bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection by RNA blot hybridization of RNA sequences homologous to the BglII-N fragment of herpes simplex virus type 2 DNA
Authors: Jenkins F J , Howett M K , Spector D J , Rapp F
Published: January 2020

RNA species, extracted at the time of peak synthesis of the alpha, beta, and gamma classes of herpes simplex virus polypeptides from lytically infected Vero cells, were examined for homology to the BglII-N fragment (map units 0.58 to 0.63) of herpes simplex virus type 2 DNA. By using northern blot analysis, two major and several minor polyadenylated RNA species showed homology to the BglII-N fragment at times corresponding to the maximum synthesis of the beta (7 h postinfection) and gamma (12 h postinfection) herpes simplex virus polypeptides. No alpha RNA homologous to the BglII-N fragment was detected.
Molecular cloning of DNA complementary to mRNA of the baculovirus Autographa californica nuclear polyhedrosis virus: location and gene products of RNA transcripts found late in infection
Authors: Adang M J , Miller L K
Published: January 2020

DNAs complementary to late Autographa californica nuclear polyhedrosis virus (AcNPV) mRNA were synthesized by reverse transcription and cloned in Escherichia coli by using pBR322 as a vector. Eleven different cDNAs were distinguished in our screening of 45 AcNPV-homologous clones. Location of the regions of cDNA homology with respect to the AcNPV physical map showed that the 11 cDNAs were dispersed throughout the genome. The most abundant cDNA insertion, representing approximately one-third of the late viral mRNAs, was homologous to the AcNPV HindIII-P,Q and EcoRI-P fragments. The direction of transcription in this region was from left to right on a linearized AcNPV physical map. Hybridization selection followed by in vitro translation showed that this region encoded a 7,200-dalton (7.2K) protein which comigrated with a minor protein found in the extracellular nonoccluded form of the virus (NOV). Similarly, the gene for polyhedrin, the major structural protein of the occluded virus form, was located, at least in part, in the HindIII-V/EcoRI-I region of the AcNPV map. The polyhedrin transcript represented approximately one-quarter of the viral polyadenylic acid-containing RNAs at 27 h postinfection. Another relatively abundant cDNA was homologous to the HindIII-A/EcoRI-C/SstI-G region, and RNA selected by this cDNA directed the synthesis of two proteins (31K and 30K). The protein products of five other cDNA-selected RNAs were identified. The HindIII-D/EcoRI-O, HindIII-C/EcoRI-D, HindIII-B1/EcoRI-E, and HindIII-B2/EcoRI-H regions of the AcNPV L-1 genome were homologous to RNAs which directed the synthesis of a 57K protein, a 25K protein, a 61K protein, and a 37K protein (plus a minor 26K protein), respectively. Late mRNA selected by a cDNA homologous to the HindIII-P/EcoRI-B region of the AcNPV map directed the synthesis of 31K and 30K proteins which comigrated with the 31K and 30K proteins translated from RNA selected by the HindIII-A/EcoRI-C/SstI-G cDNA. Three other cDNAs have not been correlated yet with specific protein products.
A locus that enhances the induction of endogenous ecotropic murine leukemia viruses is distinct from genome-length ecotropic proviruses
Authors: Horowitz J M , Risser R
Published: January 2020

The segregation of genes that enhance the induction of ecotropic murine leukemia viruses (In loci) has been compared with the segregation of ecotropic-specific nucleotide sequences in 12 low-leukemic mouse strains and 18 recombinant inbred strains. Endogenous ecotropic viruses of these strains are of genome length and structurally similar to AKR ecotropic proviruses. Low-leukemic strains of related pedigree contain ecotropic proviruses at common integration sites. Loci previously identified which enhance induction of ecotropic viruses (In genes) were correlated with the inheritance of ecotropic viral sequences in inbred low-leukemic mouse strains and in CXB recombinant inbred mouse strains. However, four BXH recombinant inbred strains were observed to possess an In gene(s) yet lack the probed envelope gene region for the corresponding endogenous ecotropic virus. These observations indicate that at least one gene that enhances ecotropic virus expression in vitro is encoded by DNA sequences outside ecotropic proviruses or by subgenomic viral sequences.
Structure and expression of c-rel, the cellular homolog to the oncogene of reticuloendotheliosis virus strain T
Authors: Chen I S , Wilhelmsen K C , Temin H M
Published: January 2020

The cellular homolog of the onc sequences in the avian retrovirus reticuloendotheliosis virus strain T (v-rel) was studied by molecular cloning and nucleic acid hybridization. In contrast to v-rel sequences, which are 1.4 kilobase pairs long, the cellular homolog, c-rel, from line 15B chickens is at least 25 kilobase pairs long, with multiple apparent introns. A 4.0-kilobase polyadenylic acid-containing RNA transcript is the primary species of c-rel RNA present in uninfected chicken cells. Sequences at the 3' end of this c-rel RNA are not present in v-rel.
Influenza Virus Temperature-Sensitive Cap (m
7
GpppNm)-Dependent Endonuclease
Authors: Ulmanen Ismo , Broni Barbara , Krug Robert M.
Published: January 2020

The first step in influenza viral mRNA synthesis is the endonucleolytic cleavage of heterologous RNAs containing cap 1 (m
7
GpppNm) structures to generate capped primers that are 10 to 13 nucleotides long, which are then elongated to form the viral mRNA chains. We examined the temperature sensitivity of these steps in vitro by using two WSN virus temperature-sensitive mutants,
ts
1 and
ts
6, which have a defect in the genome RNA segment coding for the viral PB2 protein. For these experiments, it was necessary to employ purified viral cores rather than detergent-treated virions to catalyze transcription, as preparations of detergent-treated virions contain destabilizing or inhibitory activities which render even the transcription catalyzed by wild-type virus temperature sensitive. Using purified wild-type viral cores, we found that the rates of endonucleolytic cleavage of capped primers and of overall transcription were similar at 39.5 and 33°C, the in vivo nonpermissive and permissive temperatures, respectively. In contrast, the activities of the cap-dependent endonucleases of
ts
1 and
ts
6 viral cores at 39.5°C were only about 15% of those at 33°C. The steps in transcription after endonucleolytic cleavage of the capped RNA primer were largely, if not totally, temperature insensitive, indicating that the mutations in the PB2 protein found in
ts
1 and
ts
6 virions affect only the endonuclease step. The temperature-sensitive defect is most likely in the recognition of the 5′-terminal cap 1 structure that occurs as a required first step in the endonuclease reaction: the cap-dependent binding of a specific capped primer fragment to
ts
1 viral cores was temperature sensitive under conditions in which binding to wild-type viral cores was not affected by increasing the temperature from 33 to 39.5°C. Thus, our results establish that the viral PB2 protein functions in cap recognition during the endonuclease reaction.
Improved localization of phosphorylation sites in simian virus 40 large T antigen
Authors: van Roy F , Fransen L , Fiers W
Published: January 2020

The location of phosphorylation sites in the large T antigen of simian virus 40 has been studied both by partial chemical cleavage and by partial proteolysis of various forms of large T. These included the full-size wild-type molecule with an apparent molecular weight of 88,000, deleted molecules coded for by the mutants dl1265 and dl1263, and several shortened derivatives generated by the action of a cellular protease. These molecules differed from each other by variations in the carboxy-terminal end. In contrast, a ubiquitous but minor large T form with a molecular weight of 91,000 was found to be modified in the amino-terminal half of the molecule. In addition to the phosphorylation of threonine at position 701 (K.-H. Scheidtmann et al., J. Virol. 38:59-69, 1981), two other discrete domains of phosphorylation were recognized, one at either side of the molecule. The amino-terminal region was located between positions 81 and 124 and contained both phosphothreonine and phosphoserine residues. The carboxy-terminal region was located between approximate positions 500 and 640 and contained at least one phosphoserine residue but no phosphothreonine. The presence in the phosphorylated domains of large T of known recognition sequences for different types of protein kinases is discussed, together with possible functions of large T associated with these domains.
Substrate specificity of two bacteriophage-associated endo-N-acetylneuraminidases
Authors: Kwiatkowski B , Boschek B , Thiele H , Stirm S
Published: January 2020

For Escherichia coli Bos12 (O16:K92:H-), a bacteriophage (phi 92) has been isolated which carries a depolymerase active on the K92 capsular polysaccharide. As seen under the electron microscope, phi 92 belongs to Bradley's morphology group A and is different from the phage phi 1.2 previously described (Kwiatkowski et al., J. Virol. 43:697-704, 1982), which grows on E. coli K235 (O1:K1:H-), depolymerizes colominic acid, and belongs to morphology group C. The specificity of the phi 1.2- and phi 92-associated endo-N-acetylneuraminidases has been studied with respect to the following substrates (all alkali treated, and where NeuNAc represents N-acetylneuraminic acid): (i) [-alpha-NeuNAc-(2 leads to 8)-]n (colominic acid), (ii) [-alpha-NeuNAc-(2 leads to 8)-alpha-NeuNAc-(2 leads to 9)-]n (E. coli K92 polysaccharide), and (iii) [-alpha-NeuNAc-(2 leads to 9)-]n (Neisseria meningitidis type C capsular polysaccharide). The increase in periodate consumption of these glycans upon incubation with purified phi 1.2 or phi 92 particles was measured, and the split products obtained from all substrates after exhaustive degradation were analyzed by gel chromatography. It was found that the Neisseria polysaccharide is not appreciably affected by either virus enzyme and that phi 1.2 only depolymerizes a small fraction of the K92 glycan. Colominic acid, however, is completely degraded by both agents, phi 92 yielding smaller fragments (one to six NeuNAc residues) than phi 1.2 (two to seven). Phage phi 92 additionally depolymerizes the K92 glycan, essentially to oligosaccharides of two, four, and six residues. The size distribution of these K92 oligosaccharides indicates that the phi 92 enzyme predominantly cleaves the alpha(2 leads to 8) linkages in this polymer.
Molecular genetics of herpes simplex virus. VIII. further characterization of a temperature-sensitive mutant defective in release of viral DNA and in other stages of the viral reproductive cycle
Authors: Batterson W , Furlong D , Roizman B
Published: January 2020

Previous studies have shown that cells infected with the herpes simplex virus 1(HFEM) mutant tsB7 and maintained at the nonpermissive temperature fail to accumulate viral polypeptides. Analyses of intertypic recombinants generated by marker rescue of tsB7 with herpes simplex virus 2 DNA fragments localized the mutation between 0.46 and 0.52 map units on the viral genome (Knipe et al., J. Virol. 38:539-547, 1981). In this paper we report that the mutation in tsB7 affects several aspects of the reproductive cycle of the virus at the nonpermissive temperature. Thus, (i) viral capsids accumulate at the nuclear pores and do not release viral DNA for at least 6 h postinfection at 39 degrees C. The DNA was released within 30 min after a shift to the permissive temperature. (ii) Experiments involving shifts from the permissive to the nonpermissive temperature indicated that viral protein synthesis was not sustained in cells maintained at the permissive temperature for less than 4 h. (iii) Viral DNA synthesis was delayed at the permissive temperature for as long as 8 h. Once initiated, it continued at 39 degrees C. (iv) Marker rescue of tsB7 by transfection with herpes simplex virus 1(F) DNA fragments localized the mutation to between 0.501 and 0.503 map units on the viral genome. These results are consistent with the tsB7 lesion being in a gene coding for a virion component which affects release of viral DNA from capsids and onset of viral DNA synthesis.
The influenza virus nucleoprotein synthesized from cloned DNA in a simian virus 40 vector is detected in the nucleus
Authors: Lin B C , Lai C J
Published: January 2020

We obtained DNA sequences coding for the nucleoprotein (NP) of an influenza A virus by reverse transcription of virion RNA with synthetic oligonucleotide primers. Terminal sequence analysis showed that the cloned gene contained a full-length copy of the virion RNA segment. The NP-specific DNA was inserted into the late region of a simian virus 40 vector, and the DNA recombinant was propagated in the presence of an early simian virus 40 temperature-sensitive mutant helper. Infection of African green monkey kidney cells with the recombinant produced a polypeptide immunoprecipitable with NP-specific antisera. The polypeptide product had a molecular weight of 56,000, identical to that of the nucleoprotein of influenza virus as estimated on polyacrylamide gels. The putative NP was detected in the nucleus of infected primate cells by an immunofluorescence assay. This nuclear localization of NP from recombinant DNA was similar to that seen during influenza virus infection.
Processing of the Encephalomyocarditis Virus Capsid Precursor Protein Studies in Rabbit Reticulocyte Lysates Incubated with
N
-Formyl-[
35
S]Methionine-tRNA
f
Met
Authors: Campbell Elizabeth A. , Jackson Richard J.
Published: January 2020

Translation of encephalomyocarditis virus RNA in rabbit reticulocyte lysates in the presence of
N
-formyl-[
35
S]methionine-tRNA
f
Met
revealed that a small polypeptide is cleaved from the N-terminus of the capsid protein precursor, preA, by virus-coded protease activity. Therefore, this N-terminal segment comprising the translation initiation site is not conserved in any of the mature capsid proteins.
A unique sequence in murine leukemia virus long terminal repeat functions as a termination signal for transcription in Escherichia coli
Authors: Honigman A , Panet A
Published: January 2020

Moloney murine leukemia virus DNA fragments were subcloned into two plasmids downstream of active promoters to detect termination sequences for transcription initiated at these promoters. Two of the viral DNA fragments (2,050 and 595 base pairs) were able to block transcription when inserted in one orientation but not when inserted in the other. These two viral DNA fragments contained long terminal repeat sequences. Deletion of 84 base pairs from the U-3 and R regions in the long terminal repeat removed the transcription termination activity.
Stability and in vitro DNA packaging of bacteriophages: effects of dextrans, sugars, and polyols
Authors: Serwer P , Masker W E , Allen J L
Published: January 2020

Attempts were made to increase the efficiency of infectious particle formation during the in vitro assembly of bacteriophage T7 from procapsids and DNA. It was found that dextrans and some smaller, related compounds (sucrose and sorbitol) increase this efficiency by a factor of 8 to 50. Dextrans also inhibited elevated temperature-induced emptying of DNA from bacteriophages T7, P22, and T4, suggesting that the stimulation of assembly is caused, at least in part, by the stabilization of packaged DNA in capsids. The data indicated that the sugars and polyols can slow DNA emptying from bacteriophages at elevated temperature whether they permeate the bacteriophage capsid or not. In contrast, the data suggested that permeation of some particle, probably a capsid, results in inhibition of in vitro T7 assembly.
Loss of Polyoma Virus Infectivity as a Result of a Single Amino Acid Change in a Region of Polyoma Virus Large T-Antigen Which Has Extensive Amino Acid Homology with Simian Virus 40 Large T-Antigen
Authors: Hayday Adrian C. , Chaudry Furzana , Fried Mike
Published: January 2020

The polyoma virus (Py) transformed cell line 7axB, selected by in vivo passage of an in vitro transformed cell, contains an integrated tandem array of 2.4 genomes and produces the large, middle, and small Py T-antigen species, with molecular weights of 100,000, 55,000, and 22,000, respectively (Hayday et al., J. Virol.
44
:67-77, 1982; Lania et al., Cold Spring Harbor Symp. Quant. Biol.
44
:597-603, 1980). The integrated viral and adjacent host DNA sequences have been molecularly cloned as three
Eco
RI fragments (Hayday et al.). One of these fragments (7B-M), derived from within the tandem viral sequences, is equivalent to an
Eco
RI viral linear molecule. Fragment 7B-M has been found to be transformation competent but incapable of producing infectious virus after DNA transfection (Hayday et al.). By constructing chimerae between 7B-M and Py DNA and by direct DNA sequencing, the mutation responsible for the loss of infectivity has been located to a single base change (adenine to guanine) at nucleotide 2503. This results in a conversion of an aspartic acid to a glycine in the C-terminal region of the Py large T-antigen but does not appear to affect the binding of the Py large T-antigen to Py DNA at the putative DNA replication and autoregulation binding sites. The mutation is located within a 21-amino acid homology region shared by the simian virus 40 large T-antigen (Friedmann et al., Cell
17
:715-724, 1979). These results suggest that the mutation in the 7axB large T-antigen may be involved in the active site of the protein for DNA replication.
Mechanism of Action of the Endonuclease Associated with the αβ and ββ Forms of Avian RNA Tumor Virus Reverse Transcriptase
Authors: Leis J. , Duyk G. , Johnson S. , Longiaru M. , Skalka A.
Published: January 2020

Preparations of the αβ and the ββ forms of reverse transcriptase from the Prague C strain of Rous sarcoma virus grown in chicken embryo fibroblasts, the αβ and the ββ forms of the enzyme from the B77 strain of Rous sarcoma virus grown in duck embryo fibroblasts, and the αβ form of reverse transcriptase from avian myeloblastosis virus have been analyzed. All these enzyme preparations contain a Mn
2+
-activated endonuclease activity. The ββ form of enzyme, in addition, contains a Mg
2+
-dependent endonuclease. Such an activity is barely detectable in the αβ form of enzymes. The endonuclease associated with reverse transcriptase introduces single- and double-strand breaks containing 3′ OH and 5′ P termini into RF I DNA. The conversion of RF I DNA to RF III DNA is more readily catalyzed by the ββ form of reverse transcriptase. In contrast to a recently published report by Hizi et al. (J. Virol
41
:974-981, 1982), we have failed to detect the conversion of RF I DNA to covalently closed relaxed circles (RF IV DNA) by any of the αβ form of enzymes tested. RF IV DNA was not produced by the ββ form of reverse transcriptase either. We conclude that topoisomerization is not an intrinsic activity of reverse transcriptase. Although the conversion of RF I DNA to RF II DNA was found to be rapid, the endonuclease associated with reverse transcriptase acted slowly on RF II, RF III, and RF IV DNAs. Circular and linear single-stranded DNAs were also susceptible to cleavage by the endonuclease at a rate comparable to nicking of RF I DNA. This pattern of activity suggests that the endonuclease cleaves the RF I DNA in the single-stranded regions of the DNA induced by its supercoiling. The preference of the αβ and the ββ forms of the endonuclease for viral DNA was tested with Rous-associated virus type 2 and Rous sarcoma virus transformation-defective Schmidt-Ruppin B strain DNA molecularly cloned in plasmid pBR322 and M13 DNA vectors, respectively. The rate of nicking of RF I DNA containing viral DNA or partial sequences of viral DNA with one or two tandem long terminal repeats was the same as when these sequences were not present in the host vectors. A similar lack of preference was observed with single-stranded M13 DNAs.
Site-directed deletions of Abelson murine leukemia virus define 3' sequences essential for transformation and lethality
Authors: Watanabe S M , Witte O N
Published: January 2020

Abelson murine leukemia virus (A-MuLV) encodes a single protein with tyrosine kinase activity that can transform fibroblast cell lines in vitro and lymphoid target cells in vitro and in vivo. Expression of kinase-active A-MuLV protein can result in a deleterious effect on transformed fibroblast populations, leading to cell death or selection for nonlethal mutants of the virus. These mutants retain expression of the kinase activity but have lost large portions of the carboxy terminus of the Abelson protein. To more precisely map the sequences involved in this lethal effect, we have isolated a series of site-directed deletions from a DNA clone of the P160 wild-type strain of A-MuLV. In addition, a number of unexpected, spontaneous deletions occurring during transfection of NIH 3T3 cells were isolated. These deletions result in expression of carboxy-terminal truncated forms of the A-MuLV protein ranging from 130,000 to 84,000 in molecular weight. Analysis of the transforming and lethal activities of each mutant recovered in its RNA viral form shows that the transformation-essential and lethal-essential sequences do not overlap. These data and our previous work suggest that a function carried by the carboxy-terminal region of the A-MuLV protein acts in cis with the kinase-essential region to mediate the lethal effect.
Basolateral maturation of retroviruses in polarized epithelial cells
Authors: Roth M G , Srinivas R V , Compans R W
Published: January 2020

We have investigated the maturation sites of avian and mammalian C-type retroviruses in polarized epithelial cells. Examination of thin sections of Madin Darby canine kidney cells infected with RD114 or avian reticuloendotheliosis virus revealed that these viruses mature from the basolateral membrane domains. Similar results were obtained with a continuous line of mouse mammary epithelial cells infected with Friend, Moloney, Rauscher, or Kirsten murine leukemia viruses, or Friend virus-related or Moloney virus-related mink cell focus-forming viruses. Immunofluorescence observations indicate that viral glycoproteins are inserted only at the basolateral membranes in these cells. Because of the availability of DNA and protein sequence data, and of molecularly cloned viruses, these virus systems offer advantages for molecular studies on directional transport of plasma membrane glycoproteins.
Expression of adenovirus type 12 early region 1 in KB cells transformed by recombinants containing the gene
Authors: Shiroki K , Saito I , Maruyama K , Fukui Y , Imatani Y , Oda K I , Shimojo H
Published: January 2020

The adenovirus type 12 (Ad12) early region 1 (E1) gene was introduced into KB cells by using a dominant selection vector, pSV2-gpt, and over 80 Gpt+ KB cell clones were established. Three types of recombinant DNAs (gAE1A, gARC, and gABA) were constructed. They contained the AccI-H, EcoRI-C, and BamHI-A fragments, respectively, of Ad12 DNA in pSV2-gpt. Five of 50 (10%) gABA-transformed cell clones, 12 of 18 (67%) gAE1A-transformed cell clones, and 10 of 18 (56%) gARC-transformed cell clones complemented the growth of Ad5 dl312 (deletion in E1A) and were designated as Gpt+ Ad+ cell clones. In these cell clones at their early passages, recombinant genome sequences were detected in cellular DNA and were expressed. T antigen g (the E1A gene product) was detected by immunofluorescence. The Gpt+ Ad+ cell clones supported the growth of Ad5 deletion mutants in parallel with the expression of Ad12 E1A or E1A plus E1B genes. After infection of Gpt+ Ad+ cell clones with Ad5 dl312, the early genes of dl312 were efficiently transcribed, indicating the expression of the pre-early function of the Ad12 E1A gene. Two clones each from gAE1A-,gARC-, and gABA-transformed cells were subcultured for a long period to determine the stability of the transfecting DNAs. Subculture in a nonselective medium resulted in cells which lost the transfecting DNAs. Subculture in a selective medium resulted in the selection of cells which maintained the gpt gene expression but lost the Ad12 gene expression. These results indicate that the transfecting DNA is present in an unstable state in KB cells.
Polycaryocyte formation mediated by Sindbis virus glycoproteins
Authors: Mann E , Edwards J , Brown D T
Published: January 2020

The process of cell fusion mediated by Sindbis virus membrane proteins synthesized after infection was examined. At the times after infection at which virus proteins were detectable on the cell surface, Sindbis virus-infected BHK-21 cells were found to express a fusion function after brief treatment at acid pH. In studies employing wild-type virus and temperature-sensitive mutants and testing drug or protease inhibition of virus production, we made the following observations on Sindbis virus-mediated fusion from within. (i) Fusion requires the synthesis of virus glycoproteins and their transport to the cell surface. (ii) Modification of the cell plasma membrane by polypeptides PE2 and E1 alone is not sufficient for expression of the fusion function. (iii) The proteolytic conversion of plasma membrane-associated PE2 to E2 is not essential for fusion. (iv) Glycosylation of virus plasma membrane proteins is essential for fusion. (v) The lesions of Sindbis virus temperature-sensitive mutants do not affect their ability to fuse cells.
Detection of an 85,000-dalton phosphoprotein in ts110 murine sarcoma virus-infected cells with antiserum against a v-mos peptide
Authors: Papkoff J , Hunter T
Published: January 2020

We have used an antiserum directed against a synthetic v-mos peptide (anti-C3 serum) to screen ts110 murine sarcoma virus (MuSV)-infected cells for the presence of v-mos-encoded proteins. Anti-C3 serum specifically recognized an 85,000-dalton protein doublet (P85) from [35S]methionine-labeled ts110 MuSV-infected producer cells grown at 32 degrees C, the permissive temperature for transformation. The P85 doublet was also recognized by an antiserum directed against the viral gag protein p15. P85 was present but at 2- to 10-fold-lower levels in ts110 MuSV-infected producer cells grown at 39 degrees C, the restrictive temperature for transformation. The P85gag-mos fusion product was the only v-mos protein reproducibly detected in this ts110 MuSV-transformed cell line. Immunoprecipitation of 32P-labeled cells with anti-C3 serum revealed that the upper band of the P85 doublet is phosphorylated, containing mostly phosphoserine and some phosphothreonine. Cells acutely infected with ts110 MuSV contained slightly higher levels of P85 than did the ts110 MuSV-infected producer cell line. Anti-C3 serum specifically recognized a 33,000-dalton protein (p33) in the acutely infected cells labeled with [35S]methionine. p33 was present in trace amounts and may represent a previously unidentified ts110 MuSV-encoded v-mos protein.
Organization and expression of the immediate early genes of human cytomegalovirus
Authors: Stinski M F , Thomsen D R , Stenberg R M , Goldstein L C
Published: January 2020

The immediate early genes of human cytomegalovirus were characterized according to map location, RNA transcripts, and translation products. Three regions in the large unique component (0.709 to 0.751 map units) were transcribed in the presence of an inhibitor of protein synthesis (anisomycin). A single size class of polyadenylated mRNA, 1.95 kilobases (kb), was transcribed abundantly relative to the other size classes. The predominant 1.95-kb viral RNA was transcribed from right to left on the prototype arrangement of the viral genome and spanned a region of approximately 2.8 kb (0.739 to 0.751 map units). This mRNA codes for a 75,000-dalton protein that represents the predominant immediate early protein detected in infected cells. Immunoprecipitation of viral proteins synthesized in vitro as well as in vivo demonstrated that the predominant immediate early protein is synthesized as a protein of 75,000 daltons, but is presumably modified in vivo, resulting in a broad banding pattern ranging from 75,000 to 68,000 daltons. A different immediate early viral gene (0.732 to 0.739 map units) is transcribed from left to right at relatively low levels. The 3' ends of the above viral RNAs terminate at approximately 230 base pairs apart in the region of approximately 0.739 map units. Five RNA size classes ranging from 2.25 to 1.10 kb were detected, but the 1.75-kb and 1.40-kb RNA size classes were more abundant from this region. At least four minor proteins are coded by these mRNAs, with apparent molecular weights ranging from 56,000 to 16,500. Last, a 1.95-kb mRNA was transcribed from a third region (0.709 to 0.728 map units). This viral mRNA was present at relatively low concentration and coded for a minor protein of 68,000 daltons. Since immediate early gene expression of human cytomegalovirus is dominated by the synthesis of an mRNA from the region of 0.739 to 0.751 map units that codes for the predominant immediate early protein found in the infected cell, we hypothesize that this protein is the major regulatory protein influencing the switch from restricted to extensive transcription.
Application of Denatured, Electrophoretically Separated, and Immobilized Lysates of Herpes Simplex Virus-Infected Cells for Detection of Monoclonal Antibodies and for Studies of the Properties of Viral Proteins
Authors: Braun Daniel K. , Pereira Lenore , Norrild Bodil , Roizman Bernard
Published: January 2020

We report the use of herpes simplex virus type 1 (HSV-1)- and HSV-2-infected cell polypeptides (ICPs) separated by electrophoresis in polyacrylamide gels and transferred to nitrocellulose to (i) detect monoclonal antibodies to viral polypeptides and to (ii) study the properties of the proteins with the monoclonal antibodies. Our results were as follows. (i) When the antigens were electrophoretically separated in denaturing gels and then immobilized on nitrocellulose strips, we detected a greater diversity of monoclonal antibodies to viral proteins than when we used the technique of immune precipitation of soluble, nondenatured viral antigens. The primary advantage of the technique is in the detection of nonprecipitating antibody and of antibody to poorly soluble antigens not available for reaction in preparations cleared by high-speed centrifugation before immune reaction. (ii) Studies of the viral polypeptides reactive with three monoclonal antibodies indicated that the technique can be used to investigate several properties of the antigens. Specifically, monoclonal antibody to ICP 4 confirmed the accumulation of viral protein in the nucleus and the mapping of the gene in the S component. The results showed, however, that HSV-1 and HSV-2 ICP 4 do have common antigenic determinants. The reaction of a nonprecipitating monoclonal antibody with electrophoretically separated, immobilized polypeptides contained in cytoplasmic and nuclear fractions, those chemically deglycosylated, or those specified by specific HSV-1 x HSV-2 intertypic recombinants identified the antigens reactive with the second monoclonal antibody as various forms of glycoprotein gC. Of particular interest was a set of four antigens, 39,000 to 46,500 in apparent molecular weight, reactive with each of several monoclonal antibodies. These studies showed that two polypeptides partition in the cytoplasm and two in the nucleus and that all comap with the previously mapped ICPs 35 and 37 in the region of the genome defined by the viral thymidine kinase gene on the left and the glycoprotein gA/B gene on the right. Unlike ICP 4 and gC, the four polypeptides are linked by intermolecular bisulfide bonds, inasmuch as the polypeptides were not at the expected locations upon denaturation and electrophoresis in the absence of reducing agents.
Gene Mapping of Rotavirus Double-Stranded RNA Segments by Northern Blot Hybridization: Application to Segments 7, 8, and 9
Authors: Dyall-Smith Michael L. , Azad Ahmed A. , Holmes Ian H.
Published: January 2020

Cloned DNA copies of double-stranded RNA segments 7, 8, and 9 of UK bovine rotavirus were nick-translated with [α-
32
P]ATP and hybridized to double-stranded RNA of various rotavirus strains which had been separated on long polyacrylamide gels and then transferred to
o
-aminophenylthioether paper. Specific hybridization of the UK calf clones to the separated RNA segments allowed the corresponding genes of four different rotaviruses to be rapidly determined.
Sendai Virus Envelopes Can Mediate Epstein-Barr Virus Binding to and Penetration into Epstein-Barr Virus Receptor-Negative Cells
Authors: Khélifa Ridha , Menezes José
Published: January 2020

Epstein-Barr virus (EBV) receptor-negative cells were treated with UV-inactivated Sendai virus (SV) or with reconstituted SV envelopes having a low hemolytic activity and then assayed for EBV binding or for susceptibility to EBV infection. EBV binding was assessed by using both unlabeled and fluoresceinated EBV preparations. It was found that SV or SV envelope treatment renders these cells able to bind EBV. Various experiments were performed to clarify the mechanism of this SV-induced binding. The EBV receptor-negative 1301 cells were treated with SV either at 0°C or at both 0 and 37°C successively and then examined for EBV binding at 0°C. It was thus found that when SV treatment was performed exclusively at 0°C, the target cells showed higher fluorescence intensity after their incubation with fluoresceinated EBV. In addition,
Clostridium perfringens
neuraminidase treatment of 1301 cells did not induce any EBV binding to these cells. These data indicate that EBV binding is not due to the disturbance of the cell membrane by SV envelope fusion or to the uncovering of EBV binding sites on the cells after the enzymatic action of SV neuraminidase. Moreover, bound EBV was partly eluted from SV-treated 1301 cells at 37°C, and the treatment of EBV with
C. perfringens
neuraminidase inhibited its SV-mediated binding. These data indicate that EBV binds to the hemagglutinin-neuraminidase of SV on the target cell surface and that a fraction of the bound EBV becomes irreversibly associated with the SV-treated cell membrane. Our data also show that EBV can penetrate into 1301 cells which have incorporated SV envelopes into their membrane, as demonstrated by the induction of the EBV-determined nuclear antigen by B95-8 EBV in SV envelope-treated 1301 cells.
Analysis of the protein-protein interactions in the parvovirus H-1 capsid
Authors: Paradiso P R
Published: January 2020

The structure of the icosahedral capsid of the H-1 parvovirus was probed by chemical cross-linking methods. Treatment of empty capsids with high-molecular-weight polyethylene glycols resulted in irreversible aggregation of the minor capsid protein VP1. Multimers of VP1 containing at least five and perhaps six molecules were obtained, but only with empty capsids and not with the full, DNA-containing virus. Cross-linking of the empty capsids with dimethylsuberimidate confirmed the assignments of the products formed after treatment with polyethylene glycol. With dimethylsuberimidate the most abundant product was a heterologous dimer containing VP1 and the major capsid protein VP2'. A small amount of homologous VP2' dimer was also obtained, but the majority of VP2' remained unreacted even at high concentrations of dimethylsuberimidate. The capsid proteins of the full virus, on the other hand, were completely unreactive to dimethylsuberimidate. The data suggest that the minor protein VP1 may be clustered in the capsid and perhaps composes one or two of the morphological units of the icosahedral shell.
Genome structure of HBI, a variant of acute leukemia virus MC29 with unique oncogenic properties
Authors: Bister K , Jansen H W , Graf T , Enrietto P , Hayman M J
Published: January 2020

We have analyzed the viral RNA of a variant of avian acute leukemia virus MC29, termed HBI. This virus was isolated during in vitro passage of a partially transformation-defective (td) mutant of MC29 (td10H-MC29) in chicken macrophages. While td10H-MC29 has a reduced ability to transform macrophages in vitro or to induce tumors in vivo, HBI-MC29 transforms macrophages efficiently and induces in vivo a high incidence of lymphoid tumors. Electrophoretic analysis of HBI-MC29 genomic RNA revealed that it has a complexity of 5.7 kilobases, like the RNA of wild-type (wt) MC29, and that it is 0.6 kilobases longer than the 5.1-kilobase RNA of the deletion mutant td10H-MC29. Analysis of the viral RNAs of two clonal isolates of HBI-MC29 by T1 oligonucleotide fingerprinting showed that sequences from the viral transformation-specific region, v-myc, which are deleted in td10H RNA, are present in HBI RNA. Moreover, hybridization of HBI RNA to molecularly cloned subgenomic fragments of wtMC29 proviral DNA, followed by fingerprint analysis of hybridized RNA, showed that the entire v-myc-specific RNA sequences defined previously are present. Hybridization to cloned DNA of the normal chicken locus c-myc shows a close relationship between HBI v-myc RNA and c-myc DNA, especially in the sequences which were deleted from td10H-MC29. T1 oligonucleotide maps of HBI and td10H RNAs were prepared and compared. Total conservation of the oligonucleotide pattern is observed in the overlapping v-myc regions, while the partial structural genes gag and env show some variations, most of which can be directly proven to be due to point mutations or recombination with helper viral RNAs that were analyzed in parallel. Recombination of td10H-MC29 with c-myc, followed by recombinational and mutational changes in the structural genes during passage with helper virus, could be a possible explanation for the origin of HBI.
Expression of adenovirus E1a and E1b gene products and the Escherichia coli XGPRT gene in KB cells
Authors: Babiss L E , Young C S , Fisher P B , Ginsberg H S
Published: January 2020

The recombinant plasmid pSV2-gpt, which contains the Escherichia coli XGPRT gene under the control of a simian virus 40 early promoter, was modified to contain the type 2 adenovirus (Ad2) XhoI-C (0 to 15.5 map units) restriction endonuclease fragment. Plasmid (pLB206) DNA was introduced into human KB cells by Ca2+-mediated DNA transfection, and transformants were selected in medium containing xanthine, aminopterin, and mycophenolic acid, as a consequence of expression of the dominant, selectable XGPRT gene. A series of 13 gpt+ cell lines were isolated and tested for their ability to complement Ad5 deletion mutants in E1a (H5dl312) and E1b (H5dl315). Four classes of gpt+ KB cell lines were identified, including clones constitutively expressing both E1a and E1b, only E1a, or only E1b or not expressing either E1a or E1b. DNA and RNA filter transfer hybridization analysis substantiated the conclusions that those cell lines capable of complementing viral host range mutants contained the appropriate viral DNA sequences and cytoplasmic polyadenylated RNA species. DNA filter transfer hybridization studies also revealed that the transfected vector DNA was stably integrated into chromosomal DNA in the KB transformants and the number of integrated sites ranged from 1 to 3. The gpt+ KB cell line that only expressed E1b gene functions only contained viral E1b gene sequences; those cell lines that expressed neither E1a nor E1b gene function contained only small or no regions of Ad2 DNA. When weaned off the selective medium, transformed KB cell lines stably maintained their inserted DNA in the absence of selective pressure and could easily be adapted to growth in suspension culture.
Isolation of episomal bovine papillomavirus chromatin and identification of a DNase I-hypersensitive region
Authors: Rösl F , Waldeck W , Sauer G
Published: January 2020

The investigation of papillomavirus chromatin has been hampered by the unavailability of a tissue culture system for vegetative growth of these viruses. We have used, therefore, bovine papillomavirus type 1-transformed hamster embryo fibroblasts containing 200 to 250 episomal genome equivalents per cell as a source of viral chromatin. The selectively isolated chromatin was shown to be slightly larger (80S) than the mature simian virus 40 chromatin, which was cosedimented in a sucrose density gradient. Both Fo I and Fo II were present in the bovine papillomavirus type 1 chromatin. A fast-sedimenting fraction, whose structure is still unknown, also contained oligomeric bovine papillomavirus type 1 DNA. By in situ DNase digestion of isolated nuclei and subsequent cleavage of the bovine papillomavirus type 1 DNA with various restriction endonucleases, a major DNase-hypersensitive region was detected in the chromatin. This region, comprising approximately 320 base pairs, is located between the relative physical map positions 0.88 and 0.92.
Stoichiometry of large T antigen and pp53 in complexes isolated from simian virus 40-transformed rat cells
Authors: Freed M I , Lubin I , Simmons D T
Published: January 2020

Simian virus 40-transformed cells synthesize high-molecular-weight protein complexes (22 to 30S) that consist of the virus-coded large T antigen (81,500 daltons) and the cellular antigen pp53. These complexes were partially purified from lysates of transformed rat cells by sucrose velocity sedimentation. The stoichiometry of the two proteins in the complex was studied by direct enzyme-linked immunosorbent assays, using alkaline phosphatase-conjugated anti-T and anti-pp53 monoclonal antibodies. The results from these experiments indicate that the T antigen-to-pp53 ratio in the complex is 0.87 +/- 0.27. No statistically significant differences were found in this ratio for faster- and slower-sedimenting complexes. These results from enzyme-linked immunosorbent assays and previous molecular weight estimates of the complex suggest that this complex is composed, on the average, of four molecules of T antigen and four or five molecules of pp53.
Resistance of bacteriophage H1 to restriction and modification by Bacillus subtilis R
Authors: Bron S , Luxen E , Venema G
Published: January 2020

H1, a 5-hydroxymethyluracil (HMU)-containing Bacillus subtilis bacteriophage, was neither restricted nor modified upon infection of B. subtilis R cells. In vitro, H1 DNA was not restricted by BsuR under standard conditions (200 mM salt), although the expected frequency of -GGCC- cleavage sites was approximately 250. However, four specific sites were cleaved under nonstandard conditions (low salt or high pH) or in the presence of organic solvents, like dimethyl sulfoxide and glycerol. After the substitution of thymine for HMU by DNA cloning in B. subtilis, a BsuR cleavage site was restricted and modified under standard conditions. No additional sites were detected after shotgun-cloning of about 11% of the chromosome. The nucleotide sequence of a cleavage site was found to be 5'. .C-A-Hmu-A-A-C-Hmu-Hmu-Hmu-G-G-C-C-Hmu-A-G-. . .3', which shows the presence of a bona fide BsuR (GGCC) recognition sequence, flanked by (Hmu-A)-rich sequences. The results suggested that the resistance of H1 to restriction and modification by B. subtilis R was due to (i) a strong bias against the GGCC-recognition sequence and (ii) protection of the four remaining GGCC sites as a consequence of HMU-A base pairs flanking the sites.
Provirus of M7 baboon endogenous virus: nucleotide sequence of the gag-pol region
Authors: Tamura T A
Published: January 2020

A 3,023-base nucleotide sequence of the M7 baboon endogenous virus genome, spanning the 5' noncoding region as well as the entire gag gene and part of the pol gene, is reported. Within the 562-base 5' noncoding region, a 21-base sequence complementary to the OH terminus of tRNApro is located immediately downstream from the long terminal repeat. Amino acid sequences were deduced from the 1,596 nucleotides comprising the gag gene, and the four structural gag polypeptides, p12, p15, p30, and p10, appeared to be coded contiguously. Only one termination codon interrupted the M7 gag and pol genes. The data suggest that 55 additional amino acids may be attached to the NH2 terminus of the gag precursor protein. However, such a sequence was not detected in virions or in virus-infected cells. With the exception of the p15 region, nucleotide and amino acid sequences of the gag and pol regions of M7 virus exhibited strong homologies to those of Moloney leukemia virus.
Replication of herpes simplex virus type 1 on hydroxyurea-resistant baby hamster kidney cells
Authors: Leary K , Bratton J , Francke B
Published: January 2020

Hydroxyurea-resistant (HUr) baby hamster kidney cells were isolated, subcloned, and characterized. One clonal line, which contained elevated levels of ribonucleotide reductase, lost its HU resistance during passage in the absence of the inhibitor, whereas another clonal line was stably resistant. The replication of herpes simplex virus type 1 on these cells was compared with that of the parvovirus minute virus of mice. Herpes simplex virus type 1 was found to be as sensitive to HU on both lines of HUr baby hamster kidney cells as it was on parental (HU-sensitive) cells, whereas parvovirus replication was about eight times more resistant on HUr baby hamster kidney cells compared with the parental cells. The results suggest that herpes simplex virus type 1 cannot use the cellular reductase and may code for its own.
DNA replication origin of polyoma virus: early proximal boundary
Authors: Katinka M , Yaniv M
Published: January 2020

We constructed a series of deleted polyoma genomes by Bal 31 nuclease digestion from the unique Bg/I site at nucleotide 86 on the "early" side of the origin of DNA replication. The ability of the cloned deleted genomes to replicate was tested after transfection into mouse 3T6 fibroblasts or into the polyomatransformed C127 (COP5) mouse cell line (Tyndall et al., Nucleic Acids Res. 9:6231-6251, 1981). Deletions up to nucleotide 64-had no effect on the amount of replicated DNA accumulated, but larger deletions, extending up to nucleotide 42, decreased this amount 7- to 10-fold. By nucleotide 38, the quantity of detected DNA was down 100-fold, and by nucleotide 20, no replication could be detected. The minimum origin segment does not contain any known high-affinity, large tumor antigen binding site.
Characterization of RNase H activity associated with reverse transcriptase in simian foamy virus type 1
Authors: Benzair A B , Rhodes-Feuillette A , Emanoil-Ravicovitch R , Peries J
Published: January 2020

Spumavirinae or foamy viruses have been shown to have a characteristic RNA-dependent DNA polymerase activity. We demonstrate here the existence of an RNase H activity that copurifies with the 81-kilodalton monomeric polypeptide, which carries the RNA-dependent DNA polymerase activity of simian foamy virus type 1. RNase H degrades RNA hybrid substrates; however, it does not solubilize single-stranded RNAs. Inactivation assays with heat, high levels of bivalent cations, ethidium bromide, and sodium fluoride suggest that the RNase H catalytic site could be topologically independent from the DNA polymerase catalytic site.
Cloning and cleavage site mapping of DNA from bovine herpesvirus 1 (Cooper strain)
Authors: Mayfield J E , Good P J , VanOort H J , Campbell A R , Reed D E
Published: January 2020

Sequences representative of most of the bovine herpesvirus 1 (Cooper strain) DNa were cloned in the plasmid vector pBR322 at the HindIII site. EcoRI, HpaI, and BamHI restriction endonuclease sites were mapped in each of the cloned fragments, and this information was used to construct a restriction endonuclease cleavage site map of the entire viral genome for the four enzymes.