bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a new neutralization antigenic site on poliovirus coat protein VP2
Authors: Emini E A , Jameson B A , Wimmer E
Published: January 2020

Major neutralization antigenic sites have been previously mapped by us on VP1, the largest capsid protein of poliovirus type 1. Here we report the first identification of the primary sequence of a neutralization antigenic site on capsid protein VP2. Inspection of the amino acid sequence of VP2 led to the selection and synthesis of a peptide (n = 12) that, after linking to a carrier protein, induced an antiviral neutralizing antibody response in rabbits. The response was augmented by a single subsequent inoculation of intact virus; thus, the peptide was also capable of priming the production of neutralizing antibodies. These antibodies were directed only against the site specified by the synthetic peptide. Although the VP2-specific neutralization antigenic site appears not to be strongly immunogenic in the intact virion, it can nevertheless contribute to neutralization of poliovirus. This observation may be important for the development of peptide vaccines.
Epstein-Barr virus-transformed lymphocytes produce monoclonal autoantibodies that react with antigens in multiple organs
Authors: Garzelli C , Taub F E , Scharff J E , Prabhakar B S , Ginsberg-Fellner F , Notkins A L
Published: January 2020

Peripheral blood lymphocytes from normal individuals and patients with autoimmune abnormalities such as insulin-dependent diabetes mellitus and thyroiditis were infected with Epstein-Barr virus, and the culture supernatants were tested for autoantibodies that reacted with normal tissues. Between 58 and 86% of Epstein-Barr virus-transformed cultures produced immunoglobulin M antibodies, and between 9 and 24% of the transformed cultures produced immunoglobulin G antibodies that reacted with normal tissues. Ten Epstein-Barr virus-transformed clones secreting human immunoglobulin M monoclonal autoantibodies were isolated. Four of these monoclonal autoantibodies were studied in depth and found to react with antigens in multiple organs, including thyroid, pancreas, stomach, smooth muscle, and nerves. It is concluded that Epstein-Barr virus can trigger the production of autoantibodies without infecting the target cells to which the autoantibodies are directed.
Zosteriform spread of herpes simplex virus as a model of recrudescence and its use to investigate the role of immune cells in prevention of recurrent disease
Authors: Simmons A , Nash A A
Published: January 2020

During the development of a zosteriform rash, which occurs after flank inoculation of BALB/c mice with herpes simplex virus, clinically normal skin becomes infected via nerve endings. This is analogous to the final step in the development of a recrudescent lesion, which may occur after reactivation of latent virus. Therefore, the zosteriform reaction has potential as a model with which to study the modification of such a recrudescent infection by immune processes. Using an adoptive transfer system, we confirmed that immune lymph node cells are potent in accelerating the clearance of virus from the primary site of replication (the inoculation site). This effect was T cell dependent. However, if injection of the same cell population was delayed until ganglionic infection was established, the appearance of the zosteriform rash was not prevented, and the virus titer recovered from the lower flank was not reduced. Immunoperoxidase studies showed that virus is at first highly localized to the epidermis after it emerges from nerves. As determined by conventional histology, little cellular infiltration was seen until clinical lesions were apparent. These observations indicate that recrudescent lesions appear in the presence of cell populations normally associated with rapid virus clearance; cellular immune mechanisms may be rendered ineffective owing to the lack of recruitment to the site of recrudescence until tissue breakdown instigates an inflammatory response.
Acquisition of serum antibodies to specific viral glycoproteins of parainfluenza virus 3 in children
Authors: Kasel J A , Frank A L , Keitel W A , Taber L H , Glezen W P
Published: January 2020

A radioimmunoprecipitation assay was used to study antibody responses to parainfluenza virus 3 glycoproteins in human sera. The method was not only more sensitive than the neutralization test for the detection of antibody but also provided semiquantitative assessments of the antibody response to both glycoproteins in a single assay system. Anti-hemagglutinin-neuraminidase titers were consistently higher than anti-fusion levels in the same serum specimen. Thirteen children were monitored serologically and virologically from birth until 12 months or more after their primary infection with parainfluenza virus 3. At 1 to 3 months after infection, a significant increase in the level of antibody to the hemagglutinin-neuraminidase protein developed in 12 children; of these, 9 showed rises in the level of fusion protein. In 11 of the children, antibody titers continued to rise and the geometric mean titers to the hemagglutinin-neuraminidase protein was highest in sera collected 8 to 10 months after primary infection. Reinfection as the reason for these progressive increases in antibody levels could only be confirmed for four of the children. Three other children had reinfections after the 10-month sera were obtained; in each instance the only antibody responses were to the fusion protein.
Cellular localization of c-fps gene product NCP98
Authors: Young J C , Martin G S
Published: January 2020

We compared the intracellular location of the product of the c-fps proto-oncogene, NCP98, with that of its viral homolog P140, the transforming protein of Fujinami sarcoma virus. Using the technique of biochemical subcellular fractionation, we determined that 60 to 90% of NCP98 and its associated kinase activity are in the soluble fraction of a chicken myeloblast cell line. This fractionation behavior differs from that of P140, which is found predominantly in the particulate fraction, both in Fujinami sarcoma virus-infected chicken embryo fibroblasts and in Fujinami sarcoma virus-infected myeloblasts. The fractionation behavior of NCP98 is, however, similar to that of the P140 encoded by a temperature-sensitive strain of Fujinami sarcoma virus in infected cells grown at the nonpermissive temperature. The absence of gag sequences from NCP98 is not responsible for the difference in fractionation behavior: the v-fps transforming protein of strain F36, P91, which lacks gag sequences, is also predominantly particulate. These results indicate that association with cellular structural components correlates with the transforming activity of proteins containing fps sequences.
An accessory role for Escherichia coli integration host factor: characterization of a lambda mutant dependent upon integration host factor for DNA packaging
Authors: Bear S E , Court D L , Friedman D I
Published: January 2020

Bacteriophage lambda grows lytically on Escherichia coli defective for integration host factor, a protein involved in lambda site-specific recombination and the regulation of gene expression. We report the characterization of a mutant, lambda cos154, that, unlike wild-type lambda, is defective for growth in integration host factor-defective E. coli. The cis-dominant mutation in lambda cos154 is a single base pair change in a region of hyphenated dyad symmetry close to the lambda left cohesive end; this mutation prevents DNA packaging. We propose the following two alternative roles for this site in lambda DNA packaging: (i) to bind an E. coli accessory protein required in the absence of integration host factor or (ii) to bind the phage-encoded terminase protein that is essential for DNA packaging.
Epitopes of herpes simplex virus type 1 glycoprotein gC are clustered in two distinct antigenic sites
Authors: Marlin S D , Holland T C , Levine M , Glorioso J C
Published: January 2020

Epitopes of herpes simplex virus type 1 (HSV-1) strain KOS glycoprotein gC were identified by using a panel of gC-specific, virus-neutralizing monoclonal antibodies and a series of antigenic variants selected for resistance to neutralization with individual members of the antibody panel. Variants that were resistant to neutralization and expressed an antigenically altered form of gC were designated monoclonal antibody-resistant (mar) mutants. mar mutants were isolated at frequencies of 10(-3) to 10(-5), depending on the antibody used for selection. The epitopes on gC were operationally grouped into antigenic sites by evaluating the patterns of neutralization observed when a panel of 22 antibodies was tested against 22 mar mutants. A minimum of nine epitopes was identified by this process. Three epitopes were assigned to one antigenic site (I), and six were clustered in a second complex site (II) composed of three distinct subsites, IIa, IIb, and IIc. The two antigenic sites were shown to reside in physically distinct domains of the glycoprotein, by radioimmunoprecipitation of truncated forms of gC. These polypeptides lacked portions of the carboxy terminus and ranged in size from approximately one-half that of the wild-type molecule to nearly full size. Antibodies recognizing epitopes in site II immunoprecipitated the entire series of truncated polypeptides and thereby demonstrated that site II resided in the N-terminal half of gC. Antibodies reactive with site I, however, did not immunoprecipitate fragments smaller than at least two-thirds the size of the wild-type polypeptide, suggesting that site I was located in the C-terminal portion. Sites I and II were also shown to be spatially separate on the gC polypeptide by competition enzyme-linked immunosorbent assay with monoclonal antibodies representative of different site I and site II epitopes.
Two related but differentially expressed potential membrane proteins encoded by the EcoRI Dhet region of Epstein-Barr virus B95-8
Authors: Hudson G S , Farrell P J , Barrell B G
Published: January 2020

Three mRNAs in the EcoRI Dhet region of Epstein-Barr virus B95-8 were mapped. Their 3' ends are coterminal. A latent gene containing three exons is transcribed from the ED-L1 promoter and was predicted to lead to expression of a 42-kilodalton protein. An unspliced late mRNA is produced by transcription from the ED-L1A promoter within the first intron of the above gene and was predicted to lead to expression of a 28-kilodalton protein corresponding to the C-terminal two-thirds of the 42-kilodalton protein. Both proteins would have hydrophobic N-terminal domains and highly acidic C-terminal domains and were predicted to span the cell membrane. An early promoter (ED-L2) is located in the 3' untranslated region of the above genes and was predicted to lead to expression of a 6.5-kilodalton polypeptide containing a C-terminal hydrophobic region.
Role of antibody in primary and recurrent herpes simplex virus infection
Authors: Simmons A , Nash A A
Published: January 2020

When herpes simplex virus was inoculated into the flank of a BALB/c mouse by scarification, the local replication of virus was followed by the establishment of an acute ganglionic infection. The subsequent centrifugal spread of this virus along nerves to the skin of the whole dermatome led to the development of a bandlike "zosteriform" rash. This represents a highly reproducible system in which virus travels through the nervous system synchronously in large numbers of animals. The transection of peripheral nerves at various times after infection showed that the virus had completed the round trip 60 h after inoculation into the upper flank and was detectable as infectious virus by 74 h postinfection. After the administration of virus, neutralizing but not nonneutralizing antibodies prevented the development of the zosteriform rash. The target epitopes of the protective antibodies were not confined to a single glycoprotein. Neutralizing antibody was effective even when given up to 60 h postinfection and was protective even when administered after sensory neurotomy at this time. Antibody was therefore able to prevent clinically and virologically detectable infection of the skin, presumably by acting peripherally on virus emerging from nerve endings. A quantitative estimate of the action of one of the neutralizing monoclonal antibody preparations, AP7, showed that high titers (several times higher than those normally found in immune mice) were needed to prevent this type of infection. These results are discussed in relation to antibody prophylaxis.
Genetic analysis of bovine papillomavirus type 1 trans-acting replication factors
Authors: Lusky M , Botchan M R
Published: January 2020

The establishment of bovine papillomavirus type 1 in somatic mammalian cells is mediated by extrachromosomal replication and stable maintenance of the viral genome as a multicopy nuclear plasmid. Previous studies indicated the requirement of viral gene expression for bovine papillomavirus type 1 replication and plasmid maintenance (M. Lusky and M. R. Botchan, Cell 36:391-401, 1984; Turek et al., Proc. Natl. Acad. Sci. U.S.A. 79:7914-7918, 1982). To define the viral genes which are necessary for this process, we constructed a series of specific mutations within the viral genome and assayed the resulting mutants for their ability to replicate extrachromosomally in mouse C127 cells. We report here that the bovine papillomavirus type 1 trans-acting replication factors were encoded by at least two distinct viral genes since the mutants fell into two complementation groups, rep and cop. Mutants (rep-) affecting the E1 open reading frame (ORF) failed to replicate bovine papillomavirus type 1 DNA extrachromosomally and would integrate into chromosomal DNA. We suggest that this gene product is one of the factors required to specifically preclude the integration event. Mutants (cop-) affecting the E7 ORF were maintained in the extrachromosomal state; however, the copy number of the mutant genomes was reduced 100-fold compared with that of wild-type DNA. Analysis of single-cell subclones showed that each cell contained the mutant genomes at a copy number of one to two, indicating that the cop- phenotype did not reflect a simple segregation defect. We propose that the gene defined by mutations in the E7 ORF played a crucial role in stably maintaining the copy number of the viral plasmid at high levels. Genomes with mutations in the cop and rep complementation groups, when cotransfected, rescued the wild-type phenotype, extrachromosomal replication with a high, stable copy number for both types of plasmids. Therefore, the gene products acted in trans, and the mutations were recessive to the wild-type functions. One specific rep- mutant showed a 30-fold-increased transformation efficiency when compared with that of the wild-type genome. In addition, morphological transformation mediated by the cop- mutants appeared to be unstable. These results imply that either or both of the replication functions played some role in regulating the expression of the viral transforming functions.
The S RNA segment of lymphocytic choriomeningitis virus codes for the nucleoprotein and glycoproteins 1 and 2
Authors: Riviere Y , Ahmed R , Southern P J , Buchmeier M J , Dutko F J , Oldstone M B
Published: January 2020

The lymphocytic choriomeningitis virus (LCMV) genome consists of a large RNA segment and a small RNA segment. The three major structural proteins of this virus are an internal nucleoprotein and two surface glycoproteins. Intertypic reassortants between the Armstrong and WE strains of LCMV were made to map proteins encoded by the LCMV genome segments. Using monoclonal antibodies specific for the nucleoprotein and the glycoproteins of WE and Armstrong, we showed that the small RNA segment of LCMV codes for the three major structural polypeptides.
Nucleotide sequence analysis of the BALB/c murine sarcoma virus transforming gene
Authors: Reddy E P , Lipman D , Andersen P R , Tronick S R , Aaronson S A
Published: January 2020

We determined the nucleotide sequence of the v-H-ras-related oncogene of BALB/c murine sarcoma virus. This oncogene contains an open reading frame of 189 amino acids that initiates and terminates entirely within the mouse cell-derived ras sequence. The protein encoded by this open reading frame matches the sequence predicted for the T24 human bladder carcinoma oncogene product, p21, in all but two positions. The presence of a lysine residue in position 12 of BALB/c murine sarcoma virus p21 likely accounts for its oncogenic properties.
A weakly pathogenic Rauscher spleen focus-forming virus mutant that lacks the carboxyl-terminal membrane anchor of its envelope glycoprotein
Authors: Machida C A , Bestwick R K , Kabat D
Published: January 2020

A mutant Rauscher spleen focus-forming virus (mutant 4-3) that causes mild splenic erythroblastosis in mice has a 44-base-pair deletion in the 3' region of its envelope glycoprotein (env) gene. The encoded glycoprotein terminates prematurely, lacks a hydrophobic membrane anchor, and has a shortened intracellular lifespan. An active site for causing erythroblast proliferation may occur in the undamaged amino-terminal domain of the env glycoprotein.
Neutralizing monoclonal antibodies to human rotavirus and indications of antigenic drift among strains from neonates
Authors: Coulson B S , Fowler K J , Bishop R F , Cotton R G
Published: January 2020

Cells producing neutralizing monoclonal antibodies to a serotype 3 human neonatal rotavirus strain RV-3 were derived by fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. As ascites fluid, three rotavirus-neutralizing monoclonal antibodies were characterized by hemagglutination inhibition and reacted with 17 cultivable mammalian rotaviruses representing five virus serotypes, by fluorescent focus neutralization and enzyme immunoassay. Two antibodies, Mab RV-3:1 and Mab RV-3:2, reacted with the seven serotype 3 rotaviruses only. Mab RV-3:1 was shown to bind to the outer capsid glycoprotein gp34 of rotavirus when variants of SA 11 rotavirus were used, and it therefore appears to react with the major neutralization epitope of serotype 3 rotaviruses. The antibody Mab RV-3:3 was specific for an epitope of RV-3 rotavirus not present on any other rotavirus of any serotype tested, including another neonatal isolate of identical RNA electropherotype isolated from the same ward of the same hospital as RV-3 3 months earlier. These two viruses were also distinguishable by fluorescent focus neutralization, using antiserum to RV-3 virus. Western blot analysis showed binding of Mab RV-3:3 to the trypsin cleavage product of the outer capsid protein p86 of RV-3. This suggests that antigenic drift may have occurred among neonatal rotaviruses in Melbourne. These monoclonal antibodies will be useful in serotyping assays of rotaviruses directly in stool samples, and in further analysis of antigenic variation within the serotype.
Characterization of virulent and avirulent A/chicken/Pennsylvania/83 influenza A viruses: potential role of defective interfering RNAs in nature
Authors: Bean W J , Kawaoka Y , Wood J M , Pearson J E , Webster R G
Published: January 2020

In April 1983, an influenza virus of low virulence appeared in chickens in Pennsylvania. Subsequently, in October 1983, the virus became virulent and caused high mortality in poultry. The causative agent has been identified as an influenza virus of the H5N2 serotype. The hemagglutinin is antigenically closely related to tern/South Africa/61 (H5N3) and the neuraminidase is similar to that from human H2N2 strains (e.g., A/Japan/305/57) and from some avian influenza virus strains (e.g., A/turkey/Mass/66 [H6N2]). Comparison of the genome RNAs of chicken/Penn with other influenza virus isolates by RNA-RNA hybridization indicated that all of the genes of this virus were closely related to those of various other influenza virus isolates from wild birds. Chickens infected with the virulent strain shed high concentrations of virus in their feces (10(7) 50% egg infective dose per g), and the virus was isolated from the albumin and yolk of eggs layed just before death. Virus was also isolated from house flies in chicken houses. Serological and virological studies showed that humans are not susceptible to infection with the virus, but can serve as short-term mechanical carriers. Analysis of the RNA of the viruses isolated in April and October by gel migration and RNA-RNA hybridization suggested that these strains were very closely related. Oligonucleotide mapping of the individual genes of virulent and avirulent strains showed a limited number of changes in the genome RNAs, but no consistent differences between the virulent and avirulent strains that could be correlated with pathogenicity were found. Polyacrylamide gel analysis of the early (avirulent) isolates demonstrated the presence of low-molecular-weight RNA bands which is indicative of defective-interfering particles. These RNAs were not present in the virulent isolates. Experimental infection of chickens with mixtures of the avirulent and virulent strains demonstrated that the avirulent virus interferes with the pathogenicity of the virulent virus. The results suggest that the original avirulent virus was probably derived from influenza viruses from wild birds and that the virulent strain was derived from the avirulent strain by selective adaptation rather than by recombination or the introduction of a new virus into the population. This adaptation may have involved the loss of defective RNAs, as well as mutations, and thus provides a possible model for a role of defective-interfering particles in nature.
Use of antibodies directed against synthetic peptides for identifying cDNA clones, establishing reading frames, and deducing the gene order of measles virus
Authors: Richardson C D , Berkovich A , Rozenblatt S , Bellini W J
Published: January 2020

A number of cDNA clones complementary to measles virus mRNA and 50S genome RNA have been generated. These clones have been mapped by restriction enzyme analysis and were subsequently sequenced by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). Computer analysis of these DNA sequences revealed open reading frames which potentially could code for a number of gene products. Portions of these putative polypeptides were synthesized, and rabbit antibodies directed against peptide-hemocyanin conjugates were produced. These antibodies were used to immunoprecipitate virus-specific polypeptides which were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For each of the antisera tested, a unique protein was precipitated whose migration on polyacrylamide gels corresponded to standard gene products identified by monoclonal antibodies and antisera against measles virus. By using this method, we were able to assign the coding regions of cDNA clones to specific protein products and, subsequently, to order the genes of the 3'-terminal third of measles genome RNA.
Posttranscriptional control of DHFR gene expression during adenovirus 2 infection
Authors: Yoder S S , Berget S M
Published: January 2020

The effects of productive adenovirus infection on host gene expression were studied by using a line of methotrexate-resistant HeLa cells with amplified dihydrofolate reductase (DHFR) genes. We have previously reported that synthesis of DHFR is induced threefold early in infection and is shut off late in infection (Yoder et al., Mol. Cell. Biol. 3:819-828, 1983). These changes in DHFR protein synthesis are accompanied by changes in both the steady-state cytoplasmic levels of DHFR mRNA and in the rate of appearance of DHFR mRNA in the cytoplasm. In this report, we examined the mechanism of nuclear control of DHFR mRNA levels. Transcription of DHFR-specific sequences continued at a constant rate throughout infection, representing 0.015% of the total transcriptional activity. In contrast, nuclear steady-state levels of DHFR sequences changed in correspondence to the changing rate of appearance of DHFR mRNA in the cytoplasm. That is, nuclear levels of DHFR-specific sequences rose 2.5-fold early in infection and declined to a level below that found in uninfected cells late in infection. Thus, the relative nuclear stability of DHFR sequences changed throughout the course of infection such that during the time of induction, DHFR sequences were preferentially stabilized. This stabilization was transient, however, and was no longer observed by the time of shutoff. These data indicate that posttranscriptional nuclear events are important in the regulation of DHFR gene expression by adenovirus.
Poliovirus RNA-dependent RNA polymerase and host cell protein synthesize product RNA twice the size of poliovirion RNA in vitro
Authors: Young D C , Tuschall D M , Flanegan J B
Published: January 2020

The poliovirus RNA-dependent RNA polymerase required an oligouridylate primer or a HeLa cell protein (host factor) to initiate RNA synthesis on poliovirion RNA in vitro. The polymerase synthesized template-sized product RNA in the oligouridylate-primed reaction. In the host factor-dependent reaction, the largest product RNA synthesized by the polymerase was twice the size of the template RNA. About half of the product RNA recovered from this reaction was shown to exist in the form of a snapback sequence. Time-course reactions and pulse-chase experiments showed that the product RNA was only slightly larger than the template RNA at early reaction times and that with time it increased in size to form the dimer-sized product RNA. Inhibition of the elongation reaction by adding only [alpha-32P]UTP and ATP resulted in the formation of template-sized product RNA. The dimer-sized product RNA was unaffected by phenol extraction or proteinase K treatment but was converted to template-sized molecules by S1 nuclease. Dimer-sized poliovirus RNA that was sensitive to S1 nuclease was also isolated from poliovirus-infected cells. The results from this study indicate that the labeled negative-strand product RNA synthesized in vitro was covalently linked to the positive-strand template RNA. Thus, in vitro, the primer-dependent poliovirus RNA polymerase may initiate RNA synthesis in the presence of the host factor by using the 3' end of the template RNA as a primer.
Reverse transcription of 7S L RNA by an avian retrovirus
Authors: Chen P J , Cywinski A , Taylor J M
Published: January 2020

Linial et al. isolated a quail cell line, SE21Q1b, that is transformed by a single integrated provirus of Rous sarcoma virus. Virus particles are released from these cells, but because of a provirus defect, cellular rather than viral RNA is packaged. When these virus particles are disrupted with melittin in the presence of an appropriate reaction mixture containing actinomycin D, there is significant reverse transcription of packaged cellular RNA species. We have shown that (i) cellular 7S L RNA is an efficient template; (ii) initiation is on a unique tRNA-like primer; (iii) synthesis produces a 135-base strong-stop DNA product; and (iv) after synthesis, RNase H acts to remove the 135 bases of the 7S L RNA which acted as the template. A possible facilitator of such specific transcription may be that, in the virus particles but not in the cell, the majority of the 7S L RNA species exist complexed with the tRNA, even before the disruption of the virus. From the size and sequence features of the reverse transcript of 7S L RNA, we speculate that such events may have participated in the process by which animal cell genomes have, in the course of evolution, accumulated multiple copies of Alu-like elements.
Immortalization of rat embryo fibroblasts by an adenovirus 2 mutant expressing a single functional E1a protein
Authors: Hurwitz D R , Chinnadurai G
Published: January 2020

Expression of the adenovirus E1a and E1b genes is required for transformation of nonpermissive rodent cells. Differential splicing of the E1a precursor RNA molecules results in the production of two early mRNAs, 13S and 12S, which encode a 289-amino-acid-residue (289R) and 243R protein, respectively. Previously we constructed a mutant virus, dl231, which can only produce normal 289R protein from the E1a gene. In this report we demonstrate that dl231 induced focal transformation of primary rat embryo fibroblasts at 20% of the level of wild-type virus. dl231 transformants were immortalized and produced normal levels of E1a 13S and E1b mRNAs but only minute levels of defective E1a 12S mRNA. These transformants only minimally expressed the transformation phenotype and were similar to untransformed cells. Unlike wild-type transformants, they had a more fibroblastic morphology, were contact inhibited, grew to only low saturation density, and were limited in their ability to grow in an anchorage-independent manner in soft agar. We conclude that the 289R E1a protein can mediate immortalization of primary cells and that the 243R E1a protein is required to elicit the full transformation phenotype.
A single amino acid substitution in a hydrophobic domain causes temperature-sensitive cell-surface transport of a mutant viral glycoprotein
Authors: Gallione C J , Rose J K
Published: January 2020

DNA sequences were determined for three cDNA clones encoding vesicular stomatitis virus glycoproteins from the tsO45 mutant (which encodes a glycoprotein that exhibits temperature-sensitive cell-surface transport), the wild-type parent strain, and a spontaneous revertant of tsO45. The DNA sequence analysis showed that as many as three amino acid changes could be responsible for the transport defect. By recombining the cDNA clones in vitro and expressing the recombinants in COS cells, we were able to trace the critical lesion in tsO45 to a single substitution of a polar amino acid (serine) for a hydrophobic amino acid (phenylalanine) in a hydrophobic domain. We suggest that this nonconservative substitution may block protein transport by causing protein denaturation at the nonpermissive temperature. Comparison of the predicted glycoprotein sequences from two vesicular stomatitis virus strains suggests a possible basis for the differential carbohydrate requirement in transport of the two glycoproteins.
Construction of nondefective adenovirus type 5 bearing a 2.8-kilobase hepatitis B virus DNA near the right end of its genome
Authors: Saito I , Oya Y , Yamamoto K , Yuasa T , Shimojo H
Published: January 2020

A novel helper-free adenovirus type 5 (Ad5) vector system, which utilizes a cloning site 0.2 kilobase (kb) from the right end of the genome, has been developed. To construct a nondefective Ad5 bearing the 2.8-kb DNA fragment of hepatitis B virus (HBV) at this site, we deleted the 2.1-kb nonessential E3 fragment from cloned DNA covering the right one-fourth of the Ad5 genome (76 to 100 map units), inserted the HBV DNA into this site, ligated the recombinant DNA to the rest of the Ad5 genome, and transfected the ligated DNA into human embryo kidney cells. Most of the recovered virus clones had only the E3 deletion and no HBV insertion, suggesting that a homologous recombination occurs between transfected DNAs in these cells. The isolated Ad5 virus bearing the HBV DNA (Ad5-HBL) grew without helper virus in HeLa cells as efficiently as wild-type Ad5, although the 1.9-kb major E4 transcript was detected only poorly in the early phase in the Ad5-HBL-infected cells, suggesting that the HBV DNA inserted upstream of the E4 promoter reduces the E4 transcript. HBV mRNAs transcribed from the inserted DNA were at least as abundant as Ad5 early mRNAs in the late phase of Ad5-HBL infection, but the HBV surface antigen was barely detectable in the infected-cell lysate and culture medium. This result suggests that HBV mRNAs can be transcribed from the inserted genes but no protein can be translated from the HBV mRNAs, presumably because of the translational suppression of cellular mRNAs caused by adenovirus in its late phase.
DNA rearrangement in the control region for early transcription in a human polyomavirus JC host range mutant capable of growing in human embryonic kidney cells
Authors: Miyamura T , Furuno A , Yoshiike K
Published: January 2020

A human polyomavirus JC virus (JCV) host range mutant (JC-HEK) can grow in human embryonic kidney cells, whereas the brain cell-tropic wild-type JCV strain (Mad-1) cannot; JC-HEK contains two complementing defective DNAs, JC-HEK-A and JC-HEK-B. We determined the nucleotide sequence of the putative transcriptional control region of JC-HEK-A DNA that can induce T-antigen synthesis in human embryonic kidney cells and compared it with the sequence of JCV Mad-1 DNA. The JC-HEK-A control region was found to have a complex DNA rearrangement, namely, a partial local duplication of a noncoding region generating two extra replication origins and translocation of segments from the large-T-antigen gene (415 base pairs) and the VP-1 gene (78 base pairs). In the rearranged segment, JC-HEK-A had seven sets of the sequence 5'TGGA(T)A(T)A(T)3', which is found in the simian virus 40 enhancer core, whereas JCV Mad-1 had only one set in its control region. JC-HEK-A also had a 5'TGGAAGTGTAA3' sequence resembling the adenovirus early region 1A enhancer core sequence 5'AGGAAGTGAA3'. Because the viral enhancer is host discriminatory and because another human polyomavirus, BK virus, that grows well in human embryonic kidney cells has these signals in its control region, it is likely that some of the newly acquired signals in JC-HEK play an important role in the altered host range of JCV.
Nucleotide sequence analysis of endogenous murine leukemia virus-related proviral clones reveals primer-binding sites for glutamine tRNA
Authors: Nikbakht K N , Ou C Y , Boone L R , Glover P L , Yang W K
Published: January 2020

Nucleotide sequences of the region that corresponds to the site of tRNA primer binding for a functional retrovirus were determined in five murine leukemia virus-related sequence clones from mouse chromosomal DNA, which contain a unique 170 to 200-base-pair additional internal segment in the long terminal repeats. The 3'-terminal 18-nucleotide sequence of a major glutamine tRNA isoacceptor was found to match well with the putative primer binding site: 18 of 18 in three clones, 17 of 18 in one clone, and 16 of 18 in one clone. This implies that most of these endogenous proviral sequences of the mouse genome, if replicated as retroviruses, will be different from ecotropic murine leukemia viruses and most mammalian type C retroviruses in using glutamine tRNA, rather than proline tRNA, as a primer.
Molecular relationships among serogroup B bacteriophages of Staphylococcus aureus
Authors: Stewart P R , Waldron H G , Lee J S , Matthews P R
Published: January 2020

The typing bacteriophages 55, 80, 83A, and 85 of Staphylococcus aureus, representative of the three major lytic groups of serological group B aureophages, have been examined for relatedness of their genomes and virion proteins. Phages 11 and 80 alpha were also examined to determine the relationship of phage 80 alpha to phages 11 and 80. Total genome hybridization measurements divided the phages into two groups. Phages 55 and 80, in the first group, had DNA homology of 50%. Phages 11, 80 alpha, 83A, and 85 formed a second group with 27 to 65% homology. Homology between the two groups was in the range of 14 to 22%. Phage 80 alpha is more closely related to phage 11 than to phage 80, though it is probably not a simple recombinant of phages 11 and 80. Restriction enzyme digestion and phage [32P]DNA hybridization analysis of the endonuclease-generated fragments from each phage DNA confirmed the findings of the DNA homology measurements. The endonuclease fragment patterns generated by EcoRI and HindIII were distinctive for each phage, confirming that none of the phages are closely related. Common sequences were present in most fragments from the phage DNAs when the labeled probe DNA was from a different phage in the same group. Cross-group probing of endonuclease fragments revealed both a diminished level of homology when similar sequences were present and the probable absence of some sequences. Virion proteins, examined by polyacrylamide gel electrophoresis, were similar in number and molecular weight for phages 11, 80 alpha, 83A, and 85, reflecting the DNA homology analyses. The virion proteins from phages 55 and 80, however, were more distinctive, and both differed from the phages in the other group.
Pathways of the early propagation of virulent and avirulent rabies strains from the eye to the brain
Authors: Kucera P , Dolivo M , Coulon P , Flamand A
Published: January 2020

Penetration of the central nervous system of the adult rat by the CVS strain of rabies virus and its two avirulent derivatives Av01 and Av02 has been studied by inoculation of the virus into the anterior chamber of the eye. The primary sites of penetration of CVS were (i) the intraocular parasympathetic oculomotor fibers, (ii) the retinopetal fibers of pretectal origin, and (iii) the intraocular fibers of the ophthalmic nerve. The mutant strains, however, lost the capacity to invade the two former groups of fibers, although their penetration into the trigeminal system was not impaired. Neither strain CVS nor the mutants infected primarily the intraocular adrenergic terminals and the optic nerve. Mutant strains, but not CVS, were able to infect the lens. These results indicate that the cholinergic receptor may not be the only receptor for rabies virus and that rabies virus is conveyed in the nervous system by retrograde axoplasmic flow. Strain CVS spread throughout the brain and propagated eventually back to the retina. The mutants penetrated the brain as well, but the infection was slow, involved different cerebral structures, and cleared up completely in 3 weeks, probably because of an efficient immune response.
Phosphorylation of the β′ Subunit of RNA Polymerase and Other Host Proteins upon φCd1 Infection of
Caulobacter crescentus
Authors: Hodgson David , Shapiro Lucille , Amemiya Kei
Published: January 2020

A protein kinase activity is induced early after infection of
Caulobacter crescentus
by the DNA phage φCd1. After phage infection at least 40 proteins are phosphorylated; these include DNA-binding proteins, a membrane-associated protein, and several ribosomal proteins. One of the phosphorylated DNA-binding proteins was identified as the β′ subunit of the host RNA polymerase.
Analysis of wild-derived mice for Fv-1 and Fv-2 murine leukemia virus restriction loci: a novel wild mouse Fv-1 allele responsible for lack of host range restriction
Authors: Kozak C A
Published: January 2020

Wild-derived mice originally obtained from Asia, Africa, North America, and Europe were typed for in vitro sensitivity to ecotropic murine leukemia viruses and for susceptibility to Friend virus-induced disease. Cell cultures established from some wild mouse populations were generally less sensitive to exogenous virus than were cell cultures from laboratory mice. Wild mice also differed from inbred strains in their in vitro sensitivity to the host range subgroups defined by restriction at the Fv-1 locus. None of the wild mice showed the Fv-1n or Fv-1b restriction patterns characteristic of most inbred strains, several mice resembled the few inbred strains carrying Fv-1nr, and most differed from laboratory mice in that they did not restrict either N- or B-tropic murine leukemia viruses. Analysis of genetic crosses of Mus spretus and Mus musculus praetextus demonstrated that the nonrestrictive phenotype is controlled by a novel allele at the Fv-1 locus, designated Fv-10. The wild mice were also tested for sensitivity to Friend virus complex-induced erythroblastosis to type for Fv-2. Only M. spretus was resistant to virus-induced splenomegaly and did not restrict replication of Friend virus helper murine leukemia virus. Genetic studies confirmed that this mouse carries the resistance allele at Fv-2.
Rous sarcoma virus variants that encode src proteins with an altered carboxy terminus are defective for cellular transformation
Authors: Wilkerson V W , Bryant D L , Parsons J T
Published: January 2020

The src gene of Rous sarcoma virus (v-src) and its cellular homolog, the c-src gene, share extensive sequence homology. The most notable differences between these genes reside in the region encoding the carboxy terminus of the src proteins. We constructed mutations within the 3' end of the v-src gene to determine the significance of this region to the transforming potential of the v-src protein, pp60v-src. The mutants CHdl300 and CHis1511 contain mutations that alter the last 23 amino acids of pp60v-src, whereas the mutant CHis1545-C contains a linker insertion that alters the last 11 amino acids of pp60v-src, and the mutant CHis1545-H contains a linker insertion that results in a 9-amino-acid insertion at position 415. Plasmids bearing each of these mutations were unable to transform chicken cells when introduced into these cells by DNA transfection. In addition, the structurally altered src proteins encoded by the mutants had much-reduced levels of tyrosine protein kinase activity in vivo, as measured by autophosphorylation and phosphorylation of the 34,000-Mr cellular protein, and in vitro, as determined by measuring the level of pp60src autophosphorylation. These data indicate that the carboxy-terminal amino acid sequences play an important role in maintaining the structure of the catalytic domain of pp60v-src. In contrast, the transfection of chicken cells with plasmid DNA containing a chimeric v-c-src gene resulted in morphological cell transformation and the synthesis of an enzymatically active hybrid protein. Therefore, the carboxy-terminal sequence alterations observed in the c-src protein do not alone serve to alter the functional activity of a hybrid v-c-src protein appreciably.
Identification of a herpes simplex virus function that represses late gene expression from parental viral genomes
Authors: Godowski P J , Knipe D M
Published: January 2020

The expression of herpes simplex virus gamma 2 (late) genes is inhibited before the onset of viral DNA replication. We report that the block in the expression of certain gamma 2 genes is relieved, at least in part, by defects in the beta ICP8 protein. We have examined the expression of the gamma 2 gene encoding glycoprotein C (gC) in cells infected with a temperature-sensitive ICP8 mutant. Under conditions in which viral DNA replication is inhibited, cells infected with the ICP8 mutant overproduce the gC family of mRNAs relative to the level observed in cells infected with a wild-type virus. The gC mRNA synthesized in cells infected with the ICP8 mutant virus is correctly initiated and spliced and is translated with the same relative efficiency as in cells infected with a replicating wild-type virus. These results suggest that ICP8 is involved in the negative regulation of gamma 2 genes expressed from parental viral genomes. The level of gC expression was greatest in cells infected with a replicating wild-type virus. These data suggest that DNA replication and genome amplification are not absolute requirements for gamma 2 gene expression but may facilitate full-level expression of these genes.
Defective interfering virus particles modulate virulence
Authors: Cave D R , Hendrickson F M , Huang A S
Published: January 2020

To determine whether defective interfering (DI) particles modulate virulence by initiating a cyclic pattern of virus growth in vivo, adult mice were infected with vesicular stomatitis virus (VSV), both with and without DI particles. A total of 184 mice divided into groups were inoculated intranasally. A majority of mice inoculated only with standard VSV developed paralysis, most of them between days 7 and 9. The addition of DI particles altered the development of paralysis in several ways. When there was significant protection, a few still became paralyzed on days 7 and 9. When overall mortality was unaffected or even slightly increased, the majority of mice became paralyzed between days 7 and 9 as well. Protection could not be predicted based on a single ratio of standard VSV to DI particles or on the absolute amount of DI particles inoculated. Infectious virus recovered from mouse brains at the time of paralysis and incipient death showed considerable variation, although the titer in a majority of the animals was between 10(5) and 10(7) PFU/ml. When the brains of these paralyzed mice were examined for hybridizable VSV RNA, the detection of standard VSV RNA correlated well with infectivity. The amount of DI RNA in the coinfected mice was more variable and independent of the amount of 40S RNA, although DI RNA was usually found when standard RNA was present. Survivors examined between days 14 and 21 did not contain infectious virus or any detectable viral RNA in their brains. Because these results were consistent with the hypothesis of viral cycling in vivo, rather than a gradual accumulation of total infectious virus, mice were coinfected with 10(8) PFU of standard VSV and 10(5) PFU equivalents of DI particles and sacrificed daily thereafter, irrespective of whether they developed paralysis. Infectivity measurements indicated a reproducible cycling pattern of VSV in the mouse brains with a periodicity of about 5 days. This cycling and the detection of DI RNA in brains several days after intranasal inoculation suggest that there is a dynamic continuous interaction between standard VSV and its DI particle beyond the initial site of replication as the virus population spreads into the host animal. Such cycling of virus production before the full development of specific immune responses from the host may have important implications for viral diagnostics and disease transmission.
Neuraminidase associated with coliphage E that specifically depolymerizes the Escherichia coli K1 capsular polysaccharide
Authors: Tomlinson S , Taylor P W
Published: January 2020

Plaque morphology indicated that the five Escherichia coli K1-specific bacteriophages (A to E) described by Gross et al. (R. J. Gross, T. Cheasty, and B. Rowe, J. Clin. Microbiol. 6:548-550, 1977) encode K1 depolymerase activity that is present in both the bound and free forms. The free form of the enzyme from bacteriophage E was purified 238-fold to apparent homogeneity and in a high yield from ammonium sulfate precipitates of cell lysates by a combination of CsCl density gradient ultracentrifugation, gel filtration, and anion-exchange chromatography. The enzyme complex had an apparent molecular weight of 208,000, as judged from its behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was dissociated by sodium dodecyl sulfate at 100 degrees C to yield two polypeptides with apparent molecular weights of 74,000 and 38,500. Optimum hydrolytic activity was observed at pH 5.5, and activity was strongly inhibited by Ca2+; the Km was 7.41 X 10(-3) M. Rapid hydrolysis of both the O-acetylated and non-O-acetylated forms of the K1 antigen, an alpha 2----8-linked homopolymer of N-acetylneuraminic acid, and of the meningococcus B antigen was observed. Limited hydrolysis of the E. coli K92 antigen, an N-acetylneuraminic acid homopolymer containing alternating alpha 2----8 and alpha 2----9 linkages, occurred, but the enzyme failed to release alpha 2----3-, alpha 2----6-, or alpha 2----9-linked sialic residues from a variety of other substrates.