cells. Analysis of viral DNA synthesis in vitro showed that the Ad12-DNA-protein complex replicated by using the nuclear extract from Ad12 wild-type (WT)-infected HeLa cells but not by using the nuclear extract from dl207-infected cells. In dl207-infected HeLa and KB cells, early mRNAs were detected, but late mRNAs were not detected. The mutant induced fewer transformed foci than the WT in rat 3Y1 cells. Cells transformed by dl207 could grow efficiently in fluid medium, form colonies in soft agar culture, and induce tumors in rats transplanted with the transformed cells at the same efficiency as WT-transformed cells. Tumors were induced in hamsters injected with WT virions but were not induced in hamsters injected with dl207 virions. The results indicate that the E1B 58K protein is required both for viral DNA replication in productive infection and for initiation of cell transformation, but not for maintenance of the transformed phenotype.
Adenoviral early region 4 is required for efficient viral DNA replication and for late gene expression
Authors: Weinberg D H , Ketner G
Published: January 2020

H2dl808 is a defective deletion mutant of human adenovirus 2 lacking most of transcriptional early region 4. Although the mutant can be grown in the complementing cell line W162, it is defective in human cell lines normally used to propagate adenovirus. In such nonpermissive cells, H2dl808 exhibits a severe defect in late gene expression, accumulating very small amounts of viral late messages and producing correspondingly small amounts of viral late proteins. H2dl808 also exhibits a defect in viral DNA synthesis: 24 h after infection, H2dl808-infected nonpermissive cells contain five- to sevenfold less viral DNA than those cells infected with wild-type adenovirus. H2dl808-infected nonpermissive cells eventually accumulate a significant amount of viral DNA. However, the rate of synthesis of viral proteins late in mutant infection remains much lower than that observed in wild-type infection at a time when DNA accumulation is comparable. Thus, the mutant's late protein synthesis defect is probably not due solely to its reduced accumulation of viral DNA. Finally, H2dl808 is much less efficient than wild-type virus in the inhibition of host cell protein synthesis in infections of nonpermissive cells. These observations imply roles for early region 4 products in several aspects of the viral growth cycle, including DNA replication, late gene expression, and host cell shutoff.
Interaction of West Nile virus with primary murine macrophages: role of cell activation and receptors for antibody and complement
Authors: Cardosa M J , Gordon S , Hirsch S , Springer T A , Porterfield J S
Published: January 2020

We have measured growth of West Nile virus in mouse primary peritoneal macrophages (resident, thioglycolate elicited, and Mycobacterium bovis BCG activated) and in macrophagelike (P338D1) and nonmacrophage (L929, PS clone D) cell lines infected in the absence or presence of specific antibodies (immunoglobulin G ([IgG], IgM), and complement. Monoclonal antibodies directed against Fc receptors (IgG1/2b, 2.4G2) and type 3 complement receptors (Mac-1) were used to define the role of each receptor. Virus yield depended on a balance between enhancement and neutralization and was influenced by the physiologic state of the macrophage, the receptor pathway of viral entry, the mouse strain and age of donor. BCG-activated macrophages displayed a greater ability to restrict West Nile virus than nonactivated cells only in the presence of antiviral IgM, with or without complement; the Fc receptors for various classes of IgG mediated striking enhancement. These studies identify some of the complex innate and acquired factors that determine the interaction between West Nile virus and primary macrophages in vitro.
Infection by choleraphage phi 138: bacteriophage DNA and replicative intermediates
Authors: Chowdhury R , Das J
Published: January 2020

Choleraphage phi 138 contains a linear, double-stranded, circularly permuted DNA molecule of 30 X 10(6) daltons or 45 kilobase pairs. Upon infection, the host DNA is degraded, and synthesis of phage-specific DNA is detectable 20 min after infection. The phage utilizes primarily the host DNA degradation products for its own DNA synthesis. A physical map of phi 138 DNA was constructed with the restriction endonucleases Bg/II, HindIII, and PstI. A concatemeric replicative DNA intermediate equivalent to eight mature genome lengths was identified. The concatemer was shown to be the precursor for the synthesis of mature bacteriophage DNA which is subsequently packaged by a headful mechanism.
In vitro experimental infection of primary duck hepatocyte cultures with duck hepatitis B virus
Authors: Tuttleman J S , Pugh J C , Summers J W
Published: January 2020

Duck hepatitis B virus (DHBV) obtained from the serum of congenitally infected ducks was used to infect primary duck hepatocyte cultures 1 to 4 days after plating. Virus replication was demonstrated by the appearance, beginning at 2 days after infection, of intracellular covalently closed-circular and single-stranded DHBV DNA replicative intermediates which were not present in the inoculating virus preparation. With increasing time after infection there was further amplification of intracellular relaxed circular, covalently closed-circular, and single-stranded DHBV DNA. Cultures of primary duck hepatocytes are competent for infection with DHBV only during the first 4 days of culture. Synthesis of DHBV core antigen and DHBV surface antigen was detected by immunofluorescence in 10% of the hepatocytes in culture. De novo synthesis and release of infectious virus was also demonstrated. Therefore, all stages of viral replication were carried out by these experimentally infected primary hepatocyte cultures. This system makes it possible to study DHBV replication in vitro.
Synthesis and processing of the envelope gp55-116 complex of human cytomegalovirus
Authors: Britt W J , Auger D
Published: January 2020

The envelope of human cytomegalovirus has been reported to contain between three and eight glycoproteins. Major constituents of the envelope include two abundant glycoproteins with estimated molecular weights of 55,000 (gp55) and 116,000 (gp116). These two glycoproteins have been shown to exist as a disulfide-linked complex (gp55-116) within the envelope of mature virions. Utilizing a panel of monoclonal antibodies reactive with the gp55-116 complex, we characterized the synthesis and processing of these two virion proteins. Infected cells were shown to contain two glycosylated proteins of 160,000 and 150,000 daltons as well as the mature gp55 and gp116. Pulse-chase analysis indicated that gp150 was a precursor protein of gp160. The mature gp55 and gp116 were generated, in turn, by cleavage of gp160. Antigenic and structural analysis revealed that gp55 and gp116 shared little structural homology and no detectable antigenic cross-reactivity. The results of this study are discussed in relation to the synthesis of envelope proteins of other herpesviruses.
Lack of evidence for VPg priming of poliovirus RNA synthesis in the host factor-dependent in vitro replicase reaction
Authors: Andrews N C , Baltimore D
Published: January 2020

Anti-VPg immunoprecipitable RNA labeled in vitro during a poliovirus RNA polymerase reaction was formed by the elongation of VPg-containing template fragments rather than by initiation with VPg. The reaction was dependent on a host factor (terminal uridylyl transferase). The incorporation of labeled UTP could be detected with only the host factor present.
Orientation and patching of the latent infection membrane protein encoded by Epstein-Barr virus
Authors: Liebowitz D , Wang D , Kieff E
Published: January 2020

Epstein-Barr virus is known to encode three nuclear proteins and one membrane protein (LMP) in latently infected growth-transformed cells. Studies of the plasma membrane localization and orientation of LMP by protease digestion of live cells and by immunofluorescence indicated the following. (i) At least 30% of LMP is in the plasma membrane, as opposed to other cytoplasmic membranes. (ii) A small LMP domain which corresponds to a previously proposed outer reverse turn between the first two transmembrane domains is exposed on the outer cell surface (and two other proposed outer-reverse-turn domains may be exposed), whereas all or almost all of the rest of the protein is not exposed on the outer cell surface. (iii) LMP is present in patches in the cell plasma membrane.
12-O-tetradecanoylphorbol-13-acetate stimulates phosphorylation of the 58,000-Mr form of polyomavirus middle T antigen in vivo: implications for a possible role of protein kinase C in middle T function
Authors: Matthews J T , Benjamin T L
Published: January 2020

The 58,000-Mr form (58K form) of the polyomavirus middle T antigen (mT) is a minor species distinguished by its phosphorylation in vivo on serine and by its efficient phosphorylation on tyrosine in immune complexes (B.S. Schaffhausen and T.L. Benjamin, J. Virol. 40:184-196, 1981). Here we report that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, rapidly stimulates phosphorylation of this mT species when added to cultures of wild-type polyomavirus-infected or polyomavirus-transformed 3T3 cells. Incubation with TPA leads to an accumulation of the 58K mT species to levels 1.5- to 5-fold higher than that in untreated cells within 15 min. TPA specifically stimulates phosphorylation of the 58K mT species without affecting that of the 56K species. Mapping by partial proteolysis shows that TPA-stimulated phosphorylation occurs at or near the site in 58K mT that is normally phosphorylated in the absence of TPA. A synthetic diacyl glycerol, 1-oleoyl-2-acetyl-glycerol, also specifically stimulates phosphorylation of 58K mT in vivo, while an inactive phorbol analog does not. TPA fails to induce phosphorylation of a 58K mT species encoded by certain nontransforming virus mutants with altered mT proteins that normally fail to undergo phosphorylation at the 58K site. These results indicate that the 58K form of mT is phosphorylated by or through the action of protein kinase C. TPA treatment of infected cells also leads to increased levels of 58K mT as measured in the immune complex kinase reaction, in which mT becomes phosphorylated on tyrosine by pp60c-src. These results are discussed in terms of a possible role for protein kinase C in activating mT function(s), including the formation of stable complexes with pp60c-src.
Isolation of a retroviruslike sequence from the TL locus of the C57BL/10 murine major histocompatibility complex
Authors: Pampeno C L , Meruelo D
Published: January 2020

Two retroviruslike sequences have been isolated from the TL locus of the major histocompatibility complex of C57BL/10 mice. One sequence (TLev2) hybridizes only with probes derived from the pol region of the murine leukemia provirus AKR; the other sequence (TLev1) hybridizes with gag, pol, and env AKR region probes. This 9-kilobase endogenous, TL region-associated virus (TLev1) has been further characterized. The TLev1 genome has been shown to contain murine leukemia virus-related sequences bounded by retroviruslike, VL30 long terminal repeats. Hybridization of TLev1-derived probes to mouse genomic digests reveals multiple copies which show distinct patterns compared with those observed with murine leukemia virus probes. The study of TLev1 may prove significant with respect to the interaction of retroviral sequences within the genome, expression of genes within the TL locus, and polymorphisms within the major histocompatibility complex.
Conserved regions in defective interfering viral double-stranded RNAs from a yeast virus
Authors: Lee M , Pietras D F , Nemeroff M E , Corstanje B J , Field L J , Bruenn J A
Published: January 2020

We have completely sequenced a defective interfering viral double-stranded RNA (dsRNA) from the Saccharomyces cerevisiae virus. This RNA (S14) is a simple internal deletion of its parental dsRNA, M1, of 1.9 kilobases. The 5' 964 bases of the M1 plus strand encode the type 1 killer toxin of the yeast. S14 is 793 base pairs (bp) long, with 253 bp from the 5' region of its parental plus strand and 540 bp from the 3' region. All three defective interfering RNAs derived from M1 that have been characterized so far preserve a large 3' region, which includes five repeats of a rotationally symmetrical 11-bp consensus sequence. This 11-bp sequence is not present in the 5' 1 kilobase of the parental RNA or in any of the sequenced regions of unrelated yeast viral dsRNAs, but it is present in the 3' region of the plus strand of another yeast viral dsRNA, M2, that encodes the type 2 killer toxin. The 3' region of 550 bases of the M1 plus strand, previously only partially sequenced, reveals no large open reading frames. Hence only about half of M1 appears to have a coding function.
Polyomavirus late leader region serves an essential spacer function necessary for viability and late gene expression
Authors: Adami G R , Carmichael G G
Published: January 2020

All three polyomavirus late mRNAs contain multiple tandem copies of the same nontranslated 57-nucleotide sequence, the late leader, at their 5' ends. We show here that a polyoma variant (ALM) lacking 48 central bases of the 57-base leader unit is nonviable by plaque assay and by a new method for testing virus viability, an immunofluorescence burst assay. ALM is, however, unaffected in early gene expression as measured both by indirect immunofluorescence of large T antigen and by transformation levels of rat F-111 cells. DNA replication in mouse cells is also as wild type, and the defect in ALM is complemented by an early-defective helper virus DNA. ALM does not make detectable levels of late viral proteins and is minimally 200-fold depressed in the accumulation of cytoplasmic polyadenylated late RNA. When the deleted leader sequence of ALM is replaced by a variety of procaryotic sequences, viability almost always returns. Some of the substituted leader variants produce plaques with the same apparent kinetics as wild-type viral DNA. The indication is that the sequence of the polyoma late leader is not important for late gene expression but that it has an essential spacer function on the RNA or DNA level. This spacer function is apparently necessary for late viral RNA transcription, processing, or stability.
Restricted replication of mouse hepatitis virus A59 in primary mouse brain astrocytes correlates with reduced pathogenicity
Authors: van Berlo M F , Wolswijk G , Calafat J , Koolen M J , Horzinek M C , van der Zeijst B A
Published: January 2020

Temperature-sensitive (ts) mutants of mouse hepatitis virus A59 (MHV-A59) are drastically attenuated in their pathogenic properties. Intracerebral inoculation of mice with 10(5) PFU of mutant ts342 results in prolonged infection of the central nervous system, whereas 100 PFU of wild-type virus are lethal (M. J. M. Koolen, A. D. M. E. Osterhaus, G. van Steenis, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 125:393-402, 1983). In the Sac(-) cell line ts342 grows as well at 37 degrees C (the body temperature of mice) as at 31 degrees C (the permissive temperature). There is, however, a difference in primary cultures of mouse brain astrocytes. After infection with ts342, astrocytes produced low levels of infectious virus (5.2 +/- 3.7%) compared with virus yields after infection with wild-type virus. The fraction of wild-type virus- and ts342-infected cells was similar. Electron microscopy showed in wild-type virus-infected cells abundant virions in smooth vesicles usually closely associated with a well-developed Golgi apparatus. In mutant-infected cells no mature ts342 virus particles were found. There was no difference between ts342 and wild-type virus regarding the intracellular virus-specific RNAs. In ts342-infected cells the viral glycoproteins E2 and E1 were not detectable or were barely detectable. Either the mRNAs for the glycoproteins are not translated or the proteins are rapidly broken down. Revertants of ts342 were isolated. They grew as well as wild-type virus in astrocytes, indicating that they apparently produced sufficient amounts of E2 and E1, the ts defect itself rather than a second site mutation is responsible for the defect in replication, and the ts defect acts in unison with host-cell factors. The revertants also regained the lethal properties of wild-type virus.
DNA requirements at the bacteriophage G4 origin of complementary-strand DNA synthesis
Authors: Lambert P F , Waring D A , Wells R D , Reznikoff W S
Published: January 2020

An in vivo assay was used to define the DNA requirements at the bacteriophage G4 origin of complementary-strand DNA synthesis (G4 origin). This assay made use of an origin-cloning vector, mRZ1000, a defective M13 recombinant phage deleted for its natural origin of complementary-strand DNA synthesis. The minimal DNA sequence of the G4 genome sufficient for the restoration of normal M13 growth parameters was determined to be 139 bases long, located between positions 3868 and 4007. This G4-M13 construct was also found to give rise to proper initiation of complementary-strand synthesis in vitro. The cloned DNA sequence contains all the regions of potential secondary structure which have been implicated in primase-dependent replication initiation as well as additional sequence information. To address the role of one region which potentially forms a DNA secondary structure, the DNA sequence internal to the G4 origin was altered by site-directed mutagenesis. A 3-base insertion at the AvaII site as well as a 17-base deletion between the AvaI and AvaII sites both resulted in loss of origin function. The 17-base deletion was also generated within the G4 genome and found to dramatically reduce the infectious growth rate of the resulting phage. These results are discussed with respect to the role of the G4 origin as the recognition site for primase-dependent replication initiation and its possible role in stage II replication.
Persistent infection of a temperature-sensitive G31 vesicular stomatitis virus mutant in neural and nonneural cells: biological and virological characteristics
Authors: Huprikar J , Rabinowitz S G , DalCanto M C , Rundell M K
Published: January 2020

Mouse L-929 cells (L cells), human oligodendroglioma cells, and rat glioma cells were persistently infected with vesicular stomatitis virus (VSV) mutant tsG31 and maintained for at least 4 years at 37 degrees C. The striking observation in this study was that there is a marked difference in neurovirulence among the persistent infections (PIs) derived from the three cell lines. tsG31 VSV derived from persistently infected L cells and oligodendroglioma cells remained highly virulent as assayed by intracerebral (i.c.) inoculation into 3-week-old Swiss mice. In contrast, tsG31 VSV isolated from glioma cells lost neurovirulence by passage 20. Persistently infected glioma cells were carried through more than 180 passages without reemergence of neurovirulent virus. Importantly, glioma PI virus neurovirulence was restored quickly by i.c. passage in mice and more slowly by passage through normal L cells. In contrast, the neurovirulence of L-cell PI virus was enhanced by i.c. passage in mice and slowly reduced by passage through normal glioma cells. Furthermore, no alteration in neurovirulence was observed in the case of oligodendroglioma PI virus. Although the mechanism(s) underlying the loss of virulence in glioma cells is unclear, our studies suggest that either strict temperature sensitivity or the presence of a heat-labile transcriptase or both play a major role in this phenomenon.
Structural domains of the avian erythroblastosis virus erbB protein required for fibroblast transformation: dissection by in-frame insertional mutagenesis
Authors: Ng M , Privalsky M L
Published: January 2020

Avian erythroblastosis virus (AEV) induces erythroblastosis and fibrosarcomas. The viral erbB protein is required for AEV-mediated oncogenesis. To explore the structural aspects of the v-erbB polypeptide necessary for its oncogenic function, we created a series of small in-frame insertions in different domains of the v-erbB oncogene. AEV genomes bearing lesions within the v-erbB kinase domain demonstrated a drastically decreased ability to transform avian fibroblasts, establishing a functional role for this structurally conserved oncogene domain. In contrast, mutations in the extracellular domain, between the transmembrane region and the kinase domain, or at the extreme C terminus of the v-erbB protein had no effect on AEV-mediated fibroblast transformation. One lesion within the v-erbB kinase domain, a 10-amino acid insertion, produced a temperature-sensitive mutant capable of fibroblast transformation at 36 degrees C but not at 41 degrees C, suggesting that small in-frame insertions have general utility for the in vitro creation of conditional mutants.
VA RNAs from avian and human adenoviruses: dramatic differences in length, sequence, and gene location
Authors: Larsson S , Bellett A , Akusjärvi G
Published: January 2020

Human adenoviruses encode low-molecular-weight RNAs, so-called VA RNAs, which are transcribed by RNA polymerase III. These RNAs are required for an efficient translation of viral mRNAs late after infection. The genes for the VA RNAs in the genome of CELO virus were mapped and characterized. The results showed a number of surprising differences between CELO virus and human adenovirus type 2 (Ad2). Thus, the CELO virus genome encoded only one VA RNA species, in contrast to human Ad2, which encoded two distinct species. The VA RNA from CELO virus was much shorter than the Ad2 VA RNAs (90 nucleotides compared with 160 nucleotides), and there existed no detectable primary sequence homology between them. The predicted secondary structure of CELO virus VA RNA was, however, similar to that of the Ad2 VA RNAs, implying that the folding rather than the primary sequence was the important feature for biological activity. CELO VA RNA also stimulated translation in a transient expression assay, as did the Ad2 counterparts, albeit with a much lower efficiency. The location of the gene for CELO VA RNA also differed from all previously characterized serotypes, suggesting that the genome organization of avian and human adenoviruses are different. Finally, termination of CELO VA RNA transcription occurred in a TTATT sequence which is unique as a stop signal for RNA polymerase III transcription.