bluuebunny/crossref_metadata_2025 huggingface dataset:
Site-directed mutagenesis of proteinase 3C results in a poliovirus deficient in synthesis of viral RNA polymerase
Authors: Dewalt P G , Semler B L
Published: January 2020

We used a synthetic double-stranded oligonucleotide to introduce amino acid substitutions into the proteinase 3C region of a poliovirus type 1 cDNA clone. The six different mutant viruses recovered exhibited a small-plaque phenotype when assayed on HeLa cells. Further investigation revealed that all the mutations (with the exception of one) yielded P3 region proteins that displayed altered mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A conservative Val----Ala change at amino acid 54 of the proteinase resulted in a virus that was deficient in the production of the mature viral RNA polymerase 3D. Although this mutant achieved less than one-half of the wild-type levels of RNA synthesis during the course of infection, it still grew to nearly wild-type titers.
Biochemical and immunological characterization of the duck hepatitis B virus envelope proteins
Authors: Schlicht H J , Kuhn C , Guhr B , Mattaliano R J , Schaller H
Published: January 2020

To examine the envelope proteins of duck hepatitis B virus (DHBV), which are encoded by the pre-S/S open reading frame of the viral genome, an antiserum was raised in rabbits against a fusion protein comprising most of the pre-S coding segment. By using this antiserum, viral particles could be precipitated from serum, and two pre-S proteins with molecular sizes of approximately 35 and 37 kilodaltons were detected in the sera and livers of DHBV-infected ducks after Western blotting and after biosynthetic labeling of a primary duck liver cell culture. In serum, the pre-S proteins were shown to exist predominantly in DHBV-DNA-free particles associated with a 17-kilodalton protein which, by N-terminal amino acid sequence analysis, was shown to represent the viral S protein which is encoded by the 3' proximal segment of the DHBV pre-S/S open reading frame. To compare the immunogenic potential of the S and pre-S proteins, serum particles and gel-purified S protein were used to immunize rabbits. In neither case was a significant immune response against the DHBV S protein observed. However, a good antibody titer against DHBV pre-S was obtained even after immunization with small amounts of the pre-S antigen.
Bacteriophage P22 gene 23 product acts preferentially in cis
Authors: Riggs P D , Botstein D
Published: January 2020

The expression of the P22 late operon was measured while the activator of the late operon, the product of gene 23, was provided in cis or in trans. It was found that expression of the late operon, assayed from a late-gene-lacZ gene fusion, was reduced by more than twofold when the only functional copy of gene 23 was present in trans.
Expression of a cellular gene cloned in herpes simplex virus: rabbit beta-globin is regulated as an early viral gene in infected fibroblasts
Authors: Smiley J R , Smibert C , Everett R D
Published: January 2020

We constructed nondefective herpes simplex virus type 1 recombinants bearing the intact rabbit beta-globin gene inserted into the viral gene for thymidine kinase to study the expression of a cellular gene when it is present in the viral genome during lytic viral infections. The globin promoter was activated to high levels during productive infection of Vero cells, giving rise to properly spliced and processed cytoplasmic globin transcripts. Expression of globin RNA occurred with early kinetics, was not affected by blocking viral DNA replication, and was strongly inhibited by preventing viral immediate-early protein synthesis with cycloheximide. These results support the hypothesis that temporal control of herpes simplex virus early gene expression is accomplished by mechanisms that are not restricted to viral promoters. In addition, these data show that a cellular transcript can be correctly processed and can accumulate to high levels during viral infection; this indicates that the mechanisms of virally induced shutoff of host RNA accumulation and degradation of host mRNAs do not depend on sequence-specific differentiation between host and viral RNAs. These findings also suggest that herpesviruses have considerable potential as high-capacity gene transfer vectors for a variety of applications.
Restriction of translation of capped mRNA in vitro as a model for poliovirus-induced inhibition of host cell protein synthesis: relationship to p220 cleavage
Authors: Lloyd R E , Jense H G , Ehrenfeld E
Published: January 2020

Poliovirus infection of HeLa cells results in a rapid inhibition of host protein synthesis by a mechanism that does not affect the translation of poliovirus RNA. It has been suggested that this virus-induced translational control results from inactivation of the cap-binding protein complex, and it has been shown that the 220-kilodalton component(s) (p220) of the cap-binding protein complex is cleaved in infected HeLa cells to form antigenically related polypeptides of 100 to 130 kilodaltons. We have previously described an activity in infected cells that specifically restricts translation of capped mRNA in rabbit reticulocyte lysates. Here, we describe further refinements and characterization of restriction assay. We determined that the assay is a good in vitro model for study of host cell shutoff by several criteria: (i) translation was inhibited in both instances at the step involving mRNA binding to ribosomes; (ii) translation of capped mRNA was specifically inhibited, whereas translation of poliovirus RNA was not; (iii) restriction activity appeared in infected cells with kinetics which parallel host cell shutoff; and (iv) restriction activity, like the specific inhibition of host translation, appeared in cells infected in the presence of guanidine-HCl. The restricting activity was partially purified from poliovirus-infected cells and was compared with the virus-induced p220 cleavage activity. Both activities copurified through numerous cell fractionation and biochemical fractionation procedures. However, specific restriction of capped mRNA translation in reticulocyte lysates occurred without complete cleavage of the endogenous p220.
N-terminal deletion in the src gene of Rous sarcoma virus results in synthesis of a 45,000-Mr protein with mitogenic activity
Authors: Laugier D , Marx M , Barnier J V , Poirier F , Genvrin P , Dezélée P , Calothy G
Published: January 2020

Expression of the v-src gene of Rous sarcoma virus in avian embryo neuroretina cells results in transformation and sustained proliferation of these normally resting cells. Transformed neuroretina cells are also tumorigenic upon inoculation into immunodeficient hosts. We have previously described conditional mutants of Rous sarcoma virus encoding p60v-src proteins which induce proliferation of neuroretina cells in the absence of transformation and tumorigenicity. These results suggest that p60v-src is composed of functionally distinct domains which may interact with multiple cellular targets. In this study, we describe a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion of 278 base pairs in the 5' portion of the v-src gene but which has retained the ability to induce proliferation of quail neuroretina cells. The deleted v-src gene encodes a 45,000-molecular-weight phosphoprotein which contains both phosphoserine and phosphotyrosine, is myristylated, and possesses tyrosine kinase activity indistinguishable from that of wild-type p60v-src. Molecular cloning and sequence analysis of the mutant v-src gene have shown that this deletion extends from amino acid 33 to 126 of the wild-type p60v-src. Therefore, this portion of the v-src protein is dispensable for the mitogenic activity of Rous sarcoma virus in neuroretina cells.
Small Nuclear Inclusion Protein Encoded by a Plant Potyvirus Genome Is a Protease
Authors: Carrington James C. , Dougherty William G.
Published: January 2020

Tobacco etch virus, a plant potyvirus, expresses its RNA genome as a large polyprotein precursor which undergoes extensive proteolytic processing to yield seven or more mature products. Two of these products, proteins with apparent molecular weights of 49,000 and 54,000 (49K and 54K proteins), aggregate in the form of crystalline inclusions within the nuclei of infected cells. Cell-free translation of synthetic transcripts was used to map the genes for these two products on the viral genome and to express an enzymatically active protein. The 49K protein was determined to be a viral protease responsible for several cleavages of the polyprotein, including its own autocatalytic excision. Analyses of products expressed from the 49K protein genes which were altered by deletion revealed that only the carboxyl-terminal half was required for proteolytic activity.
Enhancers and trans-acting E2 transcriptional factors of papillomaviruses
Authors: Hirochika H , Broker T R , Chow L T
Published: January 2020

The upstream regulatory regions of human papillomavirus (HPV) types 1, 6b, 7, 11, 16, and 18, bovine papillomavirus type 1, and cottontail rabbit papillomavirus were cloned into transcriptional enhancer assay plasmids which carry the simian virus 40 early promoter lacking its own enhancer and the bacterial gene encoding chloramphenicol acetyltransferase (EC 2.3.1.28) (CAT). Enhancer activity, reflected by CAT gene expression, was detected in all of the upstream regulatory regions tested only when the recombinants were cotransfected with plasmids which express an intact E2 open reading frame of HPV types 1 and 11 or bovine papillomavirus type 1. Each E2 protein stimulated the enhancer from the same virus and, to somewhat lesser degrees, also those from the heterologous viruses. Hence, the enhancer and the E2 protein are functionally conserved among papillomaviruses. There was some nonreciprocity in the extent of trans-activation in heterologous E2-enhancer interactions. Primer extension analyses demonstrated that the E2 proteins increased the abundance of CAT gene mRNA. Tandem multiplication of the HPV type 11 enhancer sequence dramatically increased its response to E2 stimulation; this is possibly relevant to the pathogenicity of papillomaviruses.
Identification of the human papillomavirus type 6b L1 open reading frame protein in condylomas and corresponding antibodies in human sera
Authors: Li C C , Shah K V , Seth A , Gilden R V
Published: January 2020

Genital warts (condylomata acuminata) are among the most frequent sexually transmitted infections. Human papillomavirus type 6 (HPV-6), which is etiologically related to a majority of these lesions, has not been propagated in tissue culture. We generated two forms of HPV-6 viral antigens: a chemically synthesized oligopeptide (referred to as the C-terminal synthetic peptide) corresponding to residues 482 to 495 of the 500-amino-acid-long L1 open reading frame (ORF), and a bacterially expressed 54-kilodalton (kDa) fusion protein containing the N-terminal 13 amino acids encoded by the lambda bacteriophage cII gene followed by one vector-insert junctional residue and 462 amino acids of the L1 ORF sequence (residues 39 to 500). The cII-L1 fusion protein was specifically recognized by an antipeptide serum directed against the N-terminal 13 amino acids derived from the cII gene, an antiserum raised against the C-terminal synthetic peptide, and a genus-specific serum prepared by immunization with disrupted viral capsids. The 54-kDa fusion protein was purified, and the sequence of its first 36 amino acids was determined and found to be as predicted by the DNA sequence. Both the genus-specific anticapsid serum and the antiserum raised against the fusion protein identified authentic L1 ORF proteins in HPV-1-induced (58 kDa) and HPV-6/11-induced (56 kDa) papillomas. The synthetic peptide antiserum recognized the 56- to 58-kDa protein in HPV-6-induced warts, but not in HPV-1- or HPV-11-infected specimens. Using the fusion protein as antigen in immunoassays, we were able to detect the corresponding antibodies in human sera.
Insertion of adenovirus type 12 DNA in the vicinity of an intracisternal A particle genome in Syrian hamster tumor cells
Authors: Lichtenberg U , Zock C , Doerfler W
Published: January 2020

In the adenovirus type 12 (Ad12)-induced hamster tumor T1111(2) about 10 Ad12 genome equivalents were integrated at different sites. One of the integrated copies proved unstable and was lost from the cellular genome or rearranged upon passage of the cell line, H1111(2), established from this tumor. This unstable site of junction between the left terminus of Ad12 DNA and hamster DNA and the preinsertion site from BHK21 hamster cells was cloned, sequenced, and analyzed. The junction site showed several peculiarities. At the left terminus of Ad12 DNA, the first 64 nucleotides were deleted. At a distance of 127 nucleotides to the left from this junction site, an internal dispersed fragment of Ad12 DNA comprising nucleotides 1290 to 1361 of the authentic Ad12 DNA sequence was inserted into cellular DNA in an inverted orientation relative to the complete Ad12 genome that was located in its vicinity. The 127-nucleotide sequence between the intact Ad12 genome and the separate 72-base-pair (bp) Ad12 DNA fragment was cellular, but it was not identical to the preinsertion sequence at this location. The sequences flanking the termini of the dispersed 72-bp Ad12 DNA fragment were characterized by direct repeats of 9 or 10 nucleotides. To the left of Ad12 nucleotide 1361 in the separate 72-bp fragment, about 620 cellular nucleotides followed which were identical at the occupied and at the preinsertion sites. It was conceivable that the separate 72-bp Ad12 DNA fragment and the cellular sequence of 127 bp to its right had been transposed en bloc from another unknown location. Abutting the 620 nucleotides of cellular DNA to the left of this block, the 3'-terminal sequence of an endogenous, intracisternal A particle (IAP) genome of hamster cells was detected. The possible significance of the proximity of an IAP sequence to an inserted Ad12 genome with respect to the transformation event, to the instability at this site, or to the transcriptional activity of this region is not known. The 620 bp of cellular DNA between the 72-bp Ad12 DNA fragment and the end of the long terminal repeat of the hamster IAP sequence was apparently of a unique type. Transcriptional activity was not found in the approximate region between nucleotides -620 (to the left) and +350 (to the right) relative to the site of Ad12 DNA insertion, but was found outside these boundaries.
3'-Azido-3'-deoxythymidine inhibits the replication of avian leukosis virus
Authors: Olsen J C , Furman P , Fyfe J A , Swanstrom R
Published: January 2020

We tested the ability of the thymidine analog 3'-azido-3'-deoxythymidine (BWA509U) to inhibit the replication of the retrovirus avian leukosis virus. Inhibition was measured with two different assays: inhibition of a single round of virus replication and inhibition of virus spread through a cell culture. With both assays, we detected inhibition of virus growth, although inhibition of a single round of virus replication required a 40-fold higher drug concentration than did inhibition of virus spread. We also detected variations in the concentrations of drug needed to inhibit virus replication in different cell types. Higher concentrations of drug were needed to inhibit virus replication in chicken embryo fibroblasts than in the continuous quail cell line QT6. Viral DNA synthesis in infected cells was shown to be inhibited in the presence of the drug. The triphosphate form of the analog acted as a competitive inhibitor of purified viral reverse transcriptase, with a Ki of 0.09 +/- 0.003 microM, and was incorporated as a chain terminator during reverse transcription of the natural viral RNA substrate in vitro.
Genetic control of capsid length in bacteriophage T4: DNA sequence analysis of petite and petite/giant mutants
Authors: Mooney D T , Stockard J , Parker M L , Doermann A H
Published: January 2020

The T4 gene 23 product (gp23) encodes the major structural protein of the mature capsid. Mutations in this gene have been described which disrupt the normal length-determining mechanism (A.H. Doermann, F.A. Eiserling, and L. Boehner, J. Virol. 12:374-385, 1973). Mutants which produce high levels of petite and giant phage (ptg) are restricted to three tight clusters in gene 23 (A.H. Doermann, A. Pao, and P. Jackson, J. Virol. 61:2823-2827, 1987). Twenty-six of these ptg mutations were cloned, and their DNA sequence alterations were determined. Each member of this set of ptg mutants arose from a single mutation, and the set defined 10 different sites at which ptg mutations can occur in gene 23. Two petite (pt) mutations in gene 23 (pt21-34 and ptE920g), which produce high frequencies of petite particles but no giants, were also sequenced. Both pt21-34 and ptE920g were shown to include multiple mutations. The phenotypes attributed to both pt and ptg mutations are discussed relative to the mechanism of capsid morphogenesis. A site-directed mutation (SD-1E) was created at the ptgNg191 site, and its phenotypic consequences were examined. Plaque morphology revertants arising from a gene 23 mutant derivative of pt21-34 and from SD-1E were isolated. A preliminary mapping of the mutation(s) responsible for their revertant phenotypes suggested that both intra- and extragenic suppressors of the petite phenotype can be isolated by this method.
The role of Moloney murine leukemia virus RNase H activity in the formation of plus-strand primers
Authors: Rattray A J , Champoux J J
Published: January 2020

On the basis of earlier studies with both detergent-disrupted virions (the endogenous reaction) and an in vitro reconstructed reaction, the RNase H activity associated with Moloney murine leukemia virus reverse transcriptase has been implicated in the generation of plus-strand RNA primers during reverse transcription. Here we used an oligonucleotide extension assay to show that the RNA primers remaining bound to the plus DNA strands initiated at the normal origin in the in vitro reaction are heterogeneous in length. This result indicates that, although a precise cleavage generates the 3' end of the priming RNA, RNase H exhibits less specificity at other break sites. During the endogenous reaction, a kinetic analysis of the synthesis of plus strands corresponding to different regions of the genome suggested that additional sites for the initiation of plus-strand DNA existed upstream of the normal origin. Direct analysis of fragments produced in the endogenous reaction, as well as in the in vitro reaction, confirmed the existence of upstream plus-strand initiation sites. Several of these sites were mapped to the nucleotide level by the oligonucleotide extension method. A comparison of the nucleotide sequences surrounding the upstream initiation sites with the sequence at the normal plus-strand origin revealed common features, which suggests a mechanism for plus-strand priming based on sequence recognition by the RNase H/reverse transcriptase protein. Although primer removal by RNase H is highly efficient for DNA fragments initiated at the normal origin, the RNA primers were inefficiently removed from the fragments initiated at the upstream sites. This result suggests that primer removal, like primer generation, involves sequence recognition by the enzyme.
Replicative intermediate of hepatitis B virus in transfected murine fibroblasts
Authors: Zelent A Z , Sells M A , Shvartsman M , Price P M , Acs G
Published: January 2020

The NIH 3T3-derived cell line psi AM22b, which carries a defective Moloney murine leukemia virus, was transfected with a plasmid carrying the neo gene and two head-to-tail copies of the hepatitis B virus (HBV) genome positioned with opposing polarities. Both the two HBV dimers and the neo gene were located between two Moloney murine leukemia virus long terminal repeats. Poly(A)+ RNAs isolated from one clone that grew in the presence of G418 contained the two major classes of HBV-specific transcripts (3.5-kilobase pregenome and 2.1-kilobase mRNAs) in approximately equivalent amounts, which was reminiscent of the profiles of viral mRNAs from the livers of infected humans and chimpanzees.
Characterization of a cDNA clone corresponding to a transcript from the Epstein-Barr virus BamHI M fragment: evidence for overlapping mRNAs
Authors: Pfitzner A J , Strominger J L , Speck S H
Published: January 2020

A 1.95-kilobase cDNA clone was isolated by screening a size-selected lambda gt10 cDNA library prepared from an Epstein-Barr virus-transformed B-cell line, IB4, with the Epstein-Barr virus BamHI M fragment. Sequence analysis revealed that this clone contains about 75% of the BMRF1 and the complete BMRF2 open reading frames. The transcript is not spliced, and the polyadenylation signal at base pair 2641 of the BamHI M fragment is used. Northern blots (RNA blots) indicate that this polyadenylation signal is used for three overlapping mRNAs. The sizes of these transcripts are 3.5, 2.6, and 1.5 kilobases.
Alterations in binding characteristics of the human immunodeficiency virus enhancer factor
Authors: Wu F , Garcia J , Mitsuyasu R , Gaynor R
Published: January 2020

Five regions of the human immunodeficiency virus (HIV) long terminal repeat (LTR) serve as binding sites for cellular proteins as demonstrated by DNase I footprinting. These include the negative regulatory, enhancer, SP1, TATA, and untranslated regions. The HIV enhancer region contains two direct repeats of a sequence, GGGACTTTCC, which is also found in the enhancer sequences of simian virus 40, cytomegalovirus, and the immunoglobulin kappa gene. To further characterize binding to the enhancer sequences in the HIV LTR, DNase I footprinting was performed using extracts prepared from several different cell lines. Extracts prepared from lymphoid cells gave altered binding over the enhancer region as compared with extracts prepared from either monocytes or HeLa cells. This altered binding in extracts prepared from lymphoid cells resulted in protection of both direct repeats in the HIV LTR in contrast to complete protection of only one direct repeat with HeLa cell extracts. When HeLa cells were treated with phorbol esters in either the presence or absence of the protein synthesis inhibitor cycloheximide, the binding characteristics over the enhancer element became similar to those seen in extracts prepared from lymphoid cells. These results suggest that phorbol esters may induce posttranslational modifications of cellular transcription factors that alter their DNA-binding characteristics.
Activation of the human beta interferon gene by the adenovirus type 12 E1B gene
Authors: Shiroki K , Toth M
Published: January 2020

The transcription of endogenous beta interferon mRNA was activated in human embryo kidney (HEK) cells infected with adenovirus type 12 (Ad12) but was activated only inefficiently or not at all in HEK cells infected with Ad5 and rc-1 (Ad5 dl312 containing the Ad12 E1A region). The analysis with Ad12 mutants showed that Ad12 E1B products, especially the 19K protein, were important for the expression of the endogenous beta interferon gene and Ad12 E1A products were not involved in the expression. The expression of exogenously transfected pIFN-CAT (a hybrid plasmid having the human beta interferon promoter fused with the CAT gene) was activated in HEK and chicken embryo fibroblast (CEF) cells infected with either Ad12 or Ad5. The analysis of cotransfection of CEF cells with pIFN-CAT and plasmids containing fragments of Ad12 or Ad5 DNA showed that Ad12 or Ad5 E1B (possibly the 19K protein) was and E1A was not involved in the expression of the exogenous pIFN-CAT.
Nonpermissivity of human peripheral blood lymphocytes to adenovirus type 2 infection
Authors: Horvath J , Weber J M
Published: January 2020

The capacity of freshly explanted human peripheral blood lymphocytes (PBL) to support the replication of human adenovirus type 2 (Ad2) was investigated. Unlike other types of human cells, PBL were found to be highly nonpermissive. Ad2 adsorbed 30 to 40% of both T and non-T cells. Virus uncoating was very slow and inefficient, resulting in a 40-fold reduction compared with HEp-2 cells. On a population basis, viral DNA synthesis was reduced 460-fold and infectious virus production was reduced 10(6)-fold. Only 0.35% of PBL produced infectious centers, yielding 0.8 PFU per infected cell. Phytohemagglutinin stimulation increased DNA synthesis 23-fold, infectious centers 11-fold, and virus yield 14-fold. We conclude that resting human PBL are highly nonpermissive to Ad2 infection and that phytohemagglutinin can only marginally lift this nonpermissiveness.
Isolation and characterization of herpes simplex virus type 1 host range mutants defective in viral DNA synthesis
Authors: Carmichael E P , Kosovsky M J , Weller S K
Published: January 2020

Cell lines were generated by cotransfection of Vero cells with pSV2neo and a plasmid containing the herpes simplex virus type 1 (HSV-1) EcoRI D fragment (coordinates 0.086 to 0.194). One such cell line (S22) contained the genes for alkaline exonuclease and several uncharacterized functions. Three mutant isolates of HSV-1 strain KOS which grew on S22 cells but not on normal Vero cells were isolated and characterized. All three mutants (hr27, hr48, and hr156) were defective in the synthesis of viral DNA and late proteins when grown in nonpermissive Vero cells. Early gene expression in cells infected with these host range mutants appeared to be normal at the nonpermissive condition. The mutations were mapped by marker rescue to a 1.5-kilobase fragment (coordinates 0.145 to 0.155). The mutation of one of these mutants, hr27, was more finely mapped to an 800-base-pair region (coordinates 0.145 to 0.151). This position of these mutations is consistent with the map location of a putative 94-kilodalton polypeptide as determined by sequence analysis (D. McGeoch, personal communication). Complementation studies demonstrated that these mutants formed a new complementation group, designated 1-36. The results presented in this report indicate that the 94-kilodalton gene product affected by these mutations may have a direct role in viral DNA synthesis.
Human immunodeficiency virus infection of monoblastoid cells: cellular differentiation determines the pattern of virus replication
Authors: Pauza C D , Galindo J , Richman D D
Published: January 2020

Stringent control of human immunodeficiency virus (HIV) replication was observed in the human monoblastoid cell line U937. A low-multiplicity infection of these cells by the LAV1 strain of HIV was productive for 2.5 days; then virus replication became restricted and no further evidence of virion production was observed. The dramatic decrease in HIV production was due in part of reduced accumulation of cytoplasmic viral RNA and occurred in the absence of evident cytopathic effects. In contrast, infected cells induced to differentiate by phorbol ester, vitamin D3, or lymphokine supernatant did not release markers of HIV despite the accumulation of significant levels of cytoplasmic viral RNA. HIV infection altered the pattern of c-myc RNA accumulation in U937 cells. Expression of this gene changes normally in response to the state of cellular differentiation; in infected cells the level of c-myc expression was correlated to the levels of viral RNA accumulation and not to cellular differentiation. These results suggest that restricted replication of HIV in monocytes might be an important mechanism of virus persistence and demonstrate a relationship between HIV replication and monocyte differentiation.
Antiviral effect of lymphokine-activated killer cells: characterization of effector cells mediating prophylaxis
Authors: Bukowski J F , Yang H , Welsh R M
Published: January 2020

Lymphokine-activated killer (LAK) cells generated by cultivation of C57BL/6 mouse spleen cells in the presence of recombinant interleukin-2 were transferred into natural killer (NK) cell-deficient suckling mouse recipients. These mice were then challenged with either murine cytomegalovirus (MCMV) or lymphocytic choriomeningitis (LCMV) and sacrificed 3 days later. No interleukin 2 infusions were given. Mice receiving as few as 5 x 10(5) LAK cells had several 100-fold decreases in spleen MCMV titers as compared with untreated mice. This treatment had no effect on spleen LCMV titers. The LAK cell cultures contained 10 to 17% NK 1.1+, 50 to 55% Lyt-2+, and 33 to 50% immunoglobulin D+ cells. Double fluorescence labeling and in vitro cytotoxicity assays with fluorescence-activated cell sorting revealed at least two mutually exclusive killer cell populations. NK 1.1+ LAK cells resembled freshly isolated activated NK cells with regard to target cell range (YAC-1 cell killing greater than L-929, P815, and EL-4 cell killing), large granular lymphocyte (LGL) morphology, and decreased ability to lyse interferon (IFN)-treated target cells. Lyt-2+ LAK cells lysed the targets mentioned above but at lower levels and without the differences in susceptibility mentioned above. These Lyt-2+ LAK cells also had a decreased ability to lyse IFN-treated targets, in contrast to classic cytotoxic T lymphocytes, which lyse IFN-treated targets far more efficiently than untreated targets. Purified populations of LAK cells obtained by fluorescence-activated cell sorting were used in the antiviral protection model. The results showed that protection against MCMV could be mediated by NK 1.1+, NK 1.1-, Lyt-2+, Lyt-2-, and IgD- populations but not by IgD+ cells. The five protective populations all had in common the LGL phenotype and cytotoxic activity in vitro. The IgD+ population did not contain LGLs, lyse target cells in vitro, or mediate an antiviral effect in vivo. These results suggest that LAK cells may be therapeutically useful against certain virus infections (MCMV) but not others (LCMV) and that despite their heterogeneity in antigenic phenotype and cytotoxic activity, their pattern of antiviral activity in vivo resembles that of NK cells, which protect against MCMV but not LCMV.
Investigation of the role of P130gag-fps in transformation: generation and use of a temperature-sensitive mutant P130gag-fps
Authors: Weinmaster G A , Hunter T
Published: January 2020

Changing Glu-1025 to Asp in Fujinami sarcoma virus P130gag-fps made the protein temperature sensitive for transformation and protein-tyrosine kinase activity. Another mutant, Phe-1073 P130gag-fps, lacking the major autophosphorylation site, has an extended latent period for transformation (G. A. Weinmaster, M. J. Zoller, M. Smith, E. Hinze, and T. Pawson, Cell 37:559-568, 1984). By introducing the Asp-1025 lesion into Phe-1073 P130gag-fps, we showed that this mutant protein is required for the maintenance of the transformed phenotype of Phe-1073 P130gag-fps-expressing cells.
Genetic polymorphism of natural Epstein-Barr virus isolates from infectious mononucleosis patients and healthy carriers
Authors: Lung M L , Chang R S , Jones J H
Published: January 2020

We analyzed Epstein-Barr virus (EBV) genomes from lymphoblastoid cell lines isolated from patients with infectious mononucleosis and from healthy subjects from California, Hawaii, and Hong Kong between 1970 and 1987. Using genetic polymorphism as epidemiological markers, we found that several genotypes of EBV cocirculate in a community and that although most EBV strains isolated from California and Southern China may be differentiated genotypically, there was no specific association between genotype and disease or time of isolation.
Identification of a common helper provirus integration site in Abelson murine leukemia virus-induced lymphoma DNA
Authors: Poirier Y , Kozak C , Jolicoeur P
Published: January 2020

Abelson murine leukemia virus induces oligoclonal pre-B lymphoma in mice. The expression of the v-abl oncogene in target cells does not appear to be sufficient for tumor induction in several mouse strains, and additional genetic events are thought to be required. We postulated that the helper Moloney murine leukemia virus might induce these events, and its potential role as an insertional mutagen was assessed by the search of a common helper provirus integration site in Abelson murine leukemia virus lymphomas. Molecular cloning of cellular sequences adjacent to Moloney proviruses enabled us to identify a cellular region, designated Ahi-1, which was found occupied by the helper proviruses in 16% of Abelson pre-B-cell lymphomas. All proviruses for which the precise integration site within Ahi-1 could be mapped were found to be in the same orientation. Ahi-1 has been mapped to mouse chromosome 10 and represents a new common proviral integration site. These data suggest that the helper virus contributes to the induction of secondary genetic events which may be important for the development of Abelson murine leukemia virus-induced pre-B-cell lymphoma.
A transcription factor from simian virus 40 chromosomes which activates the viral late promoter in vitro
Authors: Beard P , Bruggmann H
Published: January 2020

We have characterized a transcription factor, obtained from simian virus 40 (SV40) chromosomes, which activates transcription from the SV40 late promoter in vitro. The late promoter-activating factor was distinct from SV40 T antigen as judged by its behavior on chromatography on hydroxylapatite; it was not recognized by anti-T antibodies, while T antigen itself was recognized. T antigen from SV40 chromosomes, on the other hand, abolished transcription in vitro from the early promoter. In DNase I footprinting experiments, a partially purified late promoter-activating factor preparation protected a region of DNA centered on SV40 nucleotide 270, which is between the repeated 72-base-pair enhancer and the major late RNA start site. Proteins from HeLa cells did not give the same footprint at this position. Gel mobility shift assays showed that proteins from SV40-infected CV-1 cells form a complex with DNA containing this binding site. The complex has a different rate of gel migration and a higher stability than complexes formed with proteins from uninfected cells.
Functional organization of the murine leukemia virus reverse transcriptase: characterization of a bacterially expressed AKR DNA polymerase deficient in RNase H activity
Authors: Levin J G , Crouch R J , Post K , Hu S C , McKelvin D , Zweig M , Court D L , Gerwin B I
Published: January 2020

The functional organization of the murine leukemia virus reverse transcriptase was investigated by expressing a molecular clone containing AKR MuLV reverse transcriptase-coding sequences in Escherichia coli. A purified preparation of the expressed enzyme (pRT250 reverse transcriptase) consisted primarily of a 69-kilodalton protein that has normal levels of murine leukemia virus polymerase activity but 10-fold-reduced levels of RNase H compared with the viral enzyme. The deficit in RNase H activity was correlated with the absence of 60 to 65 amino acids normally present at the carboxyl end of murine leukemia virus reverse transcriptase. The results provide additional experimental evidence for the localization of polymerase and RNase H domains to the N- and C-terminal regions of reverse transcriptase, respectively.
Vesicular stomatitis virus M protein in the nuclei of infected cells
Authors: Lyles D S , Puddington L , McCreedy B J
Published: January 2020

The M protein of vesicular stomatitis virus (VSV) was localized in the nuclei and cytoplasm of VSV-infected cells by subcellular fractionation and immunofluorescence microscopy. Nuclei isolated from VSV-infected Friend erythroleukemia cells were fractionated into a nuclear membrane and a nucleoplasm fraction by DNase digestion and differential centrifugation. G protein was present in the membrane fraction, and M protein was present in the nucleoplasm fraction. Immunofluorescence detection of M protein in the nucleus required that fixed cells be permeabilized with higher concentrations of detergent than were required for detection of M protein in the cytoplasm of VSV-infected BHK cells.
DNA-binding activity associated with the v-myb oncogene product is not sufficient for transformation
Authors: Ibanez C E , Garcia A , Stober-Grässer U , Lipsick J S
Published: January 2020

The product of the v-myb oncogene of avian myeloblastosis virus is a nuclear protein with an associated DNA-binding activity. We demonstrated that the highly conserved amino-terminal domain of p48v-myb is required for its associated DNA-binding activity. This activity is not required for the nuclear localization of p48v-myb. Furthermore, the associated DNA-binding activity and nuclear localization of p48v-myb together are not sufficient for transformation.
Effects and virulences of recombinant vaccinia viruses derived from attenuated strains that express the human T-cell leukemia virus type I envelope gene
Authors: Shida H , Hinuma Y , Hatanaka M , Morita M , Kidokoro M , Suzuki K , Maruyama T , Takahashi-Nishimaki F , Sugimoto M , Kitamura R
Published: January 2020

We constructed recombinant vaccinia viruses (RVVs) that expressed human T-cell leukemia virus type I (HTLV-I) envelope glycoproteins by using attenuated vaccinia viruses (VVs) which have much lower neurovirulence than the WR strain that is extensively used as a vector. The RVV produced from the LC16mO strain, one of the attenuated VVs, elicited a high titer of anti-HTLV-I antibody in rabbits and protected them against HTLV-I infection. The env gene was inserted into the VV hemagglutinin gene. The resultant inactivation of the hemagglutinin gene led to the attenuation of VVs, but the extent of their attenuation depended on the VV strain. The propagation of LC16mO and its RVV in rabbit brain was poorer than that of LO-1, a cloned derivative of Lister strain, and its RVV, although LC16mO replicated in other organs better than did LO-1. Taken together, these results suggest that LC16mO is a good candidate as a vector for vaccination of humans.
Herpes simplex virus (HSV)-specific proliferative and cytotoxic T-cell responses in humans immunized with an HSV type 2 glycoprotein subunit vaccine
Authors: Zarling J M , Moran P A , Brewer L , Ashley R , Corey L
Published: January 2020

Studies were undertaken to determine whether immunization of humans with a herpes simplex virus type 2 (HSV-2) glycoprotein-subunit vaccine would result in the priming of both HSV-specific proliferating cells and cytotoxic T cells. Peripheral blood lymphocytes (PBL) from all eight vaccines studied responded by proliferating after stimulation with HSV-2, HSV-1, and glycoprotein gB-1. The PBL of five of these eight vaccines proliferated following stimulation with gD-2, whereas stimulation with gD-1 resulted in relatively low or no proliferative responses. T-cell clones were generated from HSV-2-stimulated PBL of three vaccinees who demonstrated strong proliferative responses to HSV-1 and HSV-2. Of 12 clones studied in lymphoproliferative assays, 9 were found to be cross-reactive for HSV-1 and HSV-2. Of the approximately 90 T-cell clones isolated, 14 demonstrated HSV-specific cytotoxic activity. Radioimmunoprecipitation-polyacrylamide gel electrophoresis analyses confirmed that the vaccinees had antibodies only to HSV glycoproteins, not to proteins which are absent in the subunit vaccine, indicating that these vaccinees had not become infected with HSV. Immunization of humans with an HSV-2 glycoprotein-subunit vaccine thus results in the priming of T cells that proliferate in response to stimulation with HSV and its glycoproteins and T cells that have cytotoxic activity against HSV-infected cells. Such HSV-specific memory T cells were detected as late as 2 years following the last boost with the subunit vaccine.
c-myc products trans-activate the adenovirus E4 promoter in EC stem cells by using the same target sequence as E1A products
Authors: Onclercq R , Gilardi P , Lavenu A , Cremisi C
Published: January 2020

Using a short-term transfection assay, we show that the E4 early adenovirus promoter is expressed to a certain extent in undifferentiated F9 and PCC4 cells, which are known to possess cellular E1A-like activity. We have also observed that c-myc products trans-activate the E4 promoter in EC stem cells and HeLa cells. Using 5' deletion mutants of the E4 promoter, we show that the same target sequence is used by c-myc and E1A. This sequence is located between positions -179 and -158 upstream of the cap site and is known to contain an activating transcription factor (ATF)-binding site. Moreover, the basal of level of activity of the deletion mutants. is related to the number of ATF binding sites. We therefore suggest that c-myc is a functional cellular homolog of the viral E1A gene and that it might correspond to one of the cellular E1A-like activities previously described for EC stem cells. We have also observed that only a c-myc plasmid coding for both p67 and 64 proteins, in contrast to one coding for p64 only, is able to trans-activate the E4 and E2A adenovirus promoter, suggesting that the p67 protein plays an essential part in activation.
Novel segregation patterns of infecting-mutant genotypes in plate complementation tests among amber mutants of bacteriophage BF23
Authors: Mizobuchi K , Nagasu T
Published: January 2020

Amber mutants of bacteriophage BF23 were classified into two functional groups, types I and II, by the yields of the infecting-mutant genotypes in plate complementation tests. Type I mutants produced their genotypes at levels more than 20% of the total progeny phages, and type II mutants did so at levels of less than 5%. Comparison of the results of plate complementation tests with those of extract complementation tests revealed that all the type I mutants were defective in the tail formation, while most type II mutants were defective in the formation of either mature heads (type IIa) or both mature heads and tails (type IIb). Since in extract complementation tests the activated phages are always of genotypes corresponding to mutations defective in only the tail formation, the plate complementation test is comparable with the extract complementation test when judged on the basis of the yield of the mutant genotypes. Of 29 complementation groups, 8 type I, 14 type IIa, and 5 type IIb mutants were identified. Previously, amber mutations of BF23 were mapped on four genetic segments. These segments were ordered in one linkage map by crosses between deletion and amber mutants.
Functional role of BK virus tumor antigens in transformation
Authors: Nakshatri H , Pater M M , Pater A
Published: January 2020

We have examined the role of the human papovavirus BK virus (BKV) tumor (T) antigen(s) in the maintenance of transformation and have identified the domain of T antigen essential for transformation. BKV-transformed BHK 21 and NIH 3T3 cells expressing antisense T-antigen RNA lose their ability to grow in soft agar, indicating the need for the continued expression of T antigen for the maintenance of the transformed phenotype. Experiments using translation termination linker insertion and deletion mutagenesis of BKV T antigen demonstrate that amino acids 356 to 384 are essential for transformation. Although BKV T antigen shares 100, 95, and 82% amino acid homology with that of simian virus 40 (SV40) for the nuclear localization signal, p53-binding domain, and DNA-binding domain, respectively, the transformation domains of BKV and SV40 T antigens share only 54% homology. Also, BKV T antigen lacks a substantial portion of the ATPase domain of SV40, and our results indicate the dispensability of the remaining portion for transformation by this protein. We suggest that the differences in the amino acids in the identified transformation domains together with the differences in the ATPase domains may account for the differences in the transformation potentials of the two proteins.
Complex between glycoproteins gI and gp63 of pseudorabies virus: its effect on virus replication
Authors: Zuckermann F A , Mettenleiter T C , Schreurs C , Sugg N , Ben-Porat T
Published: January 2020

To ascertain the biological functions of different glycoproteins that are nonessential for pseudorabies virus growth in vitro, we have constructed mutants defective in one (or a combination) of these glycoproteins and have examined various aspects of their role in the infective process. We made the following two observations. (i) Glycoproteins gI and gp63 are noncovalently complexed to each other. They are coprecipitated by antisera against either one of these glycoproteins but do not share antigenic determinants: monoclonal antibodies against gp63 do not immunoprecipitate gI from extracts of gp63- mutant-infected cells, and monoclonal antibodies against gI do not immunoprecipitate gp63 from extracts of gI- mutant-infected cells. (ii) Mutants unable to synthesize either gI or gp63 have some common biological characteristics; they have a growth advantage in primary chicken embryo fibroblasts. Furthermore, we have shown previously that in conjunction with glycoprotein gIII, gI and gp63 are necessary for the expression of virulence (T. C. Mettenleiter, C. Schreurs, F. Zuckermann, T. Ben-Porat, and A. S. Kaplan, J. Virol. 62, 2712-2717, 1988). These results show that the functional entity affecting virus replication in chicken embryo fibroblasts, as well as affecting virulence, is the complex between gI and gp63. The gI-gp63 complex of pseudorabies virus does not appear to have Fc receptor activity as does its homolog, the gI-gE complex of herpes simplex virus.
Cooperative transforming activities of ras, myc, and src viral oncogenes in nonestablished rat adrenocortical cells
Authors: MacAuley A , Pawson T
Published: January 2020

Early-passage rat adrenocortical cells were infected with Kirsten murine sarcoma virus and MMCV mouse myc virus, two retroviruses carrying the v-Ki-ras and v-myc oncogenes, respectively. Efficient morphological transformation required coinfection with the two viruses, was dependent on the presence of high serum concentrations, and was not immediately accompanied by growth in soft agar. The doubly infected cells coordinately acquired the capacity for anchorage- and serum-independent growth during passage in culture. The appearance of such highly transformed cells was correlated with the emergence of a dominant clone, as suggested by an analysis of retrovirus integration sites. These results indicate that the concerted expression of v-Ki-ras and v-myc could induce rapid morphological transformation of nonestablished adrenocortical cells but that an additional genetic or epigenetic event was required to permit full transformation by these two oncogenes. In contrast, v-src, introduced by retrovirus infection in conjunction with v-myc, rapidly induced serum- and anchorage-independent growth. Therefore, the p60v-src protein-tyrosine kinase, unlike p21v-ras, is apparently not restricted in the induction of a highly transformed phenotype in adrenocortical cells. This system provides an in vitro model for the progressive transformation of epithelial cells by dominantly acting oncogenes.
Integration of Friend murine leukemia virus into both alleles of the p53 oncogene in an erythroleukemic cell line
Authors: Hicks G G , Mowat M
Published: January 2020

The Friend virus-transformed erythroleukemic cell line DP16-9B4 has undergone a complex rearrangement of the p53 oncogene and lacks any detectable expression of the p53 protein. We report here characterization of both p53 alleles in this cell line and identify independent integrations of Friend murine leukemia virus sequences into the coding region of both alleles.
Recombinant retroviruses encoding cell surface antigens as selectable markers
Authors: Strair R K , Towle M J , Smith B R
Published: January 2020

Recombinant retroviruses are frequently used in the transfer and analysis of genes. This report describes new retrovirus vectors that incorporate a cDNA copy of a cell surface antigen to function as a selectable marker. By using techniques based on quantitative cell surface immunofluorescence, these vectors allow the rapid detection and isolation of infected cells. These vectors also allow the rapid detection of packaging cell lines producing large amounts of recombinant retroviruses. Potential applications of these vectors are demonstrated.