bluuebunny/crossref_metadata_2025 huggingface dataset:
Virion DNA of ground squirrel hepatitis virus: structural analysis and molecular cloning
Authors: Ganem D , Greenbaum L , Varmus H E
Published: January 2020

The structure of the encapsidated DNA genome of ground squirrel hepatitis virus (GSHV) has been examined by restriction endonuclease cleavage, nucleic acid hybridization, and molecular cloning. GSHV virion DNA is a relaxed circular molecule of approximately 3,200 bases in length; most molecules harbor an extensive single-stranded region which is largely confined to one-half of the genome. The full-length viral DNA strand is covalently bound to protein. The single-stranded region can be repaired in vitro by the action of the endogenous virion polymerase, exogenously added DNA polymerase from avian myeloblastosis virus, or both. Restriction enzyme cleavage of viral DNA from different isolates demonstrated that multiple variants of GSHV exist in nature. The genomes of two such strains have been cloned in Escherichia coli, and their physical maps have been determined. Nucleic acid hybridization studies revealed that the strains share sequence homology with the DNA of human hepatitis B virus. Regions homologous to the coding regions for the surface and core antigens of human hepatitis B virus have been localized on the GSHV chromosome. Molecular cloning experiments have also led to the identification of a region of the viral genome which is altered in a procaryotic host.
Single-stranded regions on unintegrated avian retrovirus DNA
Authors: Hsu T W , Taylor J M
Published: January 2020

Using chromatography on benzoylated naphthoylated DEAE-cellulose, we found that greater than 99.5% of the unintegrated linear viral DNA species detected in quail embryo cells infected with Rous sarcoma virus contained single-stranded regions, even at 16 h after infection. These regions were distributed across the genome and, on average, were primarily of plus-strand DNA. Within most of the linear viral DNA species, the minus strand was interpreted as being of genome size with two copies of the large terminal redundancy, LTR. In contrast, the plus strands in the linear viral DNA species were exclusively subgenomic.
Herpes Simplex Virus Glycoprotein gA/B: Evidence that the Infected Vero Cell Products Comap and Arise by Proteolysis
Authors: Pereira Lenore , Dondero Dale , Roizman Bernard
Published: January 2020

We recently reported (Pereira et al., Proc. Natl. Acad. Sci. U.S.A.
78
:5202-5206, 1981) that herpes simplex virus 1 and 2 glycoproteins, previously designated gA and gB, could not be differentiated by a bank of independently derived type-specific and type-common monoclonal antibodies. We also reported that from lysates of infected Vero cells, all but one monoclonal antibody precipitated gA/B glycoproteins which had faster electrophoretic mobility than the corresponding infected HEp-2 cell glycoproteins and a set of three small polypeptides which we designated g(A + B) reactive polypeptides 1, 2, and 3. Antibody H368, the single exception, failed to react with the gA/B glycoproteins or related antigens accumulating in infected Vero cells. In this paper, we report the following results. (i) The high-apparent-molecular-weight gA/B glycoproteins accumulating in infected HEp-2 cells were cleaved by a proteolytic enzyme contained in Vero cell lysates to yield more rapidly migrating proteins that were indistinguishable from authentic Vero cell gA/B glycoproteins. Like its authentic counterpart, the cleaved gA/B glycoproteins failed to react with H368 monocolonal antibody. In addition, the lysate cleaved HEp-2 cell gA/B glycoproteins into g(A + B) reactive polypeptides 2 and 3. (ii) The proteolytic activity contained in the uninfected cell lysates was inhibited by
N
-α-
p
-tosyl-
l
-lysine chloromethyl ketone and is therefore trypsin-like. (iii) Pulse-chase experiments indicated that the cleavage of gA/B glycoproteins occurred during or soon after translation but that the accumulation of g(A + B) reactive polypeptide 1 was a consequence of a delayed processing event. (iv) Analysis of herpes simplex virus 1 × herpes simplex virus 2 recombinants indicated that the determinants of type-specific immune reactivity and electrophoretic mobility of gA/B glycoproteins and g(A + B) polypeptides map near the right terminus of herpes simplex virus 1
Bam
HI-G.
Poliovirus-induced alterations in HeLa cell membrane functions
Authors: Schaefer A , Kühne J , Zibirre R , Koch G
Published: January 2020

Protein synthesis, amino acid uptake, membrane potential, cell volume, Na+ and K+ levels, and ATPase (Na+,K+ activated; EC 3.6.1.3) activity were investigated in control and poliovirus-infected HeLa cells. Inhibition of protein synthesis was first observed 60 min postinfection and reached a maximum at 120 min. The onset of protein synthesis inhibition coincided with a decrease in cell volume and with an elevation of ATPase activity in isolated HeLa cell membranes. Some 3 h after virus adsorption, ATPase activity was inhibited, the Na+-K+ gradient of the cell collapsed, both membrane potential-dependent tetraphenylphosphonium ion uptake and amino acid uptake were reduced, and the cell volume increased. These results provide further experimental support for the hypothesis that modification of the cell membrane plays an important role in the strategy of cytopathogenic viruses in the shutoff of host metabolism and cell death.
Antiserum specific for the carboxy terminus of the transforming protein of Rous sarcoma virus
Authors: Sefton B M , Walter G
Published: January 2020

An antiserum specific for the carboxy terminus of p60src, the transforming protein of Rous sarcoma virus, was produced by immunization of rabbits with a conjugate of bovine serum albumin and the synthetic peptide NH2-Tyr-Val-Leu-Glu-Val-Ala-Glu-COOH. The carboxy-terminal six amino acids of this peptide correspond in sequence to that deduced for the carboxy terminus of the p60src of the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup A. The p60src proteins of the several strains of Rous sarcoma virus and the cellular homolog of the viral transforming protein, p60c-src, comprise a polymorphic family of polypeptides. The anticarboxy-terminal serum reacted readily with the p60src proteins of three different strains of Rous sarcoma virus. In contrast, no precipitation of cellular p60c-src could be detected with this serum. This suggests that the viral p60src proteins have identical carboxy termini and that the carboxy terminus of cellular p60c-src may be different from that of viral p60src. The anticarboxy-terminal serum reacted poorly with the subpopulation of viral p60src which is present in a complex with two cellular phosphoproteins. Apparently, the presence of the two cellular proteins interferes with the recognition of p60src by the anticarboxy-terminal serum. It seems likely, therefore, that these two cellular proteins bind to the carboxy-terminal domain of p60src.
A phosphorylated basic vaccinia virion polypeptide of molecular weight 11,000 is exposed on the surface of mature particles and interacts with actin-containing cytoskeletal elements
Authors: Hiller G , Weber K
Published: January 2020

A phosphorylated vaccinia virus structural polypeptide of an apparent molecular weight of 11,000 (p11K) was isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used for antibody induction. After purification by antigen affinity chromatography, the immunoglobulins detected only one target of a rather basic nature in two-dimensional immune blotting procedures of total virion proteins. By use of a combination of biological, biochemical, and microscopic techniques, p11K could be located on the surface of those vaccinia virus particles, with "classical" morphology and a buoyant density of 1.27 g/cm3. Upon immunoprecipitation from radioactively labeled infected cells, p11K appeared to be complexed to two additional virus structural proteins, which could be tentatively identified by their molecular weights as precursors for the two major core constituents. When virus assembly was inhibited by rifampin treatment of infected cells, a great part of p11K, either free or in complexed form, was found associated with actin-containing cytoskeletal elements. The ability of p11K to interact with a not-yet-identified, microfilament-associated cellular protein may be related to previous findings showing that assembled vaccinia particles in situ are found in connection with microfilaments. A possible role for the structures precipitated by p11K-specific antibodies in early stages of particle assembly is discussed.
Specific Cleavage at Fibers of a Bacteriophage-Tail-Like Bacteriocin, Pyocin R1, by Successive Treatment with Organomercurial Compounds and Trypsin
Authors: Shimizu Yasuaki , Kumazaki Takashi , Ishii Shin-Ichi
Published: January 2020

When pretreated with the organomercurial compound that reversibly blocks the adhesion of pyocin R1 to sensitive cells, this bacteriocin became susceptible to trypsin. Trypsinization resulted in the exclusive fragmentation of subunit protein no. 2 among 20 different subunit components and the disappearance of distal knobs from the fibers as well as in irreversible inactivation.
Alkylation of T7 bacteriophage blocks superinfection exclusion
Authors: Karska-Wysocki B , Racine J F , Mamet-Bratley M D
Published: January 2020

Alkylation of T7 bacteriophage by methyl methane sulfonate blocked superinfection exclusion. This blockage could be correlated with a delay in the synthesis of phage-specific proteins. Therefore we conclude that protein synthesis directed by the primary infecting phage is required for efficient exclusion of superinfecting phage particles.
Reverse transcriptase from simian foamy virus serotype 1: purification and characterization
Authors: Benzair A B , Rhodes-Feuillette A , Emanoïl-Ravicovitch R , Peries J
Published: January 2020

Chromatography on heparin-Sepharose, known for its affinity for nucleotide-binding polypeptides, was used to purify the viral RNA-dependent DNA polymerase (reverse transcriptase) from the core polypeptides of simian foamy virus type 1. This procedure allowed the recovery of highly purified enzyme with a high specific activity. The average molecular weight of this monomeric enzyme is 81,000 and is thus comparable to that found for other known primate retroviruses. Reverse transcriptase activity of simian foamy virus type 1 requires a ribonucleotide template as a primer or otherwise a DNA with 3'-OH ends. Other optimal conditions of activity are reviewed. Heat inactivation studies led to the concept of an enzyme with two loci, one specific for the substrate and the other for the template-primer.
Caprine Arthritis-Encephalitis Virus Is Distinct from Visna and Progressive Pneumonia Viruses as Measured by Genome Sequence Homology
Authors: Roberson Susan M. , McGuire Travis C. , Klevjer-Anderson Paula , Gorham John R. , Cheevers William P.
Published: January 2020

Competitive inhibition of hybridization between
125
I-labeled caprine arthritis-encephalitis viral RNA and homologous cDNA by heterologous viral RNA shows that the caprine retrovirus shares <20% genome sequence homology with visna and progressive pneumonia viruses. These viruses, however, are indistinguishable in immunodiffusion reactions involving the major structural protein (p28).
Effect of monensin on Mason-Pfizer monkey virus glycoprotein synthesis
Authors: Chatterjee S , Bradac J A , Hunter E
Published: January 2020

The effect of the monovalent carboxylic ionophore monensin on the biosynthesis, intracellular transport, and surface expression of the glycoproteins of Mason-Pfizer monkey virus was examined. Cells treated with monensin at concentrations of 10(-7) or 10(-6) M continued to synthesize virus particles, which from electron microscopic studies appeared to bud normally from the plasma membrane of the cells. However, the particles released had an altered buoyant density in sucrose gradients and were noninfectious. These noninfectious virions had a normal complement of non-glycosylated polypeptides but showed a significantly reduced amount of glycosylated proteins. The gp70 and gp20 polypeptides appeared to be completely absent, and a heterogeneous, higher-molecular-weight protein was observed on the virions instead. Studies on intracellular protein synthesis indicated that the precursor (Pr86env) to gp70 and gp20 is synthesized normally but is not cleaved to the mature proteins. Immunofluorescence studies showed, however, that the uncleaved molecule is expressed on the cell surface. In this system, therefore, Mason-Pfizer monkey virus glycoprotein migration appears to occur in the presence of monensin, whereas the cleavage and insertion of the glycoproteins into virions are inhibited.
DNA-binding properties of a herpes simplex virus immediate early protein
Authors: Freeman M J , Powell K L
Published: January 2020

The herpes simplex virus alpha, or immediate early, protein ICP4 has been shown to be central to the control of the early stages of virus replication. The detailed mechanism of this control is unknown. In this communication we show that purified ICP4 was unable to bind to DNA even though the protein was capable of such activity in a crude extract. Addition of either infected- or uninfected-cell extracts to the purified protein restored its DNA-binding activity. These results suggest that ICP4 binds to DNA only via a component of uninfected cells.
The B95-8 isolate of Epstein-Barr virus arose from an isolate with a standard genome
Authors: Skare J , Edson C , Farley J , Strominger J L
Published: January 2020

Blot hybridization studies revealed that the deletion which characterizes the DNA from the B95-8 strain of Epstein-Barr virus was not present in the virus from which the B95-8 strain was derived (883L). The deletion event must have occurred during establishment of the B95-8 cell line or very soon afterward, since the deletion was present in Epstein-Barr virus DNA from a cell line established with B95-8 virus soon after it became available. The presence of the deletion correlates with decreased expression of the gp220 viral envelope glycoprotein.
Further characterization of a protein kinase from foot-and-mouth disease virus
Authors: Grubman M J
Published: January 2020

Acid disruption of foot-and-mouth disease virus released a protein kinase activity that sedimented at less than 7S. This enzyme was separated into three peaks of activity by ion-exchange and hydroxylapatite chromatography. Analysis of the various enzyme fractions by polyacrylamide gel electrophoresis and silver staining revealed that one of the fractions lacked the major virion structural proteins, but still contained two or three other polypeptides. This enzyme phosphorylated mainly one protein (P17) in an in vitro assay.
Genetic heterogeneity within individual bovine rotavirus isolates
Authors: Sabara M , Deregt D , Babiuk L A , Misra V
Published: January 2020

The genomic RNA patterns of six different bovine rotavirus isolates were analyzed on high-percentage polyacrylamide gels (12.5, 13.6, and 17.5%). In contrast to the RNA patterns exhibited by conventional gel systems, those on the high-percentage gels showed an improvement in segment resolution which consequently aided in the detection of extensive band splitting in these patterns. The ability to clone out various electrophoretically distinct virus subpopulations from each of the six isolates provided an explanation for the band splitting detected by the high-resolution gels. The significance of the coexistence of genetically distinct rotavirus populations within a single host is discussed.
Selective dansylation of M protein within intact influenza virions
Authors: Robertson B H , Bennett J C , Compans R W
Published: January 2020

Exposure of purified influenza virions to [14C]dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride.
Structural features of the herpes simplex virus alpha gene 4, 0, and 27 promoter-regulatory sequences which confer alpha regulation on chimeric thymidine kinase genes
Authors: Mackem S , Roizman B
Published: January 2020

Previous studies have shown that herpes simplex virus genes form three groups, alpha, beta, and gamma, whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Chimeric genes constructed by fusion of the coding and 5' nontranslated leader sequences of the thymidine kinase (TK) gene to the sequences upstream from the site of initiation of transcription of alpha genes 4 and 27 are regulated as alpha genes and are induced in cells converted to TK+ phenotype by infection with TK- virus. In alpha gene 4 (S. Mackem and B. Roizman, Proc. Natl. Acad. Sci. U.S.A. 79:4917-4921, 1982), both the promoter and the regulatory region are separable and movable. The promoter permits expression but not induction when fused to TK in the noncoding leader region of the gene. The regulator, when fused to the promoter of an expressible but noninducible portion of the natural beta TK, renders the gene inducible as an alpha gene; it consists of multiple regulatory units acting cumulatively. In this paper, we report on the precise site of initiation of transcription of alpha gene 0 within the inverted b sequences of the L component of viral DNA. We also report the following. (i) The chimeric gene consisting of the coding and 5' nontranslated leader regions of the TK gene fused to portions of the domain of alpha gene 0 extending largely upstream from the site of initiation of transcription of alpha gene 0 was regulated in the same fashion as the alpha 4- and alpha 27-TK chimeras. The regulatory region in the alpha gene 0 is largely upstream from nucleotide - 140. (ii) The promoter-regulatory regions of alpha genes 0, 4, and 27 share TATA sequences, A + T-rich (consensus) sequences occurring in regulating regions of alpha genes 0 and 4 in more than one copy, and multiple G + C-rich inverted repeats. The relation of these sequences to the function of the promoter-regulatory regions of the alpha genes is discussed.
RNA and protein encoded by MH2 virus: evidence for subgenomic expression of v-myc
Authors: Pachl C , Biegalke B , Linial M
Published: January 2020

MH2 and MC29 are highly related myc-containing avian retroviruses. We found that MH2, unlike MC29, synthesizes a 2.6-kilobase subgenomic mRNA containing myc sequences as well as sequences from the 5' end of the genome. A 57-kilodalton protein containing myc, but not gag, sequences (p57myc) was detected by hybrid selection and in vitro translation of RNA from MH2-transformed cells. Gradient separation of MH2 intracellular RNAs indicated that p57myc is encoded by the subgenomic RNA. A highly oncogenic MH2 virus variant (MH2YS3) (M. Linial, Virology 119:382-391, 1982) was shown to encode only p57myc and not P100, the previously described MH2-encoded polyprotein (Hu et al., Virology, 89:162-178, 1978). Cells transformed by subclones of this virus synthesized predominantly the 2.6-kilobase RNA rather than genomic 5.4-kilobase RNA. These results suggest that only p57myc is required for maintenance of the transformed state after MH2 infection.
Cloned mouse mammary tumor virus DNA exhibits glucocorticoid-dependent expression in simian virus 40-transformed mink cells
Authors: Owen D , Diggelmann H
Published: January 2020

Mink lung epithelial cells were transfected with two cloned mouse mammary tumor virus (MMTV) DNAs, a 9-kilobase clone derived from an unintegrated exogenous viral genome and a 14-kilobase clone containing an integrated endogenous provirus along with cellular flanking sequences. Mink lung cells were chosen because they do not contain endogenous MMTV sequences. On the basis of our observation that simian virus 40 DNA efficiently transforms these cells, we isolated cell clones containing MMTV DNA by using transformation with simian virus 40 DNA as a selective marker in cotransfection experiments. Levels of the 9-kilobase MMTV mRNA representing the entire viral genome and of the spliced 4.4-kilobase mRNA which codes for the viral envelope proteins were glucocorticoid dependent in transformed cells. Expression of low levels of Pr77gag, the precursor of the group-specific viral core proteins, and of gPr73env, the precursor of the viral envelope proteins, was also hormone dependent. We conclude that these cloned MMTV DNAs contain all the information necessary for the synthesis of normal viral RNAs and proteins. These findings also provide further evidence that the DNA sequences involved in the hormone responsiveness of MMTV expression are contained within the viral genome.
Thermostabilities of Virion Activities of Newcastle Disease Virus: Evidence that the Temperature-Sensitive Mutants in Complementation Groups B, BC, and C Have Altered HN Proteins
Authors: Peeples Mark E. , Glickman Rhona L. , Bratt Michael A.
Published: January 2020

Four virion activities of Newcastle disease virus (hemagglutinating, neuraminidase, hemolytic, and infectious activities) were examined before and after heat stress in low-salt buffer and physiological salt buffer (phosphate-buffered saline). The hemagglutinating and neuraminidase activities of the Australia-Victoria wild-type (AV-WT) strain were thermostable at both salt concentrations tested, whereas the thermostabilities of the hemolytic and infectious activities were salt dependent (thermostable in phosphate-buffered saline but not in low-salt buffer). Virions of RNA
+
temperature-sensitive (ts) mutants of AV-WT were tested for the stabilities of the four activities. Some mutants in groups B, BC, and C were as stable as AV-WT in all functions, but others were much less stable in all functions. The unstable mutants in groups B, BC, and C affirmed the assignment of the ts lesions of these mutants to the hemagglutinin/neuraminidase (HN) protein gene because HN function(s) are required for all four activities. The instability of these ts mutants was not related to their decreased virion HN protein content and was not due to physical loss of the HN protein from the virions. Three of four ts
+
plaque-forming revertants of the least stable mutant, BC2, coreverted for stability, confirming that the unstable phenotype is indeed the result of the mutation responsible for the ts phenotype. Group D mutants were approximately as stable as AV-WT in hemagglutinating, neuraminidase, and hemolytic activities; this is consistent with this group representing a lesion in a gene other than the HN protein gene. However, the infectivities of two of the three group D mutants were less stable than the infectivity of AV-WT in low-salt buffer.
Requirement for host transcription in the replication of Sindbis virus
Authors: Baric R S , Carlin L J , Johnston R E
Published: January 2020

Host cell involvement in Sindbis virus (SB) replication was examined in cells which had been treated with either actinomycin D (AMD) or alpha-amanitin (alpha-A). Treatment with these inhibitors of host transcription before infection reduced the ability of cells to support SB growth by 1 to 2 orders of magnitude, while having little or no effect on the replication of vesicular stomatitis virus. SB replication was sensitive to alpha-A in wild-type Chinese hamster ovary (CHO) cells but was resistant to alpha-A in CHOama-1 cells, a line which contains an alpha-A-resistant RNA polymerase II. A mutant of SB, SBamr, was isolated by mutagenesis followed by selection in cells which had been treated with AMD. SBamr grew normally not only in cells treated with AMD but also in alpha-A-treated cells. Our results suggest (i) that the synthesis of cellular mRNA (and presumably protein) is required for replication of SB, (ii) that prior treatment with either drug affects the same aspect of SB replication, and (iii) that mutations in the SB genome allow the virus to overcome the effect of inhibitors of host transcription.
Nucleotide sequence of the 3' end of MCF 247 murine leukemia virus
Authors: Kelly M , Holland C A , Lung M L , Chattopadhyay S K , Lowy D R , Hopkins N H
Published: January 2020

We isolated DNA clones of MCF 247, a leukemogenic, recombinant type C virus obtained from the thymus of an AKR mouse. We determined the nucleotide sequence of the viral long terminal repeat (LTR) and the 3' end of env, and we compared the sequences to corresponding sequences of the genome of Akv virus, the putative ecotropic parent of MCF 247. By analogy with Moloney leukemia virus, we identified the amino terminus of Prp15E, the C-terminal proteolytic cleavage product of env and precursor to mature virion p15E. In MCF 247 the presumptive Prp15E is encoded by a 603-nucleotide open reading frame. The majority of this sequence is identical to that of Akv. However, a recombination event near the 3' end of the Prp15E-coding region introduces nonecotropic sequences into MCF 247, and these extend to the 3' end through the U3 portion of the LTR. The U3 regions of Akv and MCF 247 are about 83% homologous. The R and U5 regions of the LTR of MCF 247 and Akv are identical. Large RNase T1-resistant oligonucleotides analyzed previously in numerous ecotropic and MCF viral genomes were located within the Akv and MCF 247 DNA sequences. The resulting precise T1 oligonucleotide maps of the 3' ends of MCF viral genomes reveal that the biologically defined, leukemogenic class I MCFs isolated from thymic neoplasms of inbred mice all share the sequence pattern seen in MCF 247, a representative of this group; they possess recombinant Prp15E genes and derive U3 from their nonecotropic parents.
Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: oriented and quantized in vitro packaging of DNA protein gp3
Authors: Bjornsti M A , Reilly B E , Anderson D L
Published: January 2020

The assembly of phage phi 29 occurs by a single pathway, and the DNA protein (DNA-gp3) of "packaging intermediates" can be obtained after DNase I interruption of in vitro complementation. A broad spectrum of DNA molecules of variable length was isolated from DNase I-treated proheads. Restriction endonuclease EcoRI digestion and electrophoretic analysis of these DNA molecules suggested that DNA-gp3 packaging was oriented with respect to the physical map and was a complex process. Proteinase K-treated exogenous DNA was not packaged. When exogenous DNA-gp3 was predigested with the restriction endonucleases BstEII. EcoRI, HpaI, and HpaII, the left-end fragments, ranging in size from 8 to 0.9 megadaltons, were selectively and efficiently packaged. During in vivo and in vitro assembly, DNA-gp3 is packaged into proheads, the "core-scaffolding" protein gp7 exits from the particles, and the DNA-filled heads assume the angular morphology of phage phi 29. The packaging of a 4.1-megadalton DNA-gp3 left-end fragment (one third of the genome) resulted in the exit of gp7 and the transition to angularity.
Expression of adenovirus type 12 E1A gene in monkey cells, using a simian virus 40 vector
Authors: Oda K , Kato H , Saito I , Sugano S , Maruyama K , Masuda M , Shiroki K , Shimojo H
Published: January 2020

Simian virus 40 (SV40) recombinants carrying the adenovirus type 12 E1A gene were constructed. The SV40 expression vector was constructed by removing most of the VP1 gene and an internal part of the intervening sequence for late 16S RNA and by joining the 5' and 3' splice sites into a small segment. The adenovirus type 12 E1A gene with or without its own promoter was inserted downstream from the SV40 late promoter and the splicing junctions. The recombinant DNA was propagated and packaged in monkey cells by cotransfection with an early temperature-sensitive mutant (tsA58) DNA as helper. Immunofluorescent staining of the monkey cells infected with the resulting virus stocks showed that up to 20% of the cells overproduced the E1A gene products in the nuclei. Two-dimensional gel electrophoresis of the products indicated that the products were very similar or identical to the authentic polypeptides synthesized in adenovirus type 12-infected human embryo kidney cells. The E1A mRNA was initiated at the SV40 late promoter irrespective of the presence of the E1A promoter and terminated at either the E1A or the SV40 polyadenylation signal. These hybrid mRNAs were correctly spliced in the E1A coding region.
Hyperproduction of adenovirus type 12 E1B gene product in monkey cells, using a simian virus 40 vector
Authors: Oda K , Fukui Y , Saito I , Masuda M , Shiroki K , Shimojo H
Published: January 2020

Simian virus 40 recombinant DNAs carrying the adenovirus type 12 E1B gene were constructed, propagated, and packaged in monkey cells. Monkey cells infected with the resulting virus stocks hyperproduced the E1B gene products in more than 80% of the cells as revealed by immunofluorescence. The products were distributed in both the nuclei and the cytoplasm, and a condensed form of fleck structure was observed in the cytoplasm. Polyacrylamide gel electrophoresis of the cell extracts and their immunoprecipitates detected the E1B-coded 19,000-molecular-weight protein but not the 50,000-molecular-weight protein. The 19,000-molecular-weight protein and the simian virus 40 VP1 protein were synthesized in nearly equal amounts.
Subcellular localization of the simian virus 40 agnoprotein
Authors: Nomura S , Khoury G , Jay G
Published: January 2020

The intracellular distribution of the simian virus 40 agnoprotein in infected cells was determined by indirect immunofluorescence and biochemical fractionation followed by indirect immunoprecipitation. The specific antibodies used in these studies were directed against either purified agnoprotein or a synthetic oligopeptide homologous to the N-terminus of the processed protein. Both procedures showed predominant localization of the agnoprotein to the cytosol and to the perinuclear region in association with the outer nuclear membrane. A minor but detectable fraction of the protein was also found in the nucleus. The definition of its subcellular distribution, as well as its high lability in vivo and affinity for nucleic acid, provide a basis for speculation on the function of this gene product.
Cycloheximide stimulates early adenovirus transcription if early gene expression is allowed before treatment
Authors: Cross F R , Darnell J E
Published: January 2020

The inhibition of protein synthesis by cycloheximide markedly stimulated adenovirus 2 early transcription (assayed in vivo and in vitro in nuclei) when the drug was added 3 h after infection of HeLa cells. The stimulation was not uniform but ranged from three- to eightfold for region 1A to 10- to 30-fold for region 2 of the adenovirus genome. The increase was complete by about 60 min after treatment. The stimulation reversed rapidly after cycloheximide was removed. Treatment with cycloheximide either before infection with wild-type adenovirus type 5 or at 3 h after infection with dl312, a mutant which fails to express early gene products in HeLa cells, both resulted in levels of transcription at or substantially below the control (infection with wild-type adenovirus without treatment). Therefore we conclude that cycloheximide treatment and previous early gene expression interact to yield the maximum levels of transcription.
Kinetics of expression of infectious ecotropic, xenotropic, and mink cell focus-forming murine leukemia virus after 5-iododeoxyuridine induction of cells from high- and low-leukemia mouse strains
Authors: Rapp U R
Published: January 2020

Endogenous murine leukemia virus (MuLV) was induced with 5-iododeoxyuridine (IdUrd) from the high-leukemia mouse strain AKR and from two low-leukemia strains, C3H/He and BALB/c. A virus-free cell line from strain AKR readily gave rise to infectious, XC-positive MuLV upon treatment with IdUrd, whereas cells from strains C3H/He and BALB/c produced replication-deficient, XC-negative MuLV. IdUrd-induced cells also produced xenotropic and mink cell focus-forming MuLV. Xenotropic virus emerged at a higher titer from both AKR and BALB/c cells during two discrete time intervals, first at day 3 after induction and a second time during spread of the induced ecotropic MuLV. Xenotropic and mink cell focus-forming MuLVs were also produced by IdUrd-induced C3H/He cells but required another round of infection in Sc-1 cells for detection. The in vitro infectivity of endogenous ecotropic MuLV isolated by IdUrd induction from C3H/He cells correlated with pathogenicity in the Fv-1-compatible, leukemia-negative mouse strain NFS/N. Thus, the virulence of endogenous ecotropic MuLV may be an important factor in determining the leukemia incidence in these inbred strains of mice.
Expression of a recombinant DNA gene coding for the vesicular stomatitis virus nucleocapsid protein
Authors: Sprague J , Condra J H , Arnheiter H , Lazzarini R A
Published: January 2020

A cDNA clone containing the entire vesicular stomatitis virus nucleocapsid gene was assembled by fusing portions of two partial clones. When the cDNA clone was inserted into a new general-purpose eucaryotic expression vector and introduced into appropriate host cells, abundant N-protein synthesis ensued. The expressed protein was indistinguishable from authentic N protein produced during vesicular stomatitis virus infections. The recombinant N protein was recognized by a polyclonal antibody and two different monoclonal antibodies and could not be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from authentic N. Our results suggest that the recombinant N protein produced in transfected cells rapidly aggregates into high-molecular-weight complexes in the absence of vesicular stomatitis virus genomic RNA.
Cytoplasmic localization of the transforming protein of Fujinami sarcoma virus: salt-sensitive association with subcellular components
Authors: Feldman R A , Wang E , Hanafusa H
Published: January 2020

Fujinami sarcoma virus (FSV) encodes a transforming protein of 130,000 daltons (P130) which is associated with a tyrosine-specific protein kinase activity. To elucidate mechanisms involved in cell transformation by FSV, we have studied the intracellular location of P130 in rat cells nonproductively infected with FSV. Immunofluorescent staining of several FSV-transformed rat cell lines with a tumor regressor antiserum specific against the fps sequences of P130 showed that the major staining was localized in the cytoplasm. Staining was also seen in cell ruffles and in some cases at areas of cell contact. The cytoplasmic location of P130 staining in cells infected with temperature-sensitive mutants of FSV was unchanged when they were grown at permissive or nonpermissive temperature. Cell fractionation of FSV-transformed cells under various conditions showed that the ionic strength used during cell fractionation had a striking effect on the distribution of P130. At 10 mM NaCl, 70% of P130 sedimented in the large granule fraction, whereas at 500 mM NaCl 70 to 90% of P130 was recovered in the cytosol fraction. Furthermore, a combination of ionic and nonionic detergents that effectively solubilized subcellular membranes was insufficient to solubilize P130 unless the salt concentration was raised. We conclude that the majority of P130 and its associated protein kinase activity are localized in the cytoplasm and that P130 is not an integral membrane protein.
Production and characterization of monoclonal antibodies against avian retrovirus reverse transcriptase
Authors: Weis J , Smith D , Burns N , Furcht L , Faras A
Published: January 2020

Monoclonal antibodies were prepared against the avian myeloblastosis virus reverse transcriptase. These monoclonal antibodies specifically immunoprecipitated the alpha and beta subunits of the reverse transcriptase molecule, as well as the Pr180gag-pol precursor protein present in virus-infected cells. In addition, these monoclonal antibodies inhibited the DNA polymerase activity associated with the reverse transcriptase molecule but not the RNase H activity. The monoclonal antibody preparations were specific for the amino-terminal portion of the protein, as determined by the immunoprecipitation of a reverse transcriptase-beta-galactosidase fusion protein produced in Escherichia coli by molecular cloning procedures.
Mutant Feline Sarcoma Proviruses Containing the Viral Oncogene (v-
fes
) and Either Feline or Murine Control Elements
Authors: Even Jos , Anderson Soni J. , Hampe Annie , Galibert Francis , Lowy Douglas , Khoury George , Sherr Charles J.
Published: January 2020

The sequences required for transformation by the Gardner-Arnstein (GA) strain of feline sarcoma virus (GA-FeSV) were defined by site-directed, in vitro mutagenesis of molecularly cloned proviral DNA. Portions of the Ga-FeSV provirus, subcloned in the plasmid pBR322, were mutagenized by deletion or frameshift at
Xho
I restriction sites flanking the nucleotide sequences presumed to encode the GA-FeSV transforming polyprotein (P108

gag-fes

). The biological activity of subgenomic and reconstructed full-genome-length molecules was assayed by transfection and focus induction in NIH 3T3 cells. Both mutant and wild-type molecules containing the intact P108

gag-fes

coding region induced foci of transformed cells at efficiencies between 10
4
and 10
5
focus-forming units per pmol of DNA; a deletion mutant lacking 3′-terminal v-
fes
sequences was completely nontransforming in parallel assays. Representative subcloned foci of transformed NIH 3T3 cells synthesized P108

gag-fes

with associated in vitro protein kinase activity. Focus-forming viruses could be rescued from transformed subclones induced by full-length proviral DNA, but not from cells transformed by subgenomic DNA lacking a 3′ long terminal repeat (LTR). It was concluded that: (i) nucleotide sequences encoding P108

gag-fes

and its associated kinase activity are responsible for transformation, (ii) the GA-FeSV 3′
env
and LTR sequences are not required for focus induction, and (iii) the 3′ LTR is necessary for rescue of infectious FeSV RNA. A chimeric DNA containing the 5′ LTR and P108

gag-fes

coding region of GA-FeSV joined to the 3′ LTR of Moloney murine sarcoma virus was both transforming and rescuable at high efficiency. Restriction analysis showed that passaged stocks of rescued transforming virus contained Moloney murine sarcoma virus U3 sequences at both proviral DNA termini, consistent with generally accepted models for LTR formation during reverse transcription. Wild-type GA-FeSV and the chimeric virus (here designated as GAHT), each rescued from NIH 3T3 cells with the same amphotropic murine leukemia virus, yielded approximately equal numbers of foci when titrated on CCL 64 mink cells. By contrast, on mouse NIH 3T3 cells, the focus-forming titer of GAHT was 1 to 2 log higher than that of FeSV. The foci induced on NIH 3T3 cells by GAHT appeared earlier and were reproducibly larger than those induced by GA-FeSV. Differences in transforming activity on NIH 3T3 cells were also found using colony formation in agar, showing that the more rapid appearance and larger size of foci formed in liquid media were not due to virus spread. These data suggest that transcriptional control signals within the viral LTR regulate the levels of the transforming gene product in a species-specific manner.
Conformational changes in Sindbis virus envelope proteins accompanying exposure to low pH
Authors: Edwards J , Mann E , Brown D T
Published: January 2020

The attachment of high multiplicities of Sindbis virus to tissue-cultured cells followed by brief treatment at low pH has been shown to produce cell fusion (fusion from without). In this report, experiments to determine the effects of low pH on the physical and biological properties of Sindbis virus are described. Exposure of purified Sindbis virions to mildly acidic conditions resulted in a rapid and irreversible alteration in particle density and sedimentation characteristics, followed by a slower loss of infectivity. Infectivity was not restored by a return to neutral pH; rather, the loss of virus infectivity seemed to be initiated by exposure to low pH but continued at neutral pH. The formation of a virus-cell complex in which virions were attached to the cell surface protected the particles from low-pH inactivation, although low pH could still expose virus functions responsible for cell fusion. Low pH was found to induce a conformational change in the E2 polypeptide of the intact virion. These results are discussed with respect to the process of Sindbis virus infection of tissue-cultured cells.
Particles of Reduced Infectivity and Deficient in Envelope Glycoproteins Are Produced in Cycloleucine-Treated B77 Avian Sarcoma Virus-Infected Chicken Embryo Fibroblasts
Authors: Stoltzfus C. Martin , Dane Richard W.
Published: January 2020

We have previously shown that the inhibition of methylation reactions by the treatment of B77 avian sarcoma virus-infected cells with medium containing cycloleucine results in an inhibition in the intracellular accumulation of the spliced subgenomic mRNA for the virion envelope protein precursor, whereas the genome-size RNA accumulates in larger than normal amounts (C. M. Stoltzfus and R. W. Dane, J. Virol.
42
:918-931, 1982). To measure the production of virus particles, we have now determined the reverse transcriptase activity in the culture fluid from infected cells treated with various concentrations of cycloleucine. The activity was somewhat greater in the fluid from the cycloleucine-treated cells than it was in the fluid from the control cells, suggesting an enhancement of particle production in the presence of cycloleucine. In contrast, the production of infectious virions, as determined by the focus assay, decreased when the cycloleucine concentration of the medium increased. We determined the polypeptide compositions of purified particles produced from infected cells treated with or without cycloleucine and labeled with [
3
H]leucine. The relative amounts of radioactivity associated with p19 and p27 were approximately the same in all of the preparations. In contrast, significant decreases were observed in the relative amounts of [
3
H]leucine radioactivity associated with the virion glycoproteins gp85 and gp37. The extent of the decrease in the ratio of gp85 to p27 was a function of the cycloleucine concentration and correlated well with the decrease in the infectivity of the virus particles. Therefore, it is probable that the observed reduction of specific infectivity results from the reduced amounts of envelope glycoproteins in the particles budding from cycloleucine-treated cells.
Characterization of the Aleutian disease virus genome and its intracellular forms
Authors: Bloom M E , Mayer L W , Garon C F
Published: January 2020

Aleutian disease virus (ADV) of mink is a nondefective parvovirus with a single-stranded DNA genome. We characterized the viral DNA forms found in infected cells prepared by a modified Hirt extraction procedure. Double-stranded DNA molecules corresponding in size to 4.8-kilobase-pair duplex monomers and 9.6-kilobase-pair duplex dimers were identified in agarose gels by blot hybridization to 32P-labeled ADV DNA. A rapidly reannealing ADV duplex monomer was isolated on a preparative scale and physically mapped with a series of restriction endonucleases. The map derived was similar to one derived from double-stranded ADV DNA produced by self-primed synthesis on virion DNA, but differed from restriction endonuclease maps reported for other parvovirus DNAs. The purified duplex monomer could be labeled with [32P]dCTP by nick translation and used as a probe in blot hybridization to detect ADV sequences in DNA from small numbers of infected cells. Additional studies indicated that double-stranded ADV DNA could first be detected at 24 h after infection.
Flat revertants of temperature-insensitive transformants induced by simian virus 40 tsA mutants lose their ability to express T-antigen
Authors: Chepelinsky A B , Chiu N H , Zannis-Hadjopoulos M , Wang S S , Martin R G
Published: January 2020

Temperature-insensitive transformants that contained simian virus 40 sequences at only one or a few sites in the rat chromosome and that were induced by a temperature-sensitive A gene mutant of simian virus 40 were used to select flat revertants (revertants that had lost the transformed phenotype). The isolation was performed at the nonpermissive temperature so as not to select against temperature-sensitive transformants. Nonetheless, all of the revertants examined had lost their ability to express the T-antigen at both temperatures, and all contained rearrangements of the integrated simian virus 40 sequences. These results are most compatible with the hypothesis that the T-antigen of simian virus 40 is required for the maintenance of the transformed state even in temperature-insensitive cell lines.
Dimethyl sulfoxide affects the amount of extrachromosomal spleen focus-forming virus DNA in murine erythroleukemia cells
Authors: Kern F G , Axelrod D E
Published: January 2020

We used Southern blot hybridization to titrate and map restriction enzyme cleavage sites of a 6.3-kilobase-pair species of extrachromosomal viral DNA found in derivatives of the 745A line of murine erythroleukemia cells, which vary in their ability to be induced to differentiate by dimethyl sulfoxide (DMSO). Greater than an eightfold variation was observed in the amount of this DNA, with the largest amounts being found in cells that were resistant to the induction of differentiation by DMSO. This increase in the level of extrachromosomal viral DNA was found to be dependent upon the continued presence of DMSO in the culture medium. The increase was shown not to be due to an immediate stimulatory effect of this agent on the synthesis or maintenance of this DNA, since cell lines sensitive to the differentiation-inducing effects of DMSO were shown to undergo a transient reduction in the amount of extrachromosomal viral DNA after the addition of DMSO to the culture medium. In addition to the 6.3-kilobase-pair linear form found in the cytoplasm, in some preparations two hybridizing bands were observed that migrated in agarose gels in the position expected of covalently closed circular species of viral DNA. Restriction enzyme mapping of the cytoplasmic linear form indicated a close relationship of this DNA to two polycythemic strains of spleen focus-forming virus that have been molecularly cloned by other workers. No obvious change in the number or arrangement of chromosomal viral sequences could be detected after treating cells with DMSO. Thus, the exposure of murine erythroleukemia cells to DMSO caused an obvious change in the amount of extrachromosomal spleen focus-forming virus DNA but no obvious change in the integration of the provirus.