bluuebunny/crossref_metadata_2025 huggingface dataset:
Some enzymatic activities associated with purified parapoxvirions
Authors: Caplen H S , Holowczak J A
Published: January 2020

Purified virions of milker's nodule virus, a parapoxvirus, were shown to contain an RNA polymerase, a nucleotide phosphohydrolase, and a protein kinase associated with or encapsulated within the DNA-containing core of the virus. In vitro, the activated viral RNA polymerase transcribed only 7 to 8% of the genome, in the form of 8S to 14S polyadenylated RNA molecules which were complementary to sequences present in milker's nodule virus DNA but not vaccinia virus DNA or DNA prepared from the host cells in which the virus was propagated. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that in vitro, the activated viral protein kinase phosphorylated viral polypeptides of 95, 60, 33.5, 15, and 13.8 kilodaltons.
Nucleotide sequence of the vaccinia virus thymidine kinase gene and the nature of spontaneous frameshift mutations
Authors: Weir J P , Moss B
Published: January 2020

Nucleotide sequencing of a 1,300-base-pair vaccinia virus DNA segment previously shown to contain a thymidine kinase (TK) gene revealed an uninterrupted reading frame of 177 codons capable of producing a polypeptide with a molecular weight of 20,102. Mapping of the TK mRNA by primer extension indicated a unique 5' end that precedes the initiation codon by only six nucleotides. Multiple 3' ends within a 10-nucleotide region, about 30 nucleotides beyond the termination codon, were located by nuclease digestion of DNA-RNA hybrids, and the length of the TK transcript, exclusive of polyadenylate, was estimated to be approximately 570 nucleotides. The region preceding the TK mRNA start site is extremely A + T rich and has sequence homologies with three other early genes. Genetic information is so compressed in this region of the DNA that the putative transcriptional regulatory sequence of the TK gene overlaps the coding sequence of a late gene. Only nine nucleotides separate the termination codon of the late gene from the initiation codon of the TK gene. Downstream, 66 nucleotides separate the TK termination codon from the apparent initiation codon of another early gene. The nature of three independent TK- mutants was revealed by nucleotide sequencing. Each has a nucleotide reiteration leading to a +1 frameshift and a nonsense codon downstream. The location of one frameshift mutation provided evidence that the first ATG is used for initiation of protein synthesis.
Construction and isolation of a transforming murine retrovirus containing the src gene of Rous sarcoma virus
Authors: Anderson S M , Scolnick E M
Published: January 2020

Recombinant murine retroviruses containing the src gene of the avian retrovirus Rous sarcoma virus were isolated. Such viruses were isolated from cells after transfection with DNAs in which the src gene was inserted into the genome of the amphotropic murine retrovirus 4070A. The isolated viruses had functional gag and pol genes, but they were all env defective since the src gene was inserted in the middle of the env gene coding region. Infectious transforming virus could be isolated only from cells transfected with DNA constructions in which the src gene was in the same polarity as that of a long terminal repeat of the amphotropic viral genome. These recombinant viruses encoded a pp60src protein with a molecular weight similar to that of the Schmidt-Ruppin strain of Rous sarcoma virus. In addition, the src protein(s) of these recombinant viruses was as active as protein kinases in the immune complex protein kinase assay. Intravenous injection of helper-independent Moloney and Friend murine leukemia virus pseudotypes of the src recombinant viruses into 6-week-old NIH Swiss mice resulted in the appearance of splenic foci within 2 weeks, splenomegaly and, later after infection (8 to 10 weeks), anemia. Infectious transforming virus could be recovered from the spleens of diseased animals. Such viruses encoded pp60src but not p21ras or mink cell focus-forming virus-related glycoproteins.
Orientation of the Cleavage Map of the 200-Kilodalton Polypeptide Encoded by the Bottom-Component RNA of Cowpea Mosaic Virus
Authors: Goldbach Rob , Rezelman Geertje
Published: January 2020

The genomic organization of the bottom-component RNA of cowpea mosaic virus was studied. In vivo, this RNA encodes at least eight different polypeptides of 170, 110, 87, 84, 60, 58, 32, and 4 kilodaltons (K), the last polypeptide representing the genome-bound protein VPg. In rabbit reticulocyte lysates, bottom-component RNA is translated into a 200K polypeptide which is then processed to give the 32 and 170K polypeptides also found in vivo. By pulse-labeling the 200K primary translation product, we now show that the 32 and 170K polypeptides are derived from the NH
2
-terminal and COOH-terminal parts of this polypeptide, respectively. Comparison of the proteolytic peptide patterns of 170K polypeptides synthesized in vitro and pulse-labeled at either the NH
2
-terminal or the COOH-terminal end with the patterns of the 170 and 110K polypeptides found in vivo demonstrates that the order within the 200K primary translation product of cowpea mosaic virus bottom-component RNA is as follows: NH
2
-32K polypeptide-58K polypeptide-VPg-24K polypeptide-87K polypeptide-COOH.
Temperature-sensitive initiation and elongation of adenovirus DNA replication in vitro with nuclear extracts from H5ts36-, H5ts149-, and H5ts125-infected HeLa cells
Authors: van Bergen B G , van der Vliet P C
Published: January 2020

Adenovirus DNA replication was studied in vitro in nuclear extracts prepared from HeLa cells infected at the permissive temperature with H5ts125, H5ts36, or H5ts149, three DNA-negative mutants belonging to two different complementation groups. At the restrictive temperature, H5ts125 extracts, containing a thermolabile 72-kilodalton DNA-binding protein, enable the formation of an initiation complex between the 82-kilodalton terminal protein precursor (pTP) and dCTP, but further elongation of this complex is inhibited. Wild-type DNA-binding protein or a 47-kilodalton chymotryptic DNA-binding fragment can complement the mutant protein in the elongation reaction. No difference in heat inactivation was observed between wild-type extracts and H5ts36 or H5ts149 extracts when the replication of terminal XbaI fragments of adenovirus type 5 DNA-terminal protein complex was studied. In contrast, the formation of a pTP-dCMP initiation complex, as well as the partial elongation reaction up to nucleotide 26, were consistently more temperature sensitive in mutant extracts. The results suggest that the H5ts36/H5ts149 gene product is required for initiation of adenovirus type 5 DNA replication and that the 72-kilodalton DNA-binding protein functions early in elongation.
Presence of leader sequences in the mRNA of mouse hepatitis virus
Authors: Lai M M , Patton C D , Baric R S , Stohlman S A
Published: January 2020

To determine the structure and the mechanism of synthesis of mouse hepatitis virus mRNA, the map positions of the large RNase T1-resistant oligonucleotides of the seven mouse hepatitis virus strain A59 intracellular mRNA species were studied. We found that all but one of the oligonucleotides were mapped at the positions within each mRNA consistent with the nested-set, stairlike structure of mouse hepatitis virus mRNA (Lai et al., J. Virol. 39:823-834). However, one oligonucleotide, 10, was mapped near the 5' ends of every mRNA and virion genomic RNA. In other words, oligonucleotide 10 and, therefore, the sequences around the 5' ends of the mRNAs are not colinear with the genomic sequences. Because this oligonucleotide is present only once in the genomic RNA, this result indicates that oligonucleotide 10 is not transcribed from multiple sites on the genomic template, but rather represents a leader RNA sequence which is joined to the body sequences of the different mRNAs during mRNA transcription. This provides the most direct evidence thus far for the presence of leader sequences in the mRNAs of mouse hepatitis virus, which is a cytoplasmic virus. Several possible mechanisms of RNA synthesis are discussed.
Nucleotide sequence of the herpes simplex virus type 2 thymidine kinase gene
Authors: Swain M A , Galloway D A
Published: January 2020

We have determined the complete nucleotide sequence of the thymidine kinase gene of herpes simplex virus (HSV) type 2 strain 333. The sequence of the thymidine kinase gene exhibits an open translational reading frame of 1,128 nucleotides encoding a protein of 376 amino acids. The DNA sequence was compared with that of the HSV type 1 thymidine kinase gene from strain MP (S. L. McKnight, Nucleic Acids Res. 8:5949-5964, 1980) and from strain CL 101 (M. J. Wagner, J. A. Sharp, and W. C. Summers, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, 1981) to assess the extent of intra- and intertypic variation for one viral gene. The nucleotides encoding the structural gene varied 1.7% between the two HSV type 1 strains and 19% between HSV type 1 and HSV type 2, which translated to differences in the amino acid sequence of the two proteins of 1.9 and 27%, respectively. The DNA encoding the 5' regulatory sequences appeared to be more conserved than the DNA coding for the structural gene, and the DNA at the 3' end of the gene was the least homologous.
Long terminal repeat enhancement of v-mos transforming activity: identification of essential regions
Authors: Wood T G , McGeady M L , Blair D G , Vande Woude G F
Published: January 2020

The transforming efficiency of recombinant DNA clones containing the Moloney sarcoma virus v-mos sequence was enhanced by introducing the Moloney sarcoma virus long terminal repeat (LTR) in either the 5' or 3' position relative to v-mos. We analyzed the polyadenylated RNA expressed in cells transformed by these recombinant DNA clones and examined the structural integrity of integrated copies of the DNA. In each case, we demonstrated the presence of v-mos containing RNA transcripts in the polyadenylated RNA and showed that these RNA transcripts are consistent with the structure of the transfected DNA. The analysis of DNA from these transformed cells showed that the relative positions of the v-mos and LTR sequences within the transfected DNA were conserved in the integrated DNA copies. These results demonstrate that a single LTR can successfully enhance the transforming activity of v-mos from either a 5' or a 3' relative position. The results from the transfection analysis of recombinant clones containing only portions of the LTR introduced 3' to v-mos demonstrate that the essential region of the LTR responsible for the enhancement of transformation is a region within the unique 3' sequences of the LTR containing the 73-base-pair tandem repeat sequence.
Intracellular forms of simian virus 40 nucleoprotein complexes. V. Enrichment for "active" simian virus 40 chromatin by differential precipitation with Mg2+
Authors: Chen S S , Hsu M T
Published: January 2020

A fraction (approximately 20%) of 70S simian virus (SV40) chromatin isolated from a sucrose gradient-purified extract of SV40-infected CV-1 cells was found to be precipitated by incubation with a buffer containing 10 mM Mg2+ at 37 degrees C. The fraction containing the Mg2+-insoluble SV40 chromatin was found also to be enriched for the following: (i) DNA replication intermediates as analyzed by short pulse with [3H]thymidine; (ii) transcription complexes as assayed by in vitro synthesis of SV40-specific RNA; (iii) newly synthesized T antigen; (iv) phosphorylated T antigen as analyzed by both in vitro and in vivo phosphorylation studies; and (v) ATP-binding activity of T antigen as assayed by affinity labeling with oxidized [alpha-32P]ATP.
Analysis of extracellular West Nile virus particles produced by cell cultures from genetically resistant and susceptible mice indicates enhanced amplification of defective interfering particles by resistant cultures
Authors: Brinton M A
Published: January 2020

[3H]uridine-labeled extracellular West Nile virus (WNV) particles produced by cell cultures obtained from genetically resistant C3H/RV and congenic susceptible C3H/HE mice were compared by sucrose density gradient centrifugation as well as by analysis of the particle RNA. Defective interfering (DI) WNV particles were observed among progeny produced during acute infections in both C3H/RV and C3H/HE cells. Although only a partial separation of standard and DI particles was achieved, the DI particles were found to be more dense than the standard virions. Particles containing several species of small RNAs consistently constituted a major proportion of the total population of virus progeny produced by C3H/RV cells, but a minor proportion of the population produced by C3H/HE cells. Decreasing the multiplicity of infection or extensive plaque purification of the WNV inoculum decreased the proportion of small RNAs found in the progeny virus. The ratio of DI particles to standard virus observed in progeny virus was determined by the cell type used to grow the virus. The ratio could be shifted by passaging virus from one cell type to the other. Homologous interference could be demonstrated with WNV produced by C3H/RV cells but not with virus produced by C3H/HE cells. Continued passage of WNV in C3H/HE cells resulted in a cycling of infectivity. However, passage in C3H/RV cells resulted in the complete loss of infectious virus. Four size classes of small viral RNA, with sedimentation coefficients of about 8, 15, 26, and 34S, were observed in the extracellular particles. A preliminary analysis of these RNAs by oligonucleotide fingerprinting indicated that the smaller RNAs were less complex than the 40S RNA and differed from each other. The data are consistent with the conclusion that WNV DI particles interfere more effectively with standard virus replication and are amplified more efficiently in C3H/RV cells than in congenic C3H/HE cells. The relevance of these findings to the further understanding of genetically controlled resistance to flaviviruses is discussed.
Nucleotide Sequence of the Envelope Gene of Gardner-Arnstein Feline Leukemia Virus B Reveals Unique Sequence Homologies with a Murine Mink Cell Focus-Forming Virus
Authors: Elder John H. , Mullins James I.
Published: January 2020

The nucleotide sequence of the envelope gene and the adjacent 3′ long terminal repeat (LTR) of Gardner-Arnstein feline leukemia virus of subgroup B (GA-FeLV-B) has been determined. Comparison of the derived amino acid sequence of the gp70-p15E polyprotein to those of several previously reported murine retroviruses revealed striking homologies between GA-FeLV-B gp70 and the gp70 of a Moloney virus-derived mink cell focus-forming virus. These homologies were located within the substituted (presumably xenotropic) portion of the mink cell focus-forming virus envelope gene and comprised amino acid sequences not present in three ecotropic virus gp70s. In addition, areas of insertions and deletions, in general, were the same between GA-FeLV-B and Moloney mink cell focus-forming virus, although the sizes of the insertions and deletions differed. Homologies between GA-FeLV-B and mink cell focus-forming virus gp70s is functionally significant in that they both possess expanded host ranges, a property dictated by gp70. The amino acid sequence of FeLV-B contains 12 Asn-X-Ser/Thr sequences, indicating 12 possible sites of N-linked glycosylation as compared with 7 or 8 for its murine counterparts. Comparison of the 3′ LTR of GA-FeLV-B to AKR and Moloney virus LTRs revealed extensive conservation in several regions including the “CCAAT” and Goldberg-Hogness (TATA) boxes thought to be involved in promotion of transcription and in the repeat region of the LTR. The inverted repeats that flanked the LTR of GA-FeLV-B were identical to the murine inverted repeats, but were one base longer than the latter. The region of U3 corresponding to the approximately 75-nucleotide “enhancer sequence” is present in GA-FeLV-B, but contains deletions relative to AKR and Moloney virus and is not repeated. An interesting pallindrome in the repeat region immediately 3′ to the U3 region was noted in all the LTRs, but was particularly pronounced in GA-FeLV-B. Possible roles for this structure are discussed.
Physiological properties of a T7-T3 recombinant bacteriophage that productively infects strains of Escherichia coli that harbor the F plasmid
Authors: Spence J L , Mooney P Q , Molineux I J
Published: January 2020

The T7-T3 recombinant phage BO2, whose isolation and physical properties are described in the accompanying paper (Molineux et al., J. Virol. 46:881-894, 1983), has been characterized by physiological means after infection of male (F plasmid-containing) and female strains of Escherichia coli. Single-step growth analyses have shown that the hybrid phage gives a burst about two-thirds that of T7 or T3 on females and about one-half that of T3 on males. This reduced burst size can be correlated with altered kinetics of macromolecular synthesis, probably at the level of transcription. The T3 insertions of BO2, designated M1 and M2, that are essential for growth of the hybrid phage on male strains, have been shown to be trans acting and can rescue T7 from F-mediated restriction. The nature of the gene products encoded by the M1 and M2 regions and their role in overcoming the abortive infection of males by T7 are discussed.
Complete sequence of the Rous sarcoma virus env gene: identification of structural and functional regions of its product
Authors: Hunter E , Hill E , Hardwick M , Bhown A , Schwartz D E , Tizard R
Published: January 2020

The amino-terminal amino acid sequences of gp85 and gp37, the envelope glycoproteins of Rous sarcoma virus (RSV), were determined. Alignment of these sequences with the amino acid sequence predicted from the complete nucleotide sequence of the Prague strain of RSV, subgroup C (PR-C), has allowed us to delineate the env gene-coding region of this virus. The coding sequences for gp85 and gp37 have been placed in an open reading frame that extends from nucleotide 5045 to nucleotide 6862 and predict sizes of 341 amino acids (36,962 molecular weight) for gp85 and 198 amino acids (21,566 molecular weight) for gp37. Carbohydrate makes a significant contribution to the observed molecular weights of these polypeptides--the amino acid sequence contains 14 potential glycosylation sites (Asn-X-Ser/Thr) in gp85 and two in gp37. Experiments aimed at estimating the number of carbohydrate side chains yielded results consistent with most or all of these sites being occupied. Although an initiation codon is located early (codon 4) in the open reading frame, it is likely that splicing yields an mRNA on which translation initiates at the same AUG as that of the gag gene to produce a nascent polypeptide in which gp85 is preceded by a 62-amino-acid-long leader peptide. This leader contains the hydrophobic sequence (signal sequence) necessary for translocation across the endoplasmic reticulum and is completely removed from the env gene product during translation. The polyprotein precursor, Pr95env, is cleaved to gp85 and gp37 at the carboxyl side of the basic sequence:-Arg-Arg-Lys-Arg-. gp85 is attached through a disulphide linkage to gp37, and although the positions of the cysteines involved in this linkage are not known, the presence of a 27-amino-acid-long hydrophobic region at the carboxy-terminus of gp37 is consistent with its role as a membrane anchor for the viral glycoprotein complex. The location of host range variable regions with respect to the possible tertiary structure of the complex is discussed.
Interaction of minute virus of mice with differentiated cells: strain-dependent target cell specificity is mediated by intracellular factors
Authors: Spalholz B A , Tattersall P
Published: January 2020

The prototype strain of minute virus of mice and the immunosuppressive strain are unable to grow lytically in each other's murine host cell type. To characterize these strain-dependent virus-host cell interactions further, we have compared the early events of both productive and restrictive infections. Each virus binds to specific receptors on the surface of both productive and restrictive cell types. Competition experiments show that both viruses recognize the same receptor on each cell type. Penetration and uncoating are presumed to be similar in both productive and restrictive infections, since incoming viral genomes are converted to parental replicative form DNA independent of the final outcome of the virus-host cell interaction. In contrast to the majority of other systems studied to date, these differences in minute virus of mice target cell specificity are not mediated at the cell surface, but by the interaction of a strain-specific viral determinant with intracellular host factors that are expressed in particular cell types as a function of differentiation. These cellular factors catalyze a step in viral replication which occurs after the initiation of viral DNA synthesis, but before the detectable expression of the viral capsid polypeptide genes.
In vitro transformation of murine pre-B lymphoid cells by Snyder-Theilen feline sarcoma virus
Authors: Pierce J H , Aaronson S A
Published: January 2020

Snyder-Theilen feline sarcoma virus (ST-FeSV) codes for a protein kinase with specificity for tyrosine residues (Barbacid et al., Proc. Natl. Acad. Sci. U.S.A. 77:5158-5163, 1980), properties analogous to those of the transforming gene product of Abelson murine leukemia virus (Witte et al., Nature (London) 283:826-831, 1980). In the present report, ST-FeSV was demonstrated to transform murine hematopoietic cells under in vitro assay conditions which detect lymphoid cell transformation by Abelson murine leukemia virus. Bone marrow colony formation was shown to require ST-FeSV, follow single-hit kinetics, and require the presence of mercaptoethanol in the agar medium. ST-FeSV-induced colonies could be established in culture as continuous cell lines that demonstrated unrestricted self-renewal capacity and leukemogenicity in vivo. The hematopoietic blast cells transformed by ST-FeSV in culture appeared to be at an early stage of B cell differentiation. They possessed Lyb 2 surface antigens, were dependent on mercaptoethanol for growth, and contained only low levels of terminal deoxynucleotidyl transferase. Moreover, a large fraction of the lines synthesized immunoglobulin mu chain in the absence of light chains. Thus, the phenotype of ST-FeSV hematopoietic transformants was indistinguishable from that of the pre-B lymphoblast transformants induced by Abelson murine leukemia virus. These findings indicate that the in vitro functional similarities in the onc gene products of ST-FeSV and Abelson murine leukemia virus may reflect a common pathway by which they exert their oncogenic potential.
Genetic mapping of the mouse oncogenes c-Ha-ras-1 and c-fes to chromosome 7
Authors: Kozak C A , Sears J F , Hoggan M D
Published: January 2020

The mouse homologs of the cellular oncogenes c-Ha-ras-1 of Harvey sarcoma virus and c-fes of feline sarcoma virus were both mapped to chromosome 7 by Southern blot analysis of hamster-mouse somatic cell hybrid DNAs.
Transcription from the BamHI J fragment of herpes simplex virus type 1 (KOS)
Authors: Betz J L , Hill T M , Pizer L I , Peake M L , Sadler J R
Published: January 2020

RNA transfer experiments (Northern analyses) were used to localize polyadenylated mRNA species made after herpes simplex virus type 1 infection to EcoRI and BamHI fragments and subfragments from the short unique region of the herpes simplex virus type 1 (KOS) genome. Three predominant early mRNAs of 2.5, 1.3, and 0.9 kilobases map in the BamHI J fragment. A detailed restriction map of the BamHI J fragment was constructed.
Specific proteolytic fragmentation of p60v-src in transformed cell lysates
Authors: Wells S K , Collett M S
Published: January 2020

Work involving the transforming protein, p60v-src, of Rous sarcoma virus has resulted in the extensive characterization of its protein structure and associated phosphotransferase activity. However, in many investigations proteolytic fragments (principally p52v-src) of the src protein are actually studied. Here, we emphasize potential problems in the interpretation of experimental results in which the proteolytic fragmentation of p60v-src may be involved and offer several means for the complete prevention of this p60v-src degradation.
Identification of four complementary RNA species in Akabane virus-infected cells
Authors: Pattnaik A K , Abraham G
Published: January 2020

The analysis of RNA extracted from purified Akabane virus demonstrated the presence of three size classes of single-stranded RNAs with sedimentation coefficients of 31S (large, L), 26S (medium, M), and 13S (small, S). Molecular weights of these RNA species were estimated to be 2.15 X 10(6), 1.5 X 10(6), and 0.48 X 10(6) for the L, M, and S RNAs, respectively. Hybridization analysis involving viral genomic RNA and RNA from virus-infected cells resulted in the identification of four virus-specific cRNA species in infected cells. These cRNAs were found to be nonpolyadenylated by their inability to bind to oligodeoxythymidylate-cellulose. Kinetic analysis of cRNA synthesis in infected cells at various times postinfection suggested that cRNA synthesis could be detected as early as 2 h postinfection and that maximal synthesis occurred at 4 to 6 h postinfection. The RNAs synthesized in infected cells could be partially resolved by sucrose density gradient centrifugation. The RNA fraction that cosedimented with the S segment of viral genomic RNA yielded two duplex RNA species when hybridized with viral genomic RNA, suggesting the presence of two small cRNA species. Specific hybridization with individual viral genomic RNAs confirmed that two species of cRNA are coded by the S RNA segment. Analysis of cRNA synthesis in the presence of the protein synthesis inhibitors cycloheximide and puromycin indicated that cycloheximide completely inhibited virus-specific RNA synthesis early and late in infection, whereas a very low level of synthesis occurred in the presence of puromycin. The inhibitory effects of these drugs were found to be reversible when the drugs were washed from the cells. It is concluded that continued protein synthesis is required for cRNA synthesis to proceed in Akabane virus-infected cells.
Different mRNAs induced by interferon in cells from inbred mouse strains A/J and A2G
Authors: Staeheli P , Colonno R J , Cheng Y S
Published: January 2020

Treatment of cells from inbred mouse strains A/J and A2G with interferon resulted in the development of different antiviral states for influenza viruses. A2G mice-derived cells that carry the resistance gene Mx were efficiently protected by interferon against influenza viruses, whereas the interferon protection against the same viruses in wild-type A/J mice-derived cells was only marginal. The two cell types, however, were equally protected by interferon against vesicular stomatitis virus and other non-orthomyxoviruses. The interferon-induced mRNAs of mouse embryonic fibroblast cells that carried either homozygous wild-type alleles or homozygous Mx alleles were compared. The isolated polysome-bound mRNAs from A/J (+/+) and A2G (Mx/Mx) cells were translated in a cell-free translation system, and the translation products were analyzed after two-dimensional gel electrophoresis. New mRNAs coding for at least eight proteins with molecular weights (MW) ranging from 30,000 to 80,000 were found in interferon-treated cells but not in control cells. Differences in the interferon-induced mRNAs from A/J and A2G cells were also found. An mRNA coding for a 72,000-MW protein was found in interferon-treated A2G cells but not in interferon-treated A/J cells. Interferon-treated A/J cells, on the other hand, contained an mRNA coding for a 65,000-MW protein that was not found in interferon-treated A2G cells. The in vitro-synthesized 65,000-MW protein efficiently bound to GMP. Cytoplasmic extracts prepared from interferon-treated A/J cells also contained a GMP-binding 65,000-MW protein that was undetectable in similarly treated A2G cells.
Generation of capsids from unstable polyoma virions
Authors: Yuen L K , Consigli R A
Published: January 2020

Polyomavirus was purified from infected mouse cell lysates under mild physiological conditions. When analyzed in a sucrose gradient, a major virus peak (240S) was identified. This sucrose-isolated virus could be divided into two populations based on its stability to CsCl gradient centrifugation. Members of the unstable population were shown to eject their DNA cores when subjected to CsCl gradient centrifugation, forming empty capsids, whereas the stable population was unaffected by the same CsCl treatment. Formaldehyde fixation of the 240S virus particles stabilized the virions and prevented ejection of DNA and generation of empty capsids. When formaldehyde-fixed 240S virus was examined with the electron microscope, only full virions were observed. These results indicate that polyoma capsids are not preformed in vivo, but instead are generated when infected cell lysates are subjected to harsh CsCl purification procedures.
Complete nucleotide sequence of the nucleoprotein gene of influenza B virus
Authors: Londo D R , Davis A R , Nayak D P
Published: January 2020

A DNA copy of influenza B/Singapore/222/79 viral RNA segment 5, containing the gene coding for the nucleoprotein (NP), has been cloned in Escherichia coli plasmid pBR322, and its nucleotide sequence has been determined. The influenza B NP gene contains 1,839 nucleotides and codes for a protein of 560 amino acids with a molecular weight of 61,593. Comparison of the influenza B NP amino acid sequence with that of influenza A NP (A/PR/8/34) reveals 37% direct homology in the aligned regions, indicating a common ancestor. However, influenza B NP has an additional 50 amino acids at its N-terminal end. As is the case with influenza A NP, influenza B NP is a basic protein, with its charged residues relatively evenly distributed rather than clustered. The structural homology suggests functional similarity between the NP of influenza A and B viruses.
Reversal of Fv-1 host range by in vitro restriction endonuclease fragment exchange between molecular clones of N-tropic and B-tropic murine leukemia virus genomes
Authors: Boone L R , Myer F E , Yang D M , Ou C Y , Koh C K , Roberson L E , Tennant R W , Yang W K
Published: January 2020

We molecularly cloned unintegrated viral DNA of the BALB/c endogenous N-tropic and B-tropic murine leukemia retroviruses and in vitro passaged N-tropic Gross (passage A) murine leukemia retroviruses. Recombinant genomes were constructed in vitro by exchanging homologous restriction enzyme fragments from N- or B-tropic parents and subsequent recloning. Infectious virus was recovered after transfection of these recombinant genomes into NIH-3T3 cells and cocultivation with the Fv-1 nonrestrictive SC-1 cells. XC plaque assays of recombinant virus progeny on Fv-ln and Fv-lb cells indicated that the Fv-l host range was determined by sequences located between the BamHI site in the p30 region of the gag gene (1.6 kilobase pairs from the left end of the map) and the HindIII site located in the pol gene (2.9 kilobase pairs from the left end of the map).
Role for the J-F intercistronic region of bacteriophages phi X174 and G4 in stability of mRNA
Authors: Hayashi M N , Hayashi M , Müller U R
Published: January 2020

A hairpin-like secondary structure in the intercistronic region between genes J and F of bacteriophages, phi X174 and G4 has been postulated to act as a transcription termination signal. We analyzed the in vivo transcripts of both phages and mutants derived from them with modifications of this hairpin structure. The phi X174 mutants appeared to fall into two groups with respect to the stability of two mRNA species. Class 1 mutants showed an mRNA profile very similar to the parental strain, whereas class 2 mutants lacked two major mRNA species normally terminated near the J-F region. The G4 mutants behaved like class 2 mutants of phi X174. Analysis of the stability of phi X174 mRNA revealed that messages specific for the genes upstream of the hairpin turn over more rapidly in class 2 mutants than in class 1 mutants. In class 1 mutants, the mRNA decay rates are similar but not identical to those of the wild-type strain. These data suggest a role for the nucleotide sequence within the J-F intercistronic region in mRNA degradation. They further imply that transcription termination occurs downstream from this site.
Terminal amino acid sequences and proteolytic cleavage sites of mouse mammary tumor virus env gene products
Authors: Henderson L E , Sowder R , Smythers G , Oroszlan S
Published: January 2020

The mature envelope glycoproteins of mouse mammary tumor virus (gp52 and gp36) were isolated by reversed-phase high-pressure liquid chromatography. The N-terminal amino acid sequence of gp36 was determined for 28 residues. The C-terminal amino acid sequences of gp52 and gp36 were determined by carboxypeptidase digestion. The N-terminal amino acid sequence of gp52 has been reported previously (L. O. Arthur et al., J. Virol. 41:414-422, 1982). These data were aligned with the predicted amino acid sequence of the env gene product obtained by translation of the DNA sequence (S. M. S. Redmond and C. Dickson, Eur. Mol. Biol. Org. J. 2:125-131, 1983). The amino acid sequences of the mature viral proteins were in agreement with the predicted amino acid sequence of the env gene product over the regions of alignment. This alignment showed the sites of proteolytic cleavages of the env gene product leading to the mature viral envelope glycoproteins. The N-terminal amino acid sequence of gp52 starts at residue 99 of the predicted structure indicating proteolytic cleavage of a signal peptide. A dipeptide (Lys-Arg) is excised between the C-terminus of gp52 and the N-terminus of gp36. The C-terminal amino acid sequence of gp36 is identical to the sequence predicted by the codons immediately preceding the termination codon for the env gene product. The data show that there is no proteolytic processing at the C-terminal of the murine mammary tumor virus env gene product and that the env gene coding region extends into the long terminal repeat.
N-acetylgalactosaminyltransferase activity involved in O-glycosylation of herpes simplex virus type 1 glycoproteins
Authors: Serafini-Cessi F , Dall'Olio F , Scannavini M , Costanzo F , Campadelli-Fiume G
Published: January 2020

We report on N-acetylgalactosaminyltransferase (UDPacetylgalactosamine--protein acetylgalactosaminyltransferase; EC 2.4.1.41) activity in herpes simplex virus type 1 (HSV-1)-infected BHK and RicR14 cells, a line of ricin-resistant BHK cells defective in N-acetylglucosaminyltransferase I. The enzyme catalyzed the transfer of [14C]N-acetylgalactosamine (GalNAc) from UDP-[14C]GalNAc into HSV glycoproteins, as identified by immunoprecipitation. The sugar was selectively incorporated into the immature forms of herpesvirus glycoproteins pgC, pgD, and gA-pgB, which are known to contain N-linked glycans of the high-mannose type. The high incorporation of [14C]GalNAc into endogenous acceptors of HSV-1-infected RicR14 cells was consistent with the accumulation of immature forms of HSV glycoproteins which occurs in these cells. Mild alkaline borohydride treatment of glycoproteins labeled via GalNAc transferase showed that the transferred GalNAc was O-linked and represented the first sugar added to the peptide backbone.
Antigenic drift in influenza virus H3 hemagglutinin from 1968 to 1980: multiple evolutionary pathways and sequential amino acid changes at key antigenic sites
Authors: Both G W , Sleigh M J , Cox N J , Kendal A P
Published: January 2020

Surveys of the antigenic properties of a wide range of variants of the H3N2 (Hong Kong) influenza virus subtype have revealed complex patterns of variants cocirculating during each of the main epidemic eras of the subtype. We determined hemagglutinin (HA) gene sequences for 14 isolates chosen to give the wildest possible spread of variant types. The addition of these data to existing HA gene sequence information for other variants provides a comprehensive picture of HA gene evolution during antigenic drift among H3N2 subtype viruses. The data reveal the existence of multiple evolutionary pathways during at least one period of development of the subtype and strikingly demonstrate that amino acid changes are limited to a small number of locations on the HA molecule during antigenic drift. The occurrence of sequential amino acid changes at key positions within these variable regions suggests that the HA structure has remained constant during subtype evolution so that only limited possibilities remain for further antigenic drift among H3N2 viruses.
Characteristics and regulation of interaction of avian retrovirus pp12 protein with viral RNA
Authors: Leis J , Jentoft J
Published: January 2020

We investigated the interaction of the avian retrovirus pp12 protein with viral RNA to assess its possible role in virion assembly. Using chemical modification techniques, we found that reagents specific for lysine or arginine residues inactivated the RNA-binding capacity of the protein. The binding of pp12 to 60S viral RNA was also strongly affected by pH (pKapp of 5.5); the affinity for viral RNA decreased by as much as 40-fold after protonation of one or more titratable groups on the protein. When the protein was cleaved by cyanogen bromide, each of the two polypeptide products bound to RNA (with low affinity), but pH dependence was lost. Thus, an intact protein was required for this effect. Since histidine and phosphoserine residues have pKa values close to the pKapp of the pp12-RNA interaction, they were studied to determine whether they were involved in this process. Each of the two histidyl residues in pp12 had pKa values of 6.2, as determined by proton nuclear magnetic resonance titrations, values too high to account for the pKapp of binding. The involvement of phosphoserine residues, which have pKa values similar to the pKapp, was investigated by removal of phosphate from pp12. When phosphate groups were chemically or enzymatically removed from the avian myeloblastosis virus, Rous sarcoma virus (Pr-C), and PR-E 95C virus pp12 proteins, the Kapp for binding 60S viral RNA was reduced 100-fold at pH 7.5. Thus, it seems possible that phosphorylation of the pp12 protein could favor viral nucleocapsid formation by increasing its affinity for the viral RNA genome. Dephosphorylation could provide for its release from the viral RNA during reverse transcription after viral infection of cells.
Virus-specific transcription in a Herpesvirus saimiri-transformed lymphoid tumor cell line
Authors: Knust E , Dietrich W , Fleckenstein B , Bodemer W
Published: January 2020

Herpesvirus saimiri-transformed lymphoid tumor cell lines contain nonintegrated covalently closed circular viral DNA molecules in high multiplicity. One of those cell lines, 1670, carries large viral DNA circles (202 kilobase pairs) with two stretches of repetitive DNA (70.8% G + C) that are interspersed between two segments of unique DNA (36% G + C). Since it was not known if there is any viral gene expression in H. saimiri-transformed cells, we initiated a study of transcription in cell line 1670. cDNA was generated by reverse transcription of cellular RNA and hybridized with cloned virion DNA fragments. The experiments indicated that appreciable transcription is restricted to a single segment of unique DNA. This sequence is present once only in the circular viral DNA and corresponds to unique DNA between map units 0.89 and 0.93 of virion DNA. By Northern blot hybridizations with labeled cloned probes of virion unique DNA, one predominant virus-specific polyadenylated transcript of, at most, 2.7 kilo-bases could be detected in tumor cell line 1670. The direction of transcription was determined by hybridization with randomly primed cDNA and, in parallel, with oligodeoxythymidylate-primed cDNA probes. Apparently, the patterns of virus-specific RNA synthesis in the H. saimiri-transformed cells are clearly distinct from the transcription program in other herpesvirus transformation systems analyzed before.
Bacteriophage SPO1 gene 27: location and nucleotide sequence
Authors: Costanzo M , Hannett N , Brzustowicz L , Pero J
Published: January 2020

Bacteriophage SPO1 gene 27, whose product is required for late gene transcription and DNA replication, has been cloned in Escherichia coli, and its complete nucleotide sequence has been determined. We infer that the product of gene 27 is a highly basic 17,518-dalton protein of 155 amino acids. The gene for this regulatory protein is transcribed from two promoters: an early promoter situated before the adjacent upstream gene 28 and a middle promoter located between genes 28 and 27.
An Escherichia coli gene required for bacteriophage P2-lambda interference
Authors: Ghisotti D , Zangrossi S , Sironi G
Published: January 2020

The gene old of bacteriophage P2 is known to (i) cause interference with phage lambda growth; (ii) kill recB- mutants of Escherichia coli after P2 infection; and (iii) determine increased sensitivity of P2 lysogenic cells to X-ray irradiation. In all of these phenomena, inhibition of protein synthesis occurs. We have isolated bacterial mutants, named pin (P2 interference), able to suppress all of the above-mentioned phenomena caused by the old+ gene product and the concurrent protein synthesis inhibition. Pin mutations are recessive, map at 12 min on the E. coli map, and identify a new gene. Satellite bacteriophage P4 does not plate on pin-3 mutant strains and causes cell lethality and protein synthesis inhibition in such mutants. P4 mutants able to grow on pin-3 strains have been isolated.
Cell receptors for baboon endogenous virus recognized by monoclonal antibodies
Authors: Thiry L , Cogniaux-LeClerc J , Olislager R , Sprecher-Goldberger S , Buekens P
Published: January 2020

Hybridomas from mice immunized with baboon endogenous virus (BaEV) from A204(M7) cells produced several antiviral monoclonal antibodies and, in addition, antibodies D-12 and E-4, which appeared to be virus specific because they reacted with BaEV but not with Mason-Pfizer virus or RD-114 virus. However, they also bound to human virus-free cells, and they did not recognize BaEV from bat or canine host cells. Cell membrane targets for these antibodies comigrated with an 18,000-dalton protein, which may contain specific determinants of BaEV receptors since antibody masking of these cell sites prevented BaEV but not Mason-Pfizer virus or RD-114 virus adsorption. However, RD-114 virus interfered with BaEV adsorption. Thus, the two viral receptors must be adjacent, but the antibody D-12 and E-4 targets are not within the active site of RD-114 virus receptor. Conversely, cell coating with BaEV from bat or canine hosts inhibited antibody D-12 binding. Noncultivated human lymphocytes and cells from fetal organs bound much less antibody D-12 than did cells from established cell lines, with a correlation between amounts of antibody D-12 acceptor sites and BaEV receptors. Thus, in vivo, BaEV infection of human cells may be inefficient. In vitro, antibody D-12 treatment of chronically infected A204(M7) cells caused intracellular accumulation of viral proteins and decreased virus release, with no such effect on RD-114 virus-producing cells. Canine cells bound antibody D-12 only if coated with BaEV from A204(M7) cells, indicating that the human determinant coadsorbed with the virions to animal cells. Possibly, determinants of cell receptors participate in BaEV maturation and become associated with the virions.
Epidermal growth factor receptor metabolism and protein kinase activity in human A431 cells infected with Snyder-Theilen feline sarcoma virus or harvey or Kirsten murine sarcoma virus
Authors: Cooper J A , Scolnick E M , Ozanne B , Hunter T
Published: January 2020

When human A431 cells, which carry high numbers of epidermal growth factor (EGF) receptors, are exposed to EGF, the total content of phosphotyrosine in cell protein is increased, the EGF receptor becomes phosphorylated at tyrosine, and new phosphotyrosine-containing 36,000- and 81,000-dalton proteins are detected. We examined the properties of A431 cells infected with Snyder-Theilen feline sarcoma virus, whose transforming protein has associated tyrosine protein kinase activity, and Harvey and Kirsten sarcoma viruses, whose transforming proteins do not. In all cases, the infected cells were more rounded and more capable of anchorage-independent growth than the uninfected cells. EGF receptors were assayed functionally by measuring EGF binding and structurally by metabolic labeling and immunoprecipitation. In no case did infection appear to alter the rate of EGF receptor synthesis, but infection reduced EGF receptor stability by about 50% for cloned Harvey sarcoma virus-infected cells and by 80% for cloned feline sarcoma virus-infected cells. The corresponding reductions in EGF binding were 70 and 90%, respectively. The proteins of feline sarcoma virus-infected A431 cells contained an increased amount of phosphotyrosine, and the 36,000- and 81,000-dalton phosphoproteins were detected. The EGF receptor was not detectably phosphorylated at tyrosine, however, unless the cells were exposed to EGF. The Harvey and Kirsten sarcoma virus-infected cells did not exhibit elevated levels of phosphotyrosine either in the total cell proteins or in the EGF receptor, nor were the 36,000- and 81,000-dalton proteins detectable. However, these phosphoproteins were found in the infected cells after EGF treatment. Thus, all of the infected A431 cells exhibited reduced EGF binding and increased degradation of EGF receptors, yet their patterns of protein phosphorylation were distinct from those of EGF-treated A431 cells.
Structure of the transforming region of human cytomegalovirus AD169
Authors: Nelson J A , Fleckenstein B , Jahn G , Galloway D A , McDougall J K
Published: January 2020

The minimum size fragment of human cytomegalovirus AD169 required to initiate transformation was determined by transfection of primary rat embryo cells with deletion fragments constructed by digestion of a cloned fragment containing the transforming region (pCM4000) with exonuclease III and S1 nuclease. The results indicate that the left-hand boundary of the minimum size sequence for transformation must reside between 490 and 318 bases from the HindIII site of pCM4000. The right-hand boundary was defined by the EcoRI site which is 20 bases from the HindIII site. The nucleotide sequence of the transforming fragment of human cytomegalovirus was determined by the chemical degradation technique of Maxam and Gilbert and with the dideoxynucleoside triphosphate chain termination method. The sequence of pCM4000 comprises 2,848 base pairs and has an A X T composition of 59.5%. Reading frame analysis of the sequence indicated the longest open reading frame was 118 amino acids in length. Northern blot analysis of polyadenylated and non-polyadenylated RNA extracted from cells at immediate early and late times after infection with human cytomegalovirus indicate that one 5.0-kilobase RNA species hybridized to pCM4000 (Jahn et al., J. Virol, in press). The direction of transcription was determined by hybridization of this transcript with M13 single-stranded probes, and S1 analysis of this RNA did not detect introns.