bluuebunny/crossref_metadata_2025 huggingface dataset:
Mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome
Authors: Wittek R , Hänggi M , Hiller G
Published: January 2020

Cell-free translation of total RNA isolated from vaccinia virus-infected cells late in infection results in a complex mixture of polypeptides. A monospecific antibody directed against one of the major structural proteins of the virus particle immunoprecipitated a single polypeptide with a molecular weight of 11,000 (11K) from this mixture. Immunoprecipitation was therefore used to identify the structural polypeptide among the in vitro translation products of RNA purified by hybridization selection to restriction fragments of the vaccinia virus genome. This allowed us to map the mRNA coding for the 11K polypeptide to the extreme left-hand end of the HindIII E fragment. Detailed transcriptional mapping of this region of the genome by nuclease S1 analysis revealed the presence of a late RNA transcribed from the rightward-reading strand. Its 5' end mapped at ca. 130 base pairs to the left of the HindIII site at the junction between the HindIII F and E fragments. The map position of this RNA coincided precisely with the map position of the late message coding for the 11K polypeptide.
Identification of immunologically cross-reactive proteins of Sindbis virus: evidence for unique conformation of E1 glycoprotein from infected cells
Authors: Wolcott J A , Wust C J , Brown A
Published: January 2020

Hyperimmune antisera to purified Sindbis (SIN) or Semliki Forest (SF) virus were used to identify alphavirus-specific and cross-reactive proteins in virions and infected cells. The hyperimmune sera participated in homologous and cross-cytolysis of alphavirus-infected cells, and the use of monospecific antisera to SIN structural proteins suggested that E1 and E2 could serve as target proteins in cytolysis. Proteins from purified virions or infected cells were extracted with Nonidet P-40, denatured by procedures for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose solid supports, and reacted with hyperimmune sera and 125I-labeled protein A (immunoblotting on denatured proteins). Alternatively, native proteins extracted by mild Nonidet P-40 treatment were precipitated with hyperimmune sera before denaturation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After immunoblotting, homologous antiserum reacted with the virus structural proteins E1, E2, capsid extracted from purified virions, and the counterparts of these proteins extracted from infected cells. In addition, PE2 and a 92,000-molecular-weight protein from infected cells reacted with homologous antiserum. These proteins were also immunoprecipitated with homologous antiserum. After immunoblotting, the Sindbis capsid protein was shown to be cross-reactive whether derived from purified virions or from infected cells; no cross-reactivity was observed with PE2 or E2 from either source, and the E1 glycoprotein was shown to be cross-reactive only when obtained from virions. However, the E1 glycoprotein could be cross-immunoprecipitated from infected cells (as well as from disrupted virions), and, in addition, capsid and a 92,000-molecular-weight protein were cross-immunoprecipitated from infected cells. These results suggest that a native conformation of the cell-associated E1 glycoproteins may be required for immunological cross-reactivity (immune precipitation), whereas virion but not cell-associated E1 retains immunological cross-reactivity after denaturation (immunoblot technique). The findings extend our previously published evidence which suggested that alphavirus maturation is accompanied by a change in immunological cross-reactivity with respect to E1.
Nucleotide sequence of noncoding regions in Rous-associated virus-2: comparisons delineate conserved regions important in replication and oncogenesis
Authors: Bizub D , Katz R A , Skalka A M
Published: January 2020

The nucleotide sequence of the regions flanking the long terminal repeat of Rous-associated virus-2 has been determined. The region analyzed spans the ends of the viral genome and includes the terminus of the env gene, the 3' noncoding region, the 5' noncoding region, and the beginning of the gag gene. These data have been compared with sequences available from other avian retroviruses. The comparisons reveal sections which are highly conserved and others which are quite variable. Sequence homologies within the conserved regions suggest details concerning the mode of origin of the src-transducing viruses. Included in the variable section is a region (XSR) found only in certain strains of Rous-derived virus. Its absence from other oncogenic viruses indicates that these sequences are not required to elicit disease.
Molecular cloning of covalently closed circular DNA of bovine leukemia virus
Authors: Kashmiri S V , Mehdi R , Ferrer J F
Published: January 2020

The two species of covalently closed circular DNA molecules of bovine leukemia virus were cloned in the lambda phage vector lambda gtWES X lambda B. Of the nine independent recombinant lambda-bovine leukemia virus clones that were analyzed, three were derived from the small and six were derived from the large circular molecules carrying, respectively, one and two copies of the long terminal repeat sequences. Comprehensive restriction endonuclease mapping of the unintegrated bovine leukemia virus and the cloned DNA molecules showed that eight of the nine clones carried viral information without any detectable deletions or insertions of more than ca. 50 base pairs. One of the nine clones, which carries a retroviral insert with one copy of the long terminal repeat, had a deletion of ca. 150 base pairs.
Further genetic localization of the transforming sequences of the p21 v-ras gene of Harvey murine sarcoma virus
Authors: Willumsen B M , Ellis R W , Scolnick E M , Lowy D R
Published: January 2020

The sequences encoding the 21-kilodalton transforming protein (p21 ras) of Harvey murine sarcoma virus have previously been localized genetically to a 1.3-kilobase segment of the viral DNA (E. H. Chang, R. W. Ellis, E. M. Scolnick, and D. R. Lowy, Science 210:1249-1251, 1980). Within this segment, DNA sequence analysis has found a single open reading frame large enough to encode the viral p21 (R. Dhar, R. W. Ellis, T. Y. Shih, S. Oroszlan, B. Shapiro, J. Maizel, D. Lowy, and E. M. Scolnick, Science 217:934-937, 1982). There are three potential in-frame ATG initiation codons at the 5' end of this open reading frame. By constructing a mutant of Harvey murine sarcoma virus DNA from which the first two ATG codons of this open reading frame have been deleted, we now show by transfection of the mutant viral DNA into NIH 3T3 cells that only the third ATG codon is necessary and sufficient for synthesis of the viral p21 and for cellular transformation.
Identification with monoclonal antibodies of glycoproteins of Marek's disease virus and herpesvirus of turkeys related to virus neutralization
Authors: Ikuta K , Ueda S , Kato S , Hirai K
Published: January 2020

By use of monoclonal antibodies cross-reactive with Marek's disease virus and herpesvirus of turkeys, three glycoproteins (for Marek's disease virus, gp115/110, gp63, and gp50; for herpesvirus of turkeys, gp115, gp62 and gp52) related to virus neutralization were identified. Immunization of chickens or rabbits with these glycoproteins purified by affinity chromatography resulted in production of neutralizing antibodies.
Oligomerization of simian virus 40 large T antigen is not necessarily repressed by temperature-sensitive A gene lesions
Authors: Montenarh M , Kohler M , Henning R
Published: January 2020

Simian virus 40 large T antigen is a multifunctional protein which exists in different molecular weight forms. According to several reports, T antigen encoded by temperature-sensitive simian virus 40 A locus mutants (tsA) is unable to oligomerize into high-molecular-weight species. To try to correlate structural and functional properties, we selected tsA58 and tsA1499, both of which are heat sensitive for lytic growth, but only tsA58 is heat sensitive for transformation. Here we report that at permissive and nonpermissive temperatures, T antigen from tsA1499-infected monkey cells retained the ability to oligomerize, whereas reported previously, tsA58 T antigen failed to oligomerize at the nonpermissive temperature. Furthermore, we studied the formation of complexes between T antigen and the cellular p53 protein (T-p53) late in infection. Corresponding to its heat-stable oligomerization properties, T antigen encoded by tsA1499 formed T-p53 complexes regardless of temperature. In contrast, tsA58 encoded T-p53 complexes, preformed at the permissive temperature, remained heat stable after shifting up to the nonpermissive temperature; but at this temperature no new T-p53 complexes arose. The mutants did not replicate viral DNA at the nonpermissive temperature, suggesting that neither the oligomerization of T antigen nor the formation of T-p53 complexes seems to be sufficient for viral DNA replication or for the expression of late viral proteins.
Studies with Bacteriophage φII. Events Following Infection of Male and Female Derivatives of
Escherichia coli
K-12
Authors: Linial Maxine , Malamy Michael H.
Published: January 2020

We studied the course of infection of the female-specific bacteriophage φII in male and female cells isogenic except for the presence of the substituted sex factor, F′lac. Both male and female cells are killed by φII; however, only limited phage replication occurs in male cells. Host macromolecular synthesis stops abruptly at 4 to 6 min after infection of male cells, and synthesis of phage components cannot be detected. Experiments with chloramphenicol indicate that phage deoxyribonucleic acid (DNA) penetrates into male cells, since protein synthesis after infection is required to stop synthesis of DNA in males. Phage DNA becomes membrane-associated in both female and male cells. In male cells, parental phage DNA does not dissociate from the membrane during the latent period as is the case with females, indicating a block in phage DNA replication. Isolation of nonrestricting F′lac mutations indicates involvement of a specific episome product in φII restriction.
Natural Occurrence of Cross-Linked Vaccinia Virus Deoxyribonucleic Acid
Authors: Berns K. I. , Silverman C.
Published: January 2020

The molecular weight of native vaccinia deoxyribonucleic acid (DNA) is 1 to 1.17 times that of native T4 DNA. Sedimentation of denatured vaccinia DNA through alkaline sucrose gradients yields an apparent molecular weight greater than twice that of denatured T4 DNA, implying that the complementary strands of vaccinia DNA do not separate upon denaturation. When alkali-denatured vaccinia DNA is neutralized, it has the physical chemical properties of native DNA when tested by sedimentation through neutral sucrose gradients, banding in CsCl, and by hydroxylapatite chromatography. We conclude that almost all mature vaccinia DNA molecules contain a small number of naturally occurring cross-links.
Synthesis, Transport, and Morphogenesis of Type 5 Adenovirus Capsid Proteins
Authors: Velicer L. F. , Ginsberg H. S.
Published: January 2020

During the period between 20 and 24 hr after infection of KB cells with type 5 adenovirus, at a time when approximately 85% of the proteins made were virus-specific, viral proteins were synthesized on polyribosomes with an average sedimentation coefficient of 200
S
. The polypeptide chains synthesized during a 1-min period of labeling with
14
C-amino acids had an average sedimentation coefficient of 3.4
S
in sucrose gradients containing 1% sodium dodecyl sulfate. Within 1 min after completion, the newly made polypeptide chains were released from polyribosomes, and the majority were transported into the nuclei within 6 min. Meanwhile, the immunological reactivity of the newly synthesized proteins also increased rapidly. During the same 6-min interval after synthesis, the single polypeptide chains assembled into multimeric proteins with average sedimentation coefficients of 6
S
, 9
S
, and 12
S
. The 6
S
and 12
S
proteins were identified immunologically as the fiber and hexon capsid proteins, respectively. The 9
S
protein was trypsin-sensitive and appeared to be the precursor of the penton; it was tentatively identified as the penton base. The penton had a sedimentation coefficient of about 10.5
S
and sedimented with the hexon in sucrose gradients. The concomitant migration of nascent proteins into the nuclei, development of the capsid proteins' immunological reactivity, and morphogenesis of the multimeric capsid proteins suggest that the single polypeptide chains or small complexes were transported into the nuclei where they assembled into mature structural proteins of the virion.
Molecular Recombination in T4 Bacteriophage Deoxyribonucleic Acid
Authors: Miller Robert C. , Kozinski Andrzej W. , Litwin Samuel
Published: January 2020

Evidence was presented to support the hypothesis that long single strands appearing at late times (15 min after infection) are produced as a result of recombination and not as a continuous elongation during the replication process. The production of long strands does not depend on the multiplicity of infection, and the first long strands appear at the time when 20 to 50 phage equivalent units of deoxyribonucleic (DNA) are synthesized, and not earlier. The addition of chloramphenicol at 5 min, which prevents molecular recombination but allows replication of DNA, prevents the formation of long, single strands. Chloramphenicol added between 8 and 10 min after infection, a time at which molecular recombination is fully expressed and covalent repair of recombinant molecules is allowed, does not prevent formation of long single strands. Cutting of single-strand DNA with a limited amount of endonuclease I allows confirmation that the fast-sedimenting characteristic of intracellular denatured DNA is caused primarily by the length of the strands, and not by the formation of aggregates. The computer simulation of two recombination models indicates the feasibility of random breakage and rejoining of molecules in generating long concatenates.
Nucleic Acid of Rubella Virus and Its Replication in Hamster Kidney Cells
Authors: Sedwick W. David , Sokol Frantisek
Published: January 2020

Ribonucleic acid (RNA) has been isolated from partially purified rubella virus preparations and fractionated by rate zonal centrifugation in sucrose density gradients. The bulk of the RNA sedimented as a sharp band with a sedimentation coefficient of 38
S
. Rubella virus RNA appears to be single-stranded on the basis of its sensitivity to the degrading action of ribonuclease. Fractionation by precipitation with 1
m
NaCl, followed by chromatography on cellulose columns, and by rate zonal centrifugation in sucrose density gradients of labeled RNA isolated from actinomycin D-treated and infected baby hamster kidney cells revealed the presence of the following virus-specific types of RNA: (i) single-stranded RNA with a heterogeneous sedimentation pattern, the 38
S
viral RNA becoming the predominant species only after long periods of labeling late after infection; (ii) double-stranded RNA with a sedimentation coefficient of 20
S
; (iii) RNA apparently composed of 20
S
double-stranded RNA and single-stranded branches. On the basis of their properties, the last two species were tentatively identified as the replicative form and the replicative intermediate of rubella virus RNA. Rubella virus RNA was infectious.
Evidence for a Single-Stranded Adenovirus-Associated Virus Genome: Isolation and Separation of Complementary Single Strands
Authors: Berns K. I. , Rose J. A.
Published: January 2020

Single-stranded adenovirus-associated virus type 2 deoxyribonucleic acid (AAV-2 DNA) has been isolated from the virion after enzymatic pretreatment of the particles by heating at 53 C for 1 hr in 0.015
m
NaCl plus 0.0015
m
sodium citrate in the presence of 1% sodium dodecyl sulfate. Double-stranded AAV-2 DNA present as a marker is not denatured by this treatment. AAV-2 single-stranded DNA is composed of two complementary species which can be separated in neutral CsCl when 5-bromodeoxyuridine has been substituted for thymidine in the DNA. The present report is the first documented instance of the separation of complementary strands of an animal virus DNA.
Ribonuclease Sensitivity of Semliki Forest Virus Nucleocapsids
Authors: Acheson Nicholas H. , Tamm Igor
Published: January 2020

Treatment of Semliki Forest virus nucleocapsids with pancreatic ribonuclease (1 μg/ml, 37 C) digests the ribonucleic acid to acid-soluble fragments; the nucleocapsid protein forms a rapidly sedimenting aggregate.
D,L-alpha-difluoromethylornithine inhibits human cytomegalovirus replication
Authors: Gibson W , van Breemen R , Fields A , LaFemina R , Irmiere A
Published: January 2020

D,L-alpha-Difluoromethylornithine (DFMO) is an inhibitor of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway. Exposure of human foreskin fibroblast cells to DFMO before their infection with human strains of cytomegalovirus (CMV) resulted in a reduction in the amount of infectious virus produced. A 3-day exposure to the drug was required to elicit maximal antiviral effect. Cells exposed to DFMO at the time of infection produced normal amounts of infectious virus. Preexposure to the drug for 1, 2, or 3 days before infection resulted in at least 10-, 100-, or 1,000-fold decreases, respectively, in the amount of infectious virus produced. This decrease paralleled the loss of intracellular spermidine and was partially spared by the addition of exogenous putrescine, spermidine, or spermine (10 microM). When added 3 days before infection, DFMO depressed production of herpes simplex virus and simian CMV, as well as wild-type and laboratory prototype strains of human CMV. Although some antiviral effect was observed at a drug concentration of 1 mM, 10 mM gave a stronger effect and was the amount routinely used. At 30 mM DFMO, growth of noninfected cells was slowed but not arrested. Studies to investigate the level at which DFMO interferes with CMV replication showed that DFMO-treated, infected cells (i) exhibit a typical CMV-specific cytopathic effect, (ii) synthesize both viral proteins and viral DNA, (iii) contain at least some capsid forms, and (iv) shed greatly reduced amounts of virus particles into the growth medium. Since CMV virions, like those of herpes simplex virus, contain the polyamines spermidine and spermine, and since DFMO essentially eliminates the pool of intracellular spermidine, the possibility is suggested that this drug may exert its antiviral effect by interfering with virus assembly, perhaps at the level of DNA packaging or capsid envelopment or both.
An oligomeric form of simian virus 40 large T-antigen is immunologically related to the cellular tumor antigen p53
Authors: Leppard K N , Crawford L V
Published: January 2020

The cellular tumor antigen p53 is bound to the simian virus 40 (SV40) large T-antigen in SV40-infected and -transformed cells. As a result, p53 can in general be immunoprecipitated by either monoclonal or polyclonal antibodies that react with large T-antigen. Despite extensive immunological characterization of both these antigens, they have not previously been found to share any antigenic determinants. We have isolated several monoclonal antibodies that bind to the human p53 protein (K. Leppard and L. V. Crawford, EMBO J. 2:1457-1464, 1983) and show here that antibody PAb1104 has distinct, intrinsic activities towards both p53 and SV40 large T-antigen. Only a subset of T-antigen is bound by PAb1104. This subset is an oligomeric form of T-antigen, as judged by its sedimentation velocity in sucrose. In contrast, all of the detectable p53 carries the PAb1104-reactive determinant. The detection of a chance cross-reactivity between two antigens that are already well characterized and which associate with one another in vivo is highly unlikely. It is possible therefore that the element of structural similarity between large T and p53 that is implied by our results has some genuine functional significance.
Effect of papaverine treatment on replication of measles virus in human neural and nonneural cells
Authors: Yoshikawa Y , Yamanouchi K
Published: January 2020

The replication of measles virus in human neural and nonneural cell lines in terms of growth and cytopathic effect was affected by treatment of the cells with papaverine, which increases endogenous cyclic AMP. Suppression of virus growth was most prominent in neuroblastoma cells, followed by that in epidermoid carcinoma and glioblastoma cells, whereas the suppressive effect was relatively weak in oligodendroglioma cells. The papaverine-induced suppression of virus growth in neuroblastoma cells was studied in detail. The suppression that occurred was dependent on the dose of papaverine and was reversible. By treatment with 10 microM papaverine, virus-cell interactions were modified as follows: (i) early replication steps such as adsorption, penetration, and uncoating of the virus were not affected; (ii) synthesis of viral RNAs, including genomic RNA and mRNA, was inhibited; (iii) translation of viral proteins from mRNA was not blocked; and (iv) glycosylation and transport of viral glycoproteins to the cell membrane were not inhibited, but phosphorylation was blocked. The significance of suppressed virus replication in neural cells is discussed in relation to the persistence mechanisms of measles virus in the central nervous system.
The primary site of replication alters the eventual site of persistent infection by polyomavirus in mice
Authors: Dubensky T W , Villarreal L P
Published: January 2020

Using DNA blot analysis, we monitored the course of polyomavirus infection in mice receiving an intranasal inoculation and compared this with the course of infection in mice receiving an intraperitoneal inoculation. Intranasal infection was characterized by an initial primary replication phase in the respiratory tract, followed by a systemic infection of the visceral organs. At 12 days postinfection, there was partial clearing of viral DNA in all organs; by 22 days postinfection, viral DNA persisted only in the lungs and kidneys, and the level of DNA slowly decreased during the next 3 months. Lungs have been a previously unrecognized site for polyomavirus persistent infection. In contrast to intranasal infection, intraperitoneal infection of mice was characterized by only three phases: an initial systemic phase in which viral DNA was found in the same respiratory and visceral organs as during intranasal infection, clearing of the virus from the organs, and ultimately, a persistent infection in the kidneys but not in the lungs. Thus, different organs became persistently infected when mice were inoculated via these different routes.
Infection of eucaryotic cells by helper-independent recombinant adenoviruses: early region 1 is not obligatory for integration of viral DNA
Authors: Van Doren K , Hanahan D , Gluzman Y
Published: January 2020

Recombinant viral genomes carrying a selectable drug resistance marker have been constructed by insertion of a hybrid gene for neomycin resistance into the helper-independent adenovirus vector, delta E1/X. The hybrid gene consists of sequences coding for the aminoglycoside 3'-phosphotransferase II from Tn5, under the control of the simian virus 40 early promoter, and renders mammalian cells resistant to the neomycin analog, G-418. Most of adenovirus early region 1 is deleted from delta E1/X (nucleotides 455 to 3330), and recombinant viral genomes carry the hybrid gene in its place. The large and small XbaI fragments of delta E1/X were ligated to the hybrid gene, and the mixture was transfected into 293 cells. Single plaques were isolated and subsequently passaged in 293 cells to produce virus stocks. The recombinant viruses efficiently rendered cultured rat (Rat2) and simian (CV1) cells resistant to G-418. Cloned cell lines selected for resistance to G-418 contained viral DNA integrated into the host cell genome, demonstrating that early region 1 is not essential for integration of the viral genome. Southern transfer experiments revealed that (i) the sites of integration in the host genome were not unique; (ii) in general, transformed CV1 cell lines contained single-copy, full-length viral genomes, colinear with the infecting virus; (iii) transformed Rat2 cell lines generally contained one to several copies of full-length viral genomes integrated colinearly with the infecting viral DNA; and (iv) three of these five lines of transformed Rat2 cell lines contained tandemly repeated viral DNA sequences in which the right and left ends of the viral genome were joined to each other.
Human immunoglobulin class and subclass specificity of Fc receptors induced by herpes simplex virus type 1
Authors: Johansson P J , Hallberg T , Oxelius V A , Grubb A , Blomberg J
Published: January 2020

Herpes simplex virus is known to induce an immunoglobulin-binding cell surface receptor in infected cells that utilizes a nonimmune mechanism. In the present paper, we report the immunoglobulin class and subclass specificity of this receptor. Of the human immunoglobulins G(IgG), IgA, IgM, and IgD, as well as the structurally related beta2 microglobulin, only IgG and its Fc portion exhibited an increased binding to herpes simplex virus-infected cells versus uninfected control cells. The IgG subclass specificity of the Fc receptor was studied in 37 radioiodinated IgG myeloma proteins representing all four subclasses. We found that IgG3 myeloma proteins did not bind to herpes simplex virus-infected cells to a greater extent than to uninfected cells. On the contrary, proteins belonging to the other subclasses exhibited an increased binding to herpes simplex virus-infected cells of the following relative magnitude: IgG4 greater than IgG1 greater than or equal to IgG2. This increment of binding could be abolished by addition of a large excess of human IgG Fc fragment. Evidence for the existence of a variable herpes simplex virus-specific binding ability between myeloma proteins belonging to the same IgG subclass was also obtained. Furthermore, we tested two other herpes simplex virus type 1 strains with a limited number of myeloma proteins with very similar results as with the herpes simplex virus type 1 F strain. Several sources of experimental artefacts were controlled, including the state of aggregation of the test proteins, the functional integrity of the Fc portion before and after radioiodination, and the subclass assignments. The implications for the biological role of the Fc receptor of herpes simplex virus are discussed.
Guanidine-resistant poliovirus mutants produce modified 37-kilodalton proteins
Authors: Anderson-Sillman K , Bartal S , Tershak D R
Published: January 2020

Eighteen spontaneous, guanidine-resistant mutants of poliovirus were obtained by plaque selection. Isoelectric focusing demonstrated charge changes in a 37-kilodalton protein, pX, among three of the mutants. The precursor of pX, NCVP5b , also exhibited charge changes among the three mutants. pX of 12 mutants was also examined by peptide mapping with Staphylococcus aureus V8 protease. Nine of the mutants presented modified maps, and seven of these maps were identical. The demonstration of mutational changes in pX in 12 of 18 mutants suggests a role for this protein in determining the guanidine trait of poliovirus and corroborates studies with foot-and-mouth disease virus.
Antiidiotype modulation of herpes simplex virus infection leading to increased pathogenicity
Authors: Kennedy R C , Adler-Storthz K , Burns J W , Henkel R D , Dreesman G R
Published: January 2020

Antiidiotype reagents that recognize idiotypic determinants associated with the combining site of monoclonal antibodies to herpes simplex virus type 2 ( HSV2 ) were used to manipulate the immune response to HSV2 in BALB/c mice. The injection of antiidiotype antibodies into mice before challenge with a 50% lethal dose of HSV2 resulted in a shorter survival time than that of mice receiving either preimmune rabbit immunoglobulin G or antiidiotype reagents against hepatitis B surface antigen before HSV2 challenge. These findings indicate that the immune response to HSV2 in mice can be modulated through idiotype- antiidiotype networks, thereby increasing the pathogenicity of HSV2 infections.
DNA rearrangement affecting expression of the BK virus transforming gene
Authors: Watanabe S , Soeda E , Uchida S , Yoshiike K
Published: January 2020

BK virus mutant pm-522, forming small turbid plaques on human cells, can transform rat or hamster cells much more efficiently than the wild-type BK virus (wt-501) does. We compared the nucleotide sequence of wt-501 HindIII C segment with that of pm-522 HindIII-C, which contains the mutation responsible for the altered plaque type and transforming capacity. The difference between the two BK viruses was the local DNA rearrangement (deletions and duplications) that had occurred in the putative control region for early transcription in pm-522 DNA. Whereas wt-501 had three sets of 68-base pair repeats (the central set had a deletion of 18 base pairs) in this region, pm-522 had one set of 68-base-pair units and two sets of shorter 37-base-pair repeats. Three BK virus mutants, forming clear large plaques like those of wt-501 but capable of transforming rat cells, were derived from the recombinant virus carrying the HindIII C segment of pm-522. These mutants had further duplications of short segments originating from the pm-522 sequence in the putative early control region.
Structure of defective DNA molecules in Epstein-Barr virus preparations from P3HR-1 cells
Authors: Cho M S , Bornkamm G W , zur Hausen H
Published: January 2020

Epstein-Barr virus (EBV), isolated from P3HR-1 cells, induces early antigen and viral capsid antigen upon infection of human B-lymphoblasts. The strong early antigen- and viral capsid antigen-inducing activity is only observed in P3HR-1 virus preparations harboring particles with defective genomes, suggesting that this biological activity is directly associated with the defective DNA population. After infection of EBV genome-carrying Raji or EBV genome-negative BJAB cells, defective genomes of P3HR-1 EBV DNA are replicated in excess, depending on the multiplicity of infecting EBV particles. Hybridization of the DNA from such infected cells with 32P-labeled EBV DNA after HindIII cleavage reveals six hypermolar fragments. Mapping of these fragments shows that they form one defective genome unit containing four nonadjacent regions (alpha, beta, gamma, and delta) of the nondefective P3HR-1 EBV DNA. Two of the segments (alpha and beta) contain ca. 17 and 13 megadaltons, respectively, from the terminal regions of the P3HR-1 genome, whereas the two smaller segments (gamma and delta) contain ca. 3.7 and 3.0 megadaltons, respectively, originating from the central portion of the genome. In the defective molecule, the regions gamma and delta are present in the opposite orientation compared with nondefective P3HR-1 EBV DNA. Tandem concatemers are formed by fusion of the alpha and beta regions. Our model suggests that tandem concatemers of three defective genome units can be packaged into virions in P3HR-1 cells.
Mutation in the matrix protein of Newcastle disease virus can result in decreased fusion glycoprotein incorporation into particles and decreased infectivity
Authors: Peeples M E , Bratt M A
Published: January 2020

Virus particles produced in eggs by the group D ts mutants of Newcastle disease virus at permissive temperature display low infectious and hemolytic activities (M.E. Peeples and M. A. Bratt , J. Virol. 42:440-446, 1982). These lower activities correlate with a decreased incorporation of F1+2 (fusion glycoprotein) into virus particles, compared with that for wild type. The incorporation of F1+2 into virus particles of the group D mutants is also lower than that for wild type when grown in chicken embryo cells in culture at either permissive or nonpermissive temperature. The infectivity of virions from these mutants correlates with the amounts of F1+2 in the virus particles, below a certain concentration, indicating that the quantity of F1+2 in virus particles is a determining factor in the infectivity of those particles. In addition, one of these mutants, D1, produces an M (matrix protein) which migrates at a faster rate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three of four revertants of D1 have coreverted to wild-type M electrophoretic mobility, associating M with the ts lesion and the other observed phenotypes. In each of these revertants, as well as in three revertants each from D2 and D3, there has been coreversion from the low specific infectious and hemolytic activities to greater, and often wild-type, activities. There is also a coreversion for F1+2 incorporation into virions. All of the revertants incorporate F1+2 into virions more efficiently than their mutant parents. The coreversions associate those phenotypes with the ts lesion and, in the case of D1, with the M lesion as well.
Murine leukemia virus mutant with a frameshift in the reverse transcriptase coding region: implications for pol gene structure
Authors: Levin J G , Hu S C , Rein A , Messer L I , Gerwin B I
Published: January 2020

The molecular defect in the nonconditional B-tropic MuLV pol mutant, clone 23 (Gerwin et al., J. Virol. 31:741-751, 1979), has been characterized by recombinant DNA technology. The entire mutant genome was cloned from an EcoRI digest of integrated cellular DNA into bacteriophage lambda Charon 4A and then subcloned at the EcoRI site of pBR322. NIH-3T3 cells transfected with the plasmid clone, termed pRTM (RTM, reverse transcriptase mutant), reproduced the properties of clone 23 virus-infected cells. In vivo ligation experiments involving cotransfection of subclones of pRTM and wild-type murine leukemia virus localized the defect in the clone 23 genome to an approximately 400-base-pair region in the pol gene between the SalI and XhoI sites. Sequence analysis of this region in the wild-type and mutant genomes revealed that the mutant has one additional C residue located 231 bases downstream of the last base of the SalI recognition site. This 1-base insertion brings three TGA termination codons into phase. Thus, the mutation in clone 23 leads to premature termination of translation, explaining the presence in clone 23 virions of a truncated polymerase with low levels of enzymatic activity. It was previously shown that the gag precursor is cleaved normally in clone 23-infected cells; therefore, if a virus-coded protease is involved in this cleavage, it must be encoded by sequences upstream of the reverse transcriptase region of the pol gene. This consideration, coupled with the observed molecular weight of the mutant polymerase and our precise determination of its C terminus, have led to a proposal for the genetic organization of the murine leukemia virus pol gene.
Electron microscopy of bacteriophage phi 6 nucleocapsid: two-dimensional image analysis
Authors: Steely H T , Lang D
Published: January 2020

Electron micrographs of negatively stained nucleocapsids isolated from intact, wild-type phi 6 bacteriophage revealed three distinct morphological forms. Two-dimensional analysis of electron micrographs of two of these forms and image averaging of all forms are consistent with a dodecahedral structure embodied in the phi 6 nucleocapsid.
Persistence of mouse hepatitis virus A59 RNA in a slow virus demyelinating infection in mice as detected by in situ hybridization
Authors: Lavi E , Gilden D H , Highkin M K , Weiss S R
Published: January 2020

Mouse hepatitis virus strain A59 produces chronic central nervous system demyelination in rodents. As late as 6 months after intracerebral inoculation of mice 4 to 6 weeks old, when infectious virus cannot be recovered and viral antigens cannot be detected in the central nervous systems and livers of these animals, primary demyelination is still evident. Using cloned virus-specific DNAs and the highly sensitive and specific technique of in situ hybridization, we have detected low levels of mouse hepatitis virus A59 RNA in the central nervous systems and livers of mice 10 months after inoculation. We suggest that viral persistence may play a role in mouse hepatitis virus A59-induced chronic demyelination.
Proteins encoded by the long terminal repeat region of mouse mammary tumor virus: identification by hybrid-selected translation
Authors: Racevskis J , Prakash O
Published: January 2020

The long terminal repeat (LTR) region of mouse mammary tumor virus (MMTV) is known to contain an open reading frame of sufficient length to code for a protein of 36,000 Mr. The coding capacity of the 3' sequences of MMTV genomic RNA has been demonstrated by in vitro translation studies, which have reported the synthesis of four related proteins: p36, p24, p21, and p18. These proteins are overlapping translation products of the same open reading frame, with the smaller ones initiating at internal methionine codons. From the predicted amino acid sequence of the LTR protein, we have selected a region likely to be antigenic, obtained a synthetic peptide of that region, and raised antiserum to the peptide. The antipeptide serum specifically immunoprecipitated all four proteins from in vitro translated genomic 3' MMTV RNA, plus an additional one of 32,000 Mr. Published sequence data of MMRV LTRs show an internal AUG codon at a position which could initiate a protein of 32,000 Mr. The three smaller in vitro translation products (p24, p21, and p18) were consistently synthesized in much greater amounts than the p36 or p32 protein. The relative amount of each in vitro synthesized protein from genomic MMTV RNA could be predicted and was in good agreement with the postulated effect of flanking nucleotides on the efficiency of the respective AUG initiation codon. Polyadenylated RNAs, isolated from various mouse tissues, were selected by hybridization to plasmid DNA containing MMTV LTR sequences immobilized on nitrocellulose. In vitro translation of hybrid-selected mRNAs isolated from BALB/c mouse lactating mammary glands and carcinogen-induced mammary tumors, followed by immunoprecipitation with antipeptide serum, revealed that only one polypeptide was synthesized by the MMTV LTR-specific mRNA, the 36,000 Mr species.
Subcellular localization of glycoproteins encoded by the viral oncogene v-fms
Authors: Anderson S J , Gonda M A , Rettenmier C W , Sherr C J
Published: January 2020

The McDonough strain of feline sarcoma virus encodes a polyprotein that is cotranslationally glycosylated and proteolytically cleaved to yield transforming glycoproteins specified by the viral oncogene v-fms. The major form of the glycoprotein (gp120fms) contains endoglycosidase H-sensitive, N-linked oligosaccharide chains lacking fucose and sialic acid, characteristic of glycoproteins in the endoplasmic reticulum. Kinetic and steady-state measurements showed that most gp120fms molecules were not converted to mature forms containing complex carbohydrate moieties. Fixed-cell immunofluorescence confirmed that the majority of v-fms-coded antigens were internally sequestered in transformed cells. Dual-antibody fluorescence performed with antibodies to intermediate filaments (IFs) showed that the IFs of transformed cells were rearranged, and their distribution coincided with that of v-fms-coded antigens. No specific disruption of actin cables was observed. The v-fms gene products cofractionated with IFs isolated from virus-transformed cells and reassociated with IFs self-assembled in vitro. A minor population of v-fms-coded molecules (gp140fms) acquired endoglycosidase H-resistant, N-linked oligosaccharide chains containing fucose and sialic acid residues, characteristic of molecules processed in the Golgi complex. Some gp140fms molecules were detected at the plasma membrane and were radiolabeled by lactoperoxidase-catalyzed iodination of live transformed cells. We suggest that v-fms-coded molecules are translated as integral transmembrane glycoproteins, most of which are inhibited in transport through the Golgi complex to the plasma membrane.
Restriction analysis and homology studies of the bovine adenovirus 7 genome
Authors: Hu S L , Battles J K , Potts D E
Published: January 2020

We located the cleavage sites for restriction endonucleases EcoRI, HindIII, and BamHI on the genome of bovine adenovirus 7. Cross-hybridization at reduced stringency revealed two regions of homology shared by the DNA of human adenovirus 2 and bovine adenoviruses 7 and 3. These regions correspond to the hexon and the IVa2 protein genes of the human adenovirus. Another region of homology shared only by the human adenovirus and bovine adenovirus 7 corresponded to the penton or the polypeptide IIIa genes. These results allowed us to align the restriction map of bovine adenovirus 7 with respect to the other adenoviruses.
Interaction of polymorphonuclear leukocytes and viruses in humans: adherence of polymorphonuclear leukocytes to respiratory syncytial virus-infected cells
Authors: Faden H , Hong J J , Ogra P L
Published: January 2020

The nature of neutrophil-respiratory syncytial virus (RSV) interaction was investigated by assessing factors that influence neutrophil adherence to RSV-infected tissue culture monolayers. The adherence of neutrophils to infected cells was directly proportional to the degree of RSV replication as evidenced by infectious virus production, cytopathological changes, or viral antigen appearance. Sixty-one percent of the neutrophils adhered to the RSV-infected cells as compared with 52.7% on noninfected monolayers (P less than 0.05). The addition of RSV-specific antibody markedly increased polymorphonuclear leukocyte adherence to 88.5% (P less than 0.001). Complement in the absence of antibody augmented polymorphonuclear leukocyte adherence, but to a lesser degree, 69.0% (P less than 0.025). Arachidonic acid metabolism appeared to play a critical role in the adherence process; thromboxane was the single most important arachidonic acid metabolite. Inhibition of thromboxane synthesis reduced antibody-dependent polymorphonuclear leukocyte adherence on RSV-infected cells to 52.3% (P less than 0.025). These observations suggest a role for neutrophils in RSV infection. It is proposed that neutrophils may participate in RSV infection at the site of viral replication through the attachment to infected cells and the subsequent release of mediators of inflammation.
pppA2'p5A' blocks vesicular stomatitis virus replication in intact cells
Authors: Alarcon B , Bugany H , Carrasco L
Published: January 2020

pppA2'p5'A blocked the production of infectious vesicular stomatitis virus in HeLa cells. When this compound was present from the beginning of infection, a selective inhibitory effect was observed in viral protein synthesis. Thus, cellular translation was not affected even after 10 h of incubation with this compound, and the bulk of viral proteins was not synthesized. However, this effect was not observed with ATP, GTP, or the core A2'p5'A. The step blocked by pppA2'p5'A is located early during virus infection, but adsorption, entry, and virus uncoating seemed to be unaffected by this compound. Analysis of the antiviral spectrum of pppA2'p5'A indicated that it is active against poliovirus, encephalomyocarditis virus, and Semliki Forest virus and shows no effect against herpes simplex virus type 1 and adenovirus type 5.
Effect of S-adenosylmethionine on human rotavirus RNA synthesis
Authors: Spencer E , García B I
Published: January 2020

The characteristics of human rotavirus-associated RNA polymerase activity have been examined in relation to the effects of ribonucleoside triphosphate analogs and S-adenosylmethionine. These effects were analyzed by testing two forms of activated virus particles: EDTA- and heat-treated virions. The former lack outer shell proteins, and activation by means of heat treatment does not introduce any apparent modification in virion structure. Virus-associated RNA polymerase shows similar properties in both preparations, suggesting that outer proteins are not directly involved in RNA synthesis. Transcription in this virus is specifically dependent on a hydrolyzable form of ATP. Such a requirement is not overcome by preincubation or by the addition of S-adenosylmethionine, suggesting a hypothetical mechanism that couples transcription to ATP hydrolysis. The addition of S-adenosylmethionine stimulated transcription and diminished the Km value not only for ATP but also for the other three ribonucleoside triphosphates. Analysis of methylated RNA products suggested that methyl groups were incorporated into all of the RNA species synthesized by virion-associated polymerase. Further analysis of those RNA molecules showed that they contained cap structures at the 5' end. The results suggest that the cap structure at the end of RNA molecules may enable RNA polymerase to elongate transcripts more efficiently, in a reaction in which the hydrolysis of ATP is involved.
Detection of multiple strains of latent herpes simplex virus type 1 within individual human hosts
Authors: Lewis M E , Leung W C , Jeffrey V M , Warren K G
Published: January 2020

One hundred and fifteen isolates of herpes simplex virus were recovered from parallel explant cultures of trigeminal and vagus ganglia and trigeminal nerve roots derived from 20 unselected human cadavers. Restriction enzyme patterns of strains recovered from 18 of 20 individuals could be differentiated from individual to individual, although all isolates from a single host were identical. Isolates from two individuals differed among themselves in the number and location of certain restriction enzyme sites.
Identification of multiple Epstein-Barr virus-induced nuclear antigens with sera from patients with rheumatoid arthritis
Authors: Sculley T B , Walker P J , Moss D J , Pope J H
Published: January 2020

By means of the protein immunoblot technique, the Epstein-Barr virus (EBV) nuclear antigen (EBNA) could be identified in a variety of EBV-transformed cell lines with anti-EBNA-positive sera from normal donors. The molecular weight of EBNA expressed in each of the cell lines varied between 70,000 and 75,000 and was dependent upon the strain of infecting virus. In contrast, 15 of 21 sera from patients with rheumatoid arthritis identified antigens in addition to EBNA. The most prominent of these antigens had molecular weights of 110,000 to 115,000 and 92,000. All of the EBV genome-positive cell lines except for QIMR-GOR and cell lines containing the P3HR-1 virus expressed these antigens. The antigens were not present in the EBV genome-negative Ramos and BJAB cell lines, nor were they identified with EBV seronegative sera, indicating that they were EBV related. There was no direct correlation between the presence of antibodies in sera to EBNA, viral capsid antigen or early antigen, and reaction with the 92,000-molecular-weight antigen in immunoblots, indicating that this antigen was distinct from previously described EBV-related antigens.