bluuebunny/crossref_metadata_2025 huggingface dataset:
Cells Persistently Infected with Newcastle Disease Virus
Authors: Thacore Harshad , Youngner Julius S.
Published: January 2020

A comparison of the replication patterns in L cells and in chick embryo (CE) cell cultures was carried out with the Herts strain of Newcastle disease virus (NDV
o
) and with a mutant (NDV
pi
) isolated from persistently infected L cells. A significant amount of virus progeny, 11 plaque-forming units (PFU)/cell, was synthesized in L cells infected with NDV
o
, but the infectivity remained cell-associated and disappeared without being detectable in the medium. In contrast, in L cells infected with NDV
pi
, progeny virus (30 PFU/cell) was released efficiently upon maturation. It is suggested that the term “covert” rather than “abortive” be used to describe the infection of L cells with NDV
o
. In both L and CE cells, the latent period of NDV
pi
was 2 to 4 hr longer than for NDV
o
. The delay in synthesis of viral ribonucleic acid (RNA) in the case of NDV
pi
coincided with the delay in the inhibition of host RNA and protein synthesis. Although both NDV
o
and NDV
pi
produced more progeny and more severe cell damage in CE cells than in L cells, the shut-off of host functions was significantly less efficient in CE cells than in L cells. Paradoxically, no detectable interferon was produced in CE cells by either of the viruses, whereas in L cells most of the interferon appeared in the medium after more than 90% of host protein synthesis was inhibited. These results suggest that the absence of induction of interferon synthesis in CE cells infected with NDV is not related to the general shut-off of host cell synthetic mechanisms but rather to the failure of some more specific event to occur. In spite of the fact that NDV
pi
RNA synthesis commenced 2 to 4 hr later than that of NDV
o
, interferon was first detected in the medium 8 hr after infection with both viruses. This finding suggests that there is no relation between viral RNA synthesis and the induction of interferon synthesis.
Identification of the Simian Virus 40 Which Replicates When Simian Virus 40-Transformed Human Cells Are Fused with Simian Virus 40-Transformed Mouse Cells or Superinfected with Simian Virus 40 Deoxyribonucleic Acid
Authors: Kit Saul , Kurimura T. , Brown McKay , Dubbs D. R.
Published: January 2020

Simian virus 40 (SV40) was rescued from heterokaryons of transformed mouse and transformed human cells. To determine whether the rescued SV40 was progeny of the SV40 genome resident in the transformed mouse cells, the transformed human cells, or both, rescue experiments were performed with mouse lines transformed by plaque morphology mutants of SV40. The transformed mouse lines that were used yielded fuzzy, small-clear, or large-clear plaques after fusion with CV-1 (African green monkey kidney) cells. The transformed human lines that were used did not release SV40 spontaneously or after fusion with CV-1 cells. From each mouse-human fusion mixture, only the SV40 resident in the transformed mouse cells was recovered. Fusion mixtures of CV-1 and transformed mouse cells yielded much more SV40 than those from transformed human and transformed mouse cells. The rate of SV40 formation was also greater from monkey-mouse than from human-mouse heterokaryons. Deoxyribonucleic acid (DNA) from SV40 strains which form fuzzy, largeclear, or small-clear plaques on CV-1 cells was also used to infect monkey (CV-1 and Vero), normal human, and transformed human cell lines. The rate of virion formation and the final SV40 yields were much higher from monkey than from normal or transformed human cells. Only virus with the plaque type of the infecting DNA was found in extracts from the infected cells. Two uncloned sublines of transformed human cells [W18 Va2(P363) and WI38 Va13A] released SV40 spontaneously. Virus yields were not appreciably enhanced by fusion with CV-1 cells. However, clonal lines of W18 Va2(P363) did not release SV40 spontaneously or after fusion with CV-1 cells. In contrast, several clonal lines of WI38 Va13A cells did continue to shed SV40 spontaneously.
Early Events in the Infection of Permissive Cells with Simian Virus 40: Adsorption, Penetration, and Uncoating
Authors: Barbanti-Brodano Giuseppe , Swetly Peter , Koprowski Hilary
Published: January 2020

The early events in the interaction of simian virus 40 (SV40) with permissive cells were investigated. Evidence is presented that 30 min after infection intact virions penetrate the nuclei of infected cells. The uncoating of the virus is carried out in the nuclei with a complete dissociation of the viral genome from the protein coat. Opening of the circular parental deoxyribonucleic acid (DNA), i.e., conversion of component I to component II of SV40 DNA, takes place after uncoating, followed by the appearance of a new component sedimenting faster than component I at alkaline
p
H.
Factors Affecting the Sensitivity of Different Viruses to Interferon
Authors: Hallum Jules V. , Thacore Harshad R. , Youngner Julius S.
Published: January 2020

When the sensitivities to interferon of Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV) were compared by the plaque reduction method in chick embryo cell cultures, NDV was found to be 45-fold more resistant than VSV. This difference was exaggerated when a multiple-cycle yield inhibition method was employed. In marked contrast, when the same viruses were tested by a single-cycle yield inhibition method, the difference in sensitivity to interferon of the two viruses was virtually eliminated. Further investigation showed that, in chick embryo cells exposed to interferon, the resistance to NDV decayed more rapidly than resistance to VSV. This finding explained the divergent results obtained with the two viruses when single- or multiple-cycle replication techniques were employed. Experiments carried out with L cells showed that cellular antiviral resistance decayed much more slowly in these cells than in chick embryo cells. Consequently, when measured by the plaque reduction method in L cells, no difference was observed in the sensitivity to interferon of VSV and NDV
pi
, a mutant of NDV which replicates efficiently in L cells. A procedure is suggested for determining the relative sensitivities to interferon of different viruses under conditions which minimize the role of decay of antiviral resistance in the host cells.
T-Even Bacteriophage-Tolerant Mutants of
Escherichia coli
B
Authors: Mathews Christopher K.
Published: January 2020

A general procedure is described for isolation of T-even phage-tolerant mutants of
Escherichia coli.
Two such mutants of
E. coli
B have been examined in some detail. These mutants adsorb T-even phages but are unable to release viable progeny. Under certain conditions, viability of the cells is completely unaffected by phage infection in one mutant, and there is but a slight decrease in colony-forming ability in the other. Phage deoxyribonucleic acid (DNA) is injected into these cells, as shown by the formation of phage-specific enzymes, but it is not degraded to acid-soluble material. Some phage DNA replication occurs in both strains. The mutants are both more resistant to ultraviolet light than is the parent strain.
Inhibition of Host Protein Synthesis During Infection of
Escherichia coli
by Bacteriophage T4
Authors: Kennell David
Published: January 2020

Two gene clusters on the
Escherichia coli
chromosome were induced at early times after T4 infection when >99% of the cells were infected: the lactose (
lac
) operon and prophage λ. Their messenger ribonucleic acid (mRNA) was detected by hybridization to φ80 d
lac
deoxyribonucleic acid (DNA) and λDNA, respectively. Synthesis of host mRNA could be initiated during the first few minutes after T4 infection, although no β-galactosidase activity could be detected. Hybridization analyses of selected fractions from sucrose gradients revealed that most of this
lac
mRNA induced at very early times of T4 infection was not associated with ribosomes. In contrast, virtually all
lac
mRNA in uninfected bacteria was associated with polysomes. This exclusion affected all host mRNA; about 70% of
E. coli
3
H-mRNA, labeled from 2 to 3 min after T4 infection, was excluded from polysomes. Infection even reduced the yield of β-galactosidase from
lac
mRNA induced before infection. Gradients from rifampicin-inhibited cells showed the normal growth of
lac
mRNA polysomes; in contrast, T4 infection prevented growth of the preinduced
lac
polysomes. It is concluded that T4 infection interferes within seconds with the reassociation of ribosomes to host mRNA.
Morphogenesis of Respiratory Syncytial Virus in a Green Monkey Kidney Cell Line (Vero)
Authors: Norrby Erling , Marusyk Halyna , Örvell Claes
Published: January 2020

The structure and morphogenesis of respiratory syncytial (RS) virus particles in a green monkey kidney cell line (Vero) were examined. Infected cells contained dense intracytoplasmic inclusions composed of filamentous structures. In places where inclusion material was associated with membranes, structural modifications were induced. There was a thickening of the membrane and an addition of projections 12 to 15 nm in length. The same changes were most frequently observed after association of isolated filamentous structures with the cytoplasmic membrane. The budding-off process was clearly visualized. The diameter of mature virus particles varied between 90 and 130 nm and that of the internal component varied between 11 and 15 nm. The similarities between ultrastructural features of cells infected with RS virus and pneumonia virus of mice are pointed out. It is proposed that these two viruses should be classified together in a third subgroup of myxoviruses.
Morphogenesis of the Nucleoprotein of Vesicular Stomatitis Virus
Authors: Zajac Barbara A. , Hummeler Klaus
Published: January 2020

Accumulation of the nucleoprotein of vesicular stomatitis virus (VSV) in the cytoplasm of BHK-21 cells and in two of four human cell lines was demonstrated. Appearance and progression of the nucleoprotein inclusions paralleled development of virus-specific immunofluorescence and production of virus progeny. The inclusions appeared early as discrete foci of filamentous material which eventually increased in size to form large masses which replaced normal cytoplasmic constituents. The filamentous strands were found in close proximity to budding virions. The inclusion material was extracted from infected cells and purified in cesium chloride gradients. The isolated filaments resembled the ribonucleoprotein isolated from purified virions. They incorporated
3
H-uridine, exhibited virus-specific complement-fixing activity, had a buoyant density of 1.32 g/cm
3
, and appeared as single wavy strands the width of which varied from 2.5 to 8.5 nm, depending on the angle of viewing.
Restricted Replication of Frog Virus 3 in Selected Variants of BHK Cells
Authors: Vilaginès R. , McAuslan B. R.
Published: January 2020

Synthesis and maturation of frog virus 3 deoxyribonucleic acid (DNA) in BHK cells and selected variants, in chick fibroblasts, and in minnow cells were compared. Wide ranges in rates of DNA synthesis and assembly of virions were found. At least three variants of BHK cells can be obtained: (i) fully permissive, characterized by rapid DNA synthesis and assembly to give a high yield of infective virus; (ii) semipermissive, in which viral DNA is synthesized slowly for extended periods of time, and the yield of infective virus is poor; and (iii) nonpermissive, in which virus adsorbs to cells and arrests host functions but viral DNA is not synthesized. Kinetics of appearance of virions and procedures for their separation from cell extracts are described.
Quantitative Determination of Neuraminidase-Active Foci in Cell Monolayer Cultures Infected with Influenza or Newcastle Disease Virus
Authors: Palese P. , Bodo G. , Tuppy H.
Published: January 2020

A synthetic neuraminidase substrate [2-(3′-methoxyphenyl)-
N
-acetyl-α-neuraminic acid] was used to detect myxo- and paramyxovirus replication in tissue culture. This method allowed estimation of viral growth even in the absence of cytopathogenic effects.
Deoxyribonucleic Acid Polymerase Associated with Rous Sarcoma Virus and Avian Myeloblastosis Virus: Properties of the Enzyme and Its Product
Authors: Garapin Axel-Claude , McDonnell Jerome P. , Levinson Warren , Quintrell Nancy , Fanshier Lois , Bishop J. Michael
Published: January 2020

Deoxyribonucleic acid (DNA) polymerase activity can be elicited in purified preparations of avian myeloblastosis virus and Rous sarcoma virus (Schmidt-Ruppin strain) by treatment with nonionic detergent. The enzyme(s) and its synthetic products appear to be virion-associated. Enzymatic activity can be inhibited by pretreatment with either ribonuclease (8- to 10-fold inhibition) or actinomycin D (twofold inhibition). By contrast, rifampin has little, if any effect. The enzyme(s) synthesizes two primary products, a ribonucleic acid (RNA):DNA hybrid and DNA which is free of RNA. The results of both zonal and equilibrium centrifugation indicate that nascent chains of DNA are associated with the 70
S
viral RNA. It is concluded that at least two enzymatic activities are under study: transcription of DNA from viral RNA, and subsequent, additional synthesis of DNA, utilizing product of the initial reaction as template.
Propagation in Human Cells of a Filterable Agent from the ST Feline Sarcoma
Authors: Chang R. Shihman , Golden H. Dean , Harrold Boyd
Published: January 2020

Nineteen lines of human fibroblasts were inoculated with a filtrate prepared from the ST feline sarcoma. Seven lines showed morphological alteration and released focus-forming activity for feline cells, 2 lines showed morphological alteration but did not release focus-forming activity, and 11 lines showed no morphological alteration and released no focus-forming agent. Morphologically altered cells appeared enlarged, hyper-refractile, and intensely stained by hematoxylin. They neither assumed a crisscross pattern nor piled up to form a visible focus. Time-lapse cinegraph showed that the morphologically altered cells did not divide and were motile. The fluid from two human fibroblast cultures, inoculated 4 and 14 weeks previously with the ST sarcoma filtrate, induced fibrosarcoma in newborn kittens.
Synthesis of Messenger Ribonucleic Acid After Bacteriophage T1 Infection
Authors: Male Carolyn J. , Christensen J. R.
Published: January 2020

Synthesis of host-specific and phage-specific messenger ribonucleic acid (mRNA) was studied in bacteria infected by unmodified (T1 · B) or modified [T1 · B(P1)] bacteriophage T1. In a “standard” infection of
Escherichia coli
B by T1 · B (no host-controlled modification involved), the rate and amount of T1 mRNA synthesis was intermediate between those values reported for infections by a virulent phage such as T4 or a temperate phage such as lambda. The initial rate of mRNA synthesis was slightly increased after T1 · B(P1) infection of
E. coli
B in comparison with T1 · B infection of the same host. Little or no phage mRNA synthesis could be detected in T1 · B infection of
E. coli
B(P1). Phage mRNA synthesis in T1 · B(P1)-infected
E. coli
B(P1) cells was approximately the same in amount as that seen in T1 · B(P1) infection of
E. coli
B. Synthesis of host-specific mRNA continued throughout the latent period in all infections studied. However, the enzyme β-galactosidase could not be induced, except after T1 · B infection of
E. coli
B(P1). In an attempt to understand the apparent differences in mRNA synthesis after infection of
E. coli
B by phages T1 · B or T1 · B(P1), the effect of altered T1 deoxyribonucleic acid (DNA) methylation on mRNA synthesis was studied. Methyl-deficient T1 DNA, made in cells infected with ultraviolet-irradiated phage T3, inhibited
14
C-uridine incorporation more strongly than normal T1. One passage of methyl-deficient T1 through
E. coli
B restored uracil incorporation rates to those seen with ordinary T1. This suggests that methylation of T1 DNA can influence the rate of phage mRNA synthesis. However, attempts to relate the difference in mRNA synthesis seen between T1 · B and T1 · B(P1) in
E. coli
B to the activity of the P1 modification gene were not conclusive.
Changes in Deoxyribonucleic Acid Synthesis Regulation in Chinese Hamster Cells Infected with Simian Virus 40
Authors: Lehman John M. , Defendi Vittorio
Published: January 2020

Infection of primary or secondary cultures of Chinese hamster embryo cells with simian virus 40 at a multiplicity of 20 to 50 induced synthesis of the virus-specific intranuclear T antigen in 80 to 90% of the cells within 48 to 72 hr. In the infected cultures, 30 to 50% more cells were recruited into deoxyribonucleic acid (DNA) synthesis than in the controls, whether or not the cultures were confluent. The newly synthesized DNA was mostly cellular, since little virus was produced (as shown by various techniques: immunofluorescence for viral antigen, virus growth curves, and isolation of viral DNA from infected cultures). Transformed cells could be detected a few weeks after infection and produced tumors when inoculated into irradiated animals. Chromosomal changes were observed soon after infection (24 hr). Initially, there was a marked increase in the proportion of polyploid cells (8 to 14%), most of which were chromosomally normal. In a few weeks, a large majority of the infected population was polyploid (30 to 50%). Thus, the polyploid cells have the ability to proliferate. Evidence is presented to suggest that polyploid cells arise by stimulation of cells in the G
1
, G
2
, or S phases to undergo two or more successive periods of DNA synthesis without an intervening mitosis. With a subsequent loss or redistribution of chromosomal material, this may lead eventually to a biologically transformed cell; thus, it is suggested that the initial event(s) relevant to transformation occurs at the level of control of cellular DNA synthesis.
Inhibition of Host Protein Synthesis During Infection of
Escherichi coli
by Bacteriophage T4
Authors: Fabricant Robert , Kennell David
Published: January 2020

Deoxyribonucleic acid (DNA)-less T2 “ghosts” were prepared by
osmotic shock and purified by KBr density gradient centrifugation.
Escherichia coli
B was treated with these ghosts in
inorganic salts-glycerol medium to see which features of phage infection could
be elicited by ghosts. At a multiplicity that was just sufficient to block
induction of β-galactosidase (EC 3.2.1.23), 89% of the bacteria were
killed and the rates of ribonucleic acid (RNA) and DNA synthesis were about 10
to 15% of normal. However, protein synthesis was almost completely blocked but
resumed after 30 min. During this period, it was possible to induce messenger
RNA (mRNA) from the lactose operon, although this mRNA could not be translated
into active β-galactosidase. These results suggest to us that the viable
cells surviving ghost infection synthesize nucleic acids at close to a normal
rate but are temporarily blocked in protein synthesis. The continued formation
of untranslated host mRNA mimics the pattern of bacterial synthesis just after
whole-phage infection, and is consistent with the interpretation that the
immediate block in the initiation of host translation by these viruses is due to
their attachment.
Relative Antiviral Resistance Induced in Homologous and Heterologous Cells by Cross-Reacting Interferons
Authors: Stewart William E. , Lockart Royce Z.
Published: January 2020

Human cells incubated with human interferon become more resistant to vesicular stomatitis virus (VSV) than to Semliki Forest virus (SFV); monkey cells treated with monkey interferon become more resistant to SFV than to VSV. However, monkey cells incubated with human interferon developed relative antiviral activity identical to that induced by homologous interferon, and human cells developed characteristic human interferon-induced relative antiviral activity when exposed to monkey interferon. Therefore, cross-reacting interferons induce the relative antiviral activity characteristic of the interferon-treated cell rather than the cell of the interferon's origin. This relationship supports the hypothesis that interferon is not itself antiviral but rather induces cells to develop their own antiviral activity.
Translation and processing of mouse hepatitis virus virion RNA in a cell-free system
Authors: Denison M R , Perlman S
Published: January 2020

The first event after infection with mouse hepatitis virus strain A59 (MHV-A59) is presumed to be the synthesis of an RNA-dependent RNA polymerase from the input genomic RNA. The synthesis and processing of this putative polymerase protein was studied in a cell-free translation system utilizing 60S RNA from MHV-A59 virions. The polypeptide products of this reaction included two major species of 220 and 28 kilodaltons. Kinetics experiments indicated that both p220 and p28 appeared after 60 min of incubation and that protein p28 was synthesized initially as the N-terminal portion of a larger precursor protein. When the cell-free translation products were labeled with N-formyl[35S]methionyl-tRNAi, p28 was the predominant radioactive product, confirming its N-terminal location within a precursor protein. Translation in the presence of the protease inhibitors leupeptin and ZnCl2 resulted in the disappearance of p28 and p220 and the appearance of a new protein, p250. This product, which approached the maximal size predicted for a protein synthesized from genomic RNA, was not routinely detected in the absence of inhibitors even under conditions which optimized the translation reaction for elongation of proteins. Subsequent chelation of ZnCl2 resulted in the partial cleavage of the precursor protein and the reappearance of p28. One-dimensional peptide mapping with Staphylococcus aureus V-8 protease confirmed the precursor-product relationship of p250 and p28. The results show that MHV virion RNA, like many other viral RNAs, is translated into a large polyprotein, which is cleaved soon after synthesis into smaller, presumably functional proteins. This is in marked contrast to the synthesis of other MHV proteins, in which minimal proteolytic processing occurs.
Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs
Authors: Weitz M , Baroudy B M , Maloy W L , Ticehurst J R , Purcell R H
Published: January 2020

The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV-infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs.
Differential transcription from the long terminal repeats of integrated avian leukosis virus DNA
Authors: Herman S A , Coffin J M
Published: January 2020

In avian leukosis virus-induced lymphoma and erythroblastosis, the expression of the proto-oncogenes c-myc and c-erbB is activated by downstream or readthrough transcripts initiated within integrated proviral DNA. To determine the relative abundance of viral RNAs extending into the downstream cellular sequences independently of the effects that may be exerted by specific sites of proviral integration, we examined the RNA of infected avian fibroblasts. Using a nuclease protection strategy to detect downstream, readthrough, and normal viral RNAs and to distinguish them from each other, we found that transcripts initiated within the 3' long terminal repeat, i.e., downstream transcripts, were undetectable in infected fibroblasts and could not have amounted to more than 1 to 2% of the total viral RNA. However, readthrough RNAs, which are transcripts initiated within the 5' long terminal repeat and extended beyond the viral polyadenylation site into the downstream cellular DNA, were present at relatively high levels, making up approximately 15% of the total viral RNA.
Nucleotide sequence of human endogenous retrovirus genome related to the mouse mammary tumor virus genome
Authors: Ono M , Yasunaga T , Miyata T , Ushikubo H
Published: January 2020

We determined the complete nucleotide sequence of the human endogenous retrovirus genome HERV-K10 isolated as the sequence homologous to the Syrian hamster intracisternal A-particle (type A retrovirus) genome. HERV-K10 is 9,179 base pairs long with long terminal repeats of 968 base pairs at both ends; a sequence 290 base pairs long, however, was found to be deleted. It was concluded that a composite genome having the 290-base-pair fragment is the prototype HERV-K provirus gag (666 codons), protease (334 codons), pol (937 codons), and env (618 codons) genes. The size of the protease gene product of HERV-K is essentially the same as that of A- and D-type oncoviruses but nearly twice that of other retroviruses. A comparison of the deduced amino acid sequences encoded by the pol region showed HERV-K to be closely related to types A and D retroviruses and even more so to type B retrovirus. It was noted that the env gene product of HERV-K structurally resembles the mouse mammary tumor virus (type B retrovirus) env protein, and the possible expression of the HERV-K env gene in human breast cancer cells is discussed.
Human respiratory syncytial virus glycoprotein G expressed from a recombinant vaccinia virus vector protects mice against live-virus challenge
Authors: Stott E J , Ball L A , Young K K , Furze J , Wertz G W
Published: January 2020

Recombinant vaccinia virus vectors were constructed which expressed the major surface glycoprotein G of human respiratory syncytial (RS) virus. The biological activity of the G protein expressed from these vectors was assayed. Inoculation of rabbits with live recombinant virus induced high titers of antibody which specifically immunoprecipitated RS virus G protein and was capable of neutralizing RS virus infectivity. Immunization of mice by either the intranasal or the intraperitoneal route with recombinant virus that expressed only the G protein resulted in complete protection of the lower respiratory tract upon subsequent challenge with live RS virus.
DNA Synthesis in Tobacco Mosaic Virus-Infected
Nicotiana tabacum
Protoplasts and Regeneration of Persistently Infected Calli
Authors: Stram Yehuda , Weissbach Arthur
Published: January 2020

Tobacco mosaic virus infection of
Nicotiana tabacum
mesophyll protoplasts did not affect the pattern of chloroplast or total cellular DNA synthesis for at least 120 h when compared with that of mock-infected cells. Calli derived from infected protoplasts often showed large amounts of tobacco mosaic virus RNA and coat protein.
Production of guanidine-resistant and -dependent poliovirus mutants from cloned cDNA: mutations in polypeptide 2C are directly responsible for altered guanidine sensitivity
Authors: Pincus S E , Wimmer E
Published: January 2020

cDNA fragments representing the region in polypeptide 2C containing mutations in a guanidine-resistant or -dependent mutant were cloned into the wild-type background of an infectious clone. Transfection of COS-1 cells with these plasmids yielded viruses that were either completely resistant to 2.0 mM guanidine hydrochloride or dependent on this concentration of drug for growth.
Oligomerization of herpes simplex virus glycoprotein B
Authors: Claesson-Welsh L , Spear P G
Published: January 2020

Glycoprotein B (gB) specified by herpes simplex virus can be extracted from virions or infected cells in the form of detergent-stable, heat-dissociable oligomers. The composition of the oligomers and requirements for their formation were investigated. Evidence is presented that the faster-migrating forms of the oligomers are homodimers of gB. Dimerization was shown to occur within minutes of polypeptide synthesis and did not depend on glycosylation, the expression of other viral proteins, or virion morphogenesis. The multiple, electrophoretically distinct forms of gB dimers differ in extent or rate of N-linked oligosaccharide processing and also have other differences that influence electrophoretic mobility.
Construction and properties of a viable herpes simplex virus 1 recombinant lacking coding sequences of the alpha 47 gene
Authors: Mavromara-Nazos P , Ackermann M , Roizman B
Published: January 2020

The five alpha genes, alpha 0, alpha 4, alpha 22, alpha 27, and alpha 47, are the first set of herpes simplex virus 1 genes to be transcribed and expressed in productively infected cells. We report here the construction of a viral recombinant from which all of the coding sequences of the alpha 47 gene were deleted. In addition to the alpha 47 protein, infected cell lysates did not contain detectable amounts of two polypeptide bands with apparent molecular weights of 18,000 and 21,000 which could be specified by a gene whose regulatory domain and 5' transcribed noncoding sequences overlap with the coding sequences of the alpha 47 gene. The alpha 47- virus grew as well as the wild-type parent virus in Vero, baby hamster kidney, and Rat-1 cell lines.
The L2 open reading frame of human papillomavirus type 1a encodes a minor structural protein carrying type-specific antigens
Authors: Komly C A , Breitburd F , Croissant O , Streeck R E
Published: January 2020

The proteins encoded by the open reading frames of papillomavirus genomes and the minor polypeptides detected in purified virions are still poorly defined. We show here by its expression in Escherichia coli that the open reading frame L2 of human papillomavirus type 1a codes for a minor structural protein of Mr 76,000. Antisera raised against a truncated L2-beta-galactosidase fusion protein in which the conserved N-terminal region of L2 is missing are type specific for human papillomavirus type 1 virions and are reactive at high dilutions. Expression of the L2-encoded type-specific antigens thus provides a powerful new tool for the identification of papillomaviruses.
Fluctuation of simian virus 40 (SV40) super T-antigen expression in tumors induced by SV40-transformed mouse mammary epithelial cells
Authors: Butel J S , Wong C , Evans B K
Published: January 2020

Higher-molecular-weight forms of the simian virus 40 (SV40) large tumor antigen (T-Ag), designated super T-Ag, are commonly found in SV40-transformed rodent cells. We examined the potential role of super T-Ag in neoplastic progression by using a series of clonal SV40-transformed mouse mammary epithelial cell lines. We confirmed an association between the presence of super T-Ag and cellular anchorage-independent growth in methylcellulose. However, tumorigenicity in nude mice did not correlate with the expression of super T-Ag. In the tumors that developed in nude mice, super T-Ag expression fluctuated almost randomly. Cell surface iodination showed that super T-Ag molecules were transported to the epithelial cell surface. The biological functions of super T-Ag remain obscure, but it is clear that it is not important for tumorigenicity by SV40-transformed mouse mammary epithelial cells. Super T-Ag may be most important as a marker of genomic rearrangements by the resident viral genes in transformed cells.
Cellular mRNA translation is blocked at both initiation and elongation after infection by influenza virus or adenovirus
Authors: Katze M G , DeCorato D , Krug R M
Published: January 2020

During influenza virus infection, protein synthesis is maintained at high levels and a dramatic switch from cellular to viral protein synthesis occurs despite the presence of high levels of functional cellular mRNAs in the cytoplasm of infected cells (M. G. Katze and R. M. Krug, Mol. Cell. Biol. 4:2198-2206, 1984). To determine the step at which the block in cellular mRNA translation occurs, we compared the polysome association of several representative cellular mRNAs (actin, glyceraldehyde-3-phosphate dehydrogenase, and pHe7 mRNAs) in infected and uninfected HeLa cells. We showed that most of these cellular mRNAs remained polysome associated after influenza viral infection, indicating that the elongation of the proteins encoded by these cellular mRNAs was severely inhibited. Because the polysomes containing these cellular mRNAs did not increase in size but either remained the same size or decreased in size, the initiation step in cellular protein synthesis must also have been defective. Several control experiments established that the cellular mRNAs sedimenting in the polysome region of sucrose gradients were in fact associated with polyribosomes. Most definitively, puromycin treatment of infected cells caused the dissociation of polysomes and the release of cellular, as well as viral, mRNAs from the polysomes, indicating that the cellular mRNAs were associated with polysomes that were capable of forming at least a single peptide bond. A similar analysis was performed with HeLa cells infected by adenovirus, which also dramatically shuts down cellular protein synthesis. Again, it was found that most of the cellular mRNAs, which were translatable in reticulocyte extracts, remained associated with polysomes and that there was a combined initiation-elongation block to cellular protein synthesis. In cells infected by both adenovirus and influenza virus, influenza viral mRNAs were on larger polysomes than were several late adenoviral mRNAs with comparably sized coding regions. In addition, after influenza virus superinfection of cells infected by the adenovirus mutant dl331, a situation in which there is a limitation in the amount of functional initiation factor eIF-2 (M. G. Katze, B. M. Detjen, B. Safer, and R. M. Krug, Mol. Cell. Biol. 6:1741-1750, 1986), influenza viral mRNAs, but not late adenoviral mRNAs, were on polysomes. These results indicate that influenza viral mRNAs are better initiators of translation than are late adenoviral mRNAs.
Progressive reorganization of the host cell cytoskeleton during adenovirus infection
Authors: Staufenbiel M , Epple P , Deppert W
Published: January 2020

Infection of cells with adenovirus lead to a characteristic reorganization of all cytoskeleton systems, starting with alterations at the microtubuli of the cells. During this progress, the flat, extended, and polar morphology of the cytoskeleton became nonpolar and rounder. These rearrangements were initiated before the appearance of adenovirus structural proteins hexon and fiber, as well as before the shutoff of host protein synthesis. We conclude that these alterations reflect a specific reorganization rather than an unorganized breakdown of the cell during adenovirus infection.
Nonecotropic murine leukemia viruses in BALB/c and NFS/N mice: characterization of the BALB/c Bxv-1 provirus and the single NFS endogenous xenotrope
Authors: Hoggan M D , O'Neill R R , Kozak C A
Published: January 2020

We used hybridization probes that react specifically with xenotropic and mink cell focus-forming virus envelope sequences to characterize the nonecotropic proviruses of BALB/c and NFS/N mice. Analysis of somatic cell hybrids with different BALB/c chromosomes showed that the 9 xenotropic and more than 20 MCF virus-related proviral sequences in this mouse were present on more than nine BALB/c chromosomes. Multiple copies were found on chromosomes 1, 4, 7, 12, and probably 11, and the copies found on a single chromosome were not identical by restriction enzyme mapping. We also identified and characterized the proviral sequences that give rise to infectious xenotropic virus in both BALB/c and NFS/N mice. BALB/c contains the major locus for induction of infectious virus in inbred mice, Bxv-1, which is on chromosome 1. We showed that this locus contains a single xenotropic provirus on an 18-kilobase HindIII fragment. Restriction enzyme analysis of a hybrid cell DNA that contains only the Bxv-1 xenotropic provirus showed that the Bxv-1 provirus contains restriction enzyme sites characteristic of the infectious virus induced from BALB/c fibroblasts. The Bxv-1 provirus and its flanking sequences also contain the same restriction sites as the provirus thought to contribute U3 long terminal repeat sequences to leukemogenic (class I) AKR MCF viruses. Analysis of cell hybrids made with the nonvirus-inducible strain NFS/N showed that the single xenotropic virus env gene of NFS mice, here termed Nfxv-1, is not on chromosome 1. Unlike that of Bxv-1, the restriction map of Nfxv-1 does not resemble that of any known infectious xenotropic virus including xenotropic viruses isolated from NFS mice. These data suggest that Bxv-1, but not Nfxv-1, is a full-length xenotropic provirus that can be transcribed directly to produce infectious virus.
Effects of S-adenosylhomocysteine and analogs on Epstein-Barr virus-induced transformation, expression of the Epstein-Barr virus capsid antigen, and methylation of Epstein-Barr virus DNA
Authors: Long W K , Fronko G E , Lindemeyer R G , Wu B , Henderson E E
Published: January 2020

S-Adenosylhomocysteine was found to have no effect on Epstein-Barr virus-induced transformation of B-lymphocytes and to stimulate viral capsid antigen expression only slightly in the FF41-1 cell line. In contrast, the S-adenosylhomocysteine analogs sinefungin and S-isobutyladenosine inhibited Epstein-Barr virus transformation and induced a significant increase in the numbers of cells expressing the viral capsid antigen. An inverse relationship between levels of viral DNA methylation and gene expression was demonstrated.
Localization of neutralizing regions of the envelope gene of feline leukemia virus by using anti-synthetic peptide antibodies
Authors: Elder J H , McGee J S , Munson M , Houghten R A , Kloetzer W , Bittle J L , Grant C K
Published: January 2020

We synthesized 27 synthetic peptides corresponding to approximately 80% of the sequences encoding gp70 and p15E of Gardner-Arnstein feline leukemia virus (FeLV) subtype B. The peptides were conjugated to keyhole limpet hemocyanin and injected into rabbits for preparation of antipeptide antisera. These sera were then tested for their ability to neutralize a broad range of FeLV isolates in vitro. Eight peptides elicited neutralizing responses against subtype B isolates. Five of these peptides corresponded to sequences of gp70 and three to p15E. The ability of these antipeptide antisera to neutralize FeLV subtypes A and C varied. In certain circumstances, failure to neutralize a particular isolate corresponded to sequence changes within the corresponding peptide region. However, four antibodies which preferentially neutralized the subtype B viruses were directed to epitopes in common with Sarma subtype C virus. These results suggest that distal changes in certain subtypes (possibly glycosylation differences) alter the availability of certain epitopes in one virus isolate relative to another. We prepared a "nest" of overlapping peptides corresponding to one of the neutralizing regions of gp70 and performed slot blot analyses with both antipeptide antibodies and a monoclonal antibody which recognized this epitope. We were able to define a five-amino-acid sequence required for reactivity. Comparisons were made between an anti-synthetic peptide antibody and a monoclonal antibody reactive to this epitope for the ability to bind both peptide and virus, as well as to neutralize virus in vitro. Both the anti-synthetic peptide and the monoclonal antibodies bound peptide and virus to high titers. However, the monoclonal antibody had a 4-fold-higher titer against virus and a 10-fold-higher neutralizing titer than did the anti-synthetic peptide antibody. Competition assays were performed with these two antibodies adjusted to equivalent antivirus titers against intact virions affixed to tissue culture plates. The monoclonal antibody had a greater ability to compete for virus binding, which suggested that differences in neutralizing titers may relate to the relative affinities of these antisera for the peptide conformation in the native structure.
Primer-dependent synthesis of covalently linked dimeric RNA molecules by poliovirus replicase
Authors: Lubinski J M , Ransone L J , Dasgupta A
Published: January 2020

Poliovirus-specific RNA-dependent RNA polymerase (replicase, 3Dpol) was purified from HeLa cells infected with poliovirus. The purified enzyme preparation contained two proteins of apparent molecular weights 63,000 and 35,000. The 63,000-Mr polypeptide was virus-specific RNA-dependent RNA polymerase, and the 35,000-Mr polypeptide was of host origin. Both polypeptides copurified through five column chromatographic steps. The purified enzyme preparation catalyzed synthesis of covalently linked dimeric RNA products from a poliovirion RNA template. This reaction was absolutely dependent on added oligo(U) primer, and the dimeric product appeared to be made of both plus- and minus-strand RNA molecules. Experiments with 5' [32P]oligo(U) primer and all four unlabeled nucleotides suggest that the viral replicase elongates the primer, copying the poliovirion RNA template (plus strand), and the newly synthesized minus strand snaps back on itself to generate a template-primer structure which is elongated by the replicase to form covalently linked dimeric RNA molecules. Kinetic studies showed that a partially purified preparation of poliovirus replicase contains a nuclease which can cleave the covalently linked dimeric RNA molecules, generating template-length RNA products.
Transforming and mitogenic effects of avian leukemia virus E26 on chicken hematopoietic cells and fibroblasts, respectively, correlate with level of expression of the provirus
Authors: Jurdic P , Benchaibi M , Gandrillon O , Samarut J
Published: January 2020

We have investigated the effect of E26, an avian leukemia retrovirus, on the growth properties of chicken embryo fibroblasts (CEFs). E26-infected CEFs were not transformed, according to several transformation parameters, but exhibited an activated growth in vitro. They started to grow without latency in serum-supplemented medium, maintained long-term growth in regular or low-serum medium, and could grow when seeded at low cell density in low-serum medium. We compared the integration and the level of expression of the proviral DNA in E26-infected CEFs and E26-transformed hematopoietic cells. An average of two provirus copies were found in each kind of cells. However, whereas high contents of both viral mRNA and E26-specific protein products were found in transformed hematopoietic cells, we detected only low amounts of viral mRNA and no E26 protein in infected CEFs. These data show that the level of expression of the E26 provirus is lower in CEFs than in hematopoietic cells. They suggest that transformation efficiency of the virus depends on its level of expression.
Induction of simian acquired immune deficiency syndrome (SAIDS) with a molecular clone of a type D SAIDS retrovirus
Authors: Heidecker G , Lerche N W , Lowenstine L J , Lackner A A , Osborn K G , Gardner M B , Marx P A
Published: January 2020

We have isolated a molecular clone of the full-length integrated provirus of simian acquired immune deficiency syndrome retrovirus serotype 1 (SRV-1) from a fatal case of simian acquired immune deficiency syndrome in a juvenile rhesus macaque. An integrated SRV-1 provirus was cloned, sequenced, and found to contain four large open reading frames encoding gag-precursor protein, protease, polymerase, and envelope. The proviral clone was transfected into D17 canine osteosarcoma cells and found to produce infectious virus. A comparison of the sequences of this clone with a noninfectious clone showed 20 differences, resulting in 10 amino acid changes. Also, a cluster of exchanges, short insertions, and deletions in the 5' leader sequences resulted in extension of the tRNA(Lys) primer-binding site from 14 to 19 nucleotides. Virus isolated from transfected cells was shown to be infectious and pathogenic, resulting in disease that followed the same time course and mortality as disease induced by uncloned, in vitro cultivated virus isolated from diseased animals. These results unequivocally show that a type D retrovirus (SRV-1) causes a fatal immunosuppressive syndrome in rhesus monkeys.
Diverse wild mouse origins of xenotropic, mink cell focus-forming, and two types of ecotropic proviral genes
Authors: Kozak C A , O'Neill R R
Published: January 2020

We analyzed wild mouse DNAs for the number and type of proviral genes related to the env sequences of various murine leukemia viruses (MuLVs). Only Mus species closely related to laboratory mice carried these retroviral sequences, and the different subclasses of viral env genes tended to be restricted to specific taxonomic groups. Only Mus musculus molossinus carried proviral genes which cross-reacted with the inbred mouse ecotropic MuLV env gene. The ecotropic viral env sequence associated with the Fv-4 resistance gene was found in the Asian mice M. musculus molossinus and Mus musculus castaneus and in California mice from Lake Casitas (LC). Both M. musculus castaneus and LC mice carried many additional Fv-4 env-related proviruses, two of which are common to both mouse populations, which suggests that these mice share a recent common ancestry. Xenotropic and mink cell focus-forming (MCF) virus env sequences were more widely dispersed in wild mice than the ecotropic viral env genes, which suggests that nonecotropic MuLVs were integrated into the Mus germ line at an earlier date. Xenotropic MuLVs represented the major component of MuLV env-reactive genes in Asian and eastern European mice classified as M. musculus molossinus, M. musculus castaneus, and Mus musculus musculus, whereas Mus musculus domesticus from western Europe, the Mediterranean, and North America contained almost exclusively MCF virus env copies. M. musculus musculus mice from central Europe trapped near the M. musculus domesticus/M. musculus musculus hybrid zone carried multiple copies of both types of env genes. LC mice also carried both xenotropic and MCF viral env genes, which is consistent with the above conclusion that they represent natural hybrids of M. musculus domesticus and M. musculus castaneus.