bluuebunny/crossref_metadata_2025 huggingface dataset:
Virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions
Authors: Holland J J , de la Torre J C , Steinhauer D A , Clarke D , Duarte E , Domingo E
Published: January 2020

Monoclonal antibody-resistant mutants have been widely used to estimate virus mutation frequencies. We demonstrate that standard virion neutralization inevitably underestimates monoclonal antibody-resistant mutant genome frequencies of vesicular stomatitis virus, due to phenotypic masking-mixing when wild-type (wt) virions are present in thousandsfold greater numbers. We show that incorporation of antibody into the plaque overlay medium (after virus penetration at 37 degrees C) can provide accurate estimates of genome frequencies of neutral monoclonal antibody-resistant mutant viruses in wt clones. By using this method, we have observed two adjacent G----A base transition frequencies in the I3 epitope to be of the order of 10(-4) in a wt glycine codon. This appears to be slightly lower than the frequencies observed at other sites for total (viable and nonviable) virus genomes when using a direct sequence approach.
Molecularly cloned simian immunodeficiency virus SIVagm3 is highly divergent from other SIVagm isolates and is biologically active in vitro and in vivo
Authors: Baier M , Werner A , Cichutek K , Garber C , Müller C , Kraus G , Ferdinand F J , Hartung S , Papas T S , Kurth R
Published: January 2020

Simian immunodeficiency viruses have been isolated from African green monkeys originating from Ethiopia. A molecular clone, termed SIVagm3, was found to be highly divergent from SIVagmTYO-1 in terms of its restriction map and partial nucleotide sequence. A premature stop codon present in the transmembrane protein of SIVagm TYO-1 was absent in SIVagm3. SIVagm3 was biologically active in vitro and in vivo and displayed characteristics reminiscent of the wild-type virus. Biological activity was demonstrated by seroconversion of juvenile African green monkeys and Macaca nemestrina after inoculation. In contrast to antibody reactivity mainly directed against env proteins in naturally infected African green monkeys. African green monkeys and M. nemestrina infected with the cloned virus showed antibody reactivity directed against all major proteins as demonstrated by immunoblot analysis. The availability of a biologically fully competent molecular clone of SIVagm allows us now to address various pertinent questions in an animal model system which should help to understand features of human immunodeficiency virus infection in human beings.
Adenoviruses with nonidentical terminal sequences are viable
Authors: Lippé R , Graham F L
Published: January 2020

Adenovirus genomes consist of linear DNA molecules containing inverted terminal repeat sequences (ITRs) of 100 to 200 base pairs. The importance of identical termini for viability of adenoviruses was investigated. The viral strains used in this study were wild-type adenovirus type 5 (Ad5) and a variant Ad2 strain with termini which were distinct from those of all other human adenoviruses sequenced to date. A hybrid virus (sub54), obtained by recombination between Ad2 and Ad5, derived the left 42 to 52% of its genome from Ad2 and the right 58 to 48% from Ad5. Southern blotting analysis with labeled oligodeoxynucleotides indicated that both Ad2 and Ad5 ITRs were present in sub54 viral DNA preparations, and successive plaque purifications of sub54 demonstrated that viruses with nonidentical terminal sequences were viable but were rapidly converted to viruses with identical ends. Cloning of the sub54 genome as a bacterial plasmid supported the observations made by analysis of sub54 virion DNA. A plasmid, pFG154, was isolated which contained the entire adenovirus genome with an Ad2 ITR at the left terminus covalently linked to an Ad5 ITR at the right terminus. Upon transfection of mammalian cells with pFG154, viral progeny were obtained which had all possible combinations of termini, thus confirming that molecules with nonidentical termini are viable. Pure populations of viruses with nonidentical termini could not be isolated, suggesting efficient repair of one end with the opposite terminus used as a template. A model for this process is proposed involving strand displacement replication and emphasizing the importance of panhandle formation (annealing of terminal sequences) as a replicative intermediate.
Topoisomerase I sites cluster asymmetrically at the ends of the simian virus 40 core origin of replication
Authors: Tsui S , Anderson M E , Tegtmeyer P
Published: January 2020

In vivo, topoisomerase I cleavage sites are located predominantly on the strands of simian virus 40 DNA that are the templates for discontinuous synthesis (S.E. Porter and J.J. Champoux, Mol. Cell. Biol. 9:541-550, 1989). This arrangement of sites suggests that topoisomerase I may associate with replication complexes in unique functional orientations at replication forks. We have mapped topoisomerase I cleavage sites in the simian virus 40 origin of replication in vitro under conditions suitable for DNA replication. Numerous sites cluster in the inverted repeat and AT-rich domains at the ends of the core origin and are arranged on the same strands that are cut most frequently in vivo. We propose that cleavage at these sites would allow bidirectional extension of the replication bubble induced by T antigen within the core origin of replication early in the initiation of DNA synthesis. A mutational analysis of the topoisomerase I sites confirms the importance of positions -4 to -1 and +1 in the consensus sequence 5'-A/T-A/G-A/T-T-break-G/A-3'. Surprisingly, more distant nucleotide positions also influence topoisomerase I sites in the inverted repeat and AT-rich domains of the core origin. The effects of distant sequences could be mediated by direct interactions with topoisomerase I or by the conformation of DNA in the core origin.
Priming for rotavirus neutralizing antibodies by a VP4 protein-derived synthetic peptide
Authors: Arias C F , Garcia G , Lopez S
Published: January 2020

In the rotavirus SA11 surface protein VP4, the trypsin cleavage sites associated with the enhancement of infectivity are flanked by two amino acid regions that are highly conserved among different rotaviruses. We have tested the ability of synthetic peptides that mimic these two regions to induce and prime for a rotavirus neutralizing antibody response in mice. After the peptide immunization schedule, both peptides induced peptide antibodies, but neither was able to induce virus antibodies, as measured by an enzyme-linked immunosorbent assay or a neutralization assay. However, when the peptide-inoculated mice were subsequently injected with intact SA11 virus, a rapid and high neutralizing antibody response was observed in mice that had previously received the peptide comprising amino acids 220 to 233 of the VP4 protein. This neutralizing activity was serotype specific; however, this peptide was also able to efficiently prime the immune system of mice for a neutralizing antibody response to the heterotypic rotavirus ST3 when the ST3 virus was used for the secondary inoculation.
Fv-4 resistance gene: a truncated endogenous murine leukemia virus with ecotropic interference properties
Authors: Ikeda H , Sugimura H
Published: January 2020

Fv-4 is a mouse gene which controls susceptibility to infection by ecotropic murine leukemia virus (MuLV). We previously cloned part of an endogenous MuLV associated with the resistance allele of the Fv-4 gene (Fv-4r). In this report, we describe an extended clone of the Fv-4r allele consisting of a 17-kilobase DNA fragment containing the retroviral sequence and its 5'-flanking sequence. The new DNA clone contains a truncated MuLV with delta pol-env-long terminal repeat sequences but no other MuLV-reactive sequence within 13 kilobases upstream of the truncated MuLV. Transfection of this clone into mouse cells led to transcription of Fv-4 env mRNA, expression of the Fv-4r-specific MuLV envelope protein, and resistance to infection with ecotropic MuLV but not amphotropic and dualtropic MuLVs. Restriction of ecotropic viruses appears to occur at or before viral cDNA synthesis. This result is consistent with a model of receptor interference for Fv-4 restriction. Our data also suggest that the 5' non-MuLV sequence is important for biological function, since a DNA clone which lacks most of the 5'-flanking sequence did not efficiently confer the resistance phenotype.
Transcriptional analysis of minute virus of mice P4 promoter mutants
Authors: Ahn J K , Gavin B J , Kumar G , Ward D C
Published: January 2020

A series of 5' deletion, internal deletion, and linker-scanning mutants of the minute virus of mice P4 promoter were constructed and analyzed for transcriptional activity in nuclear extracts of mouse A92L fibroblasts. A GC box and a TATA box essential for in vitro transcription from the P4 promoter were localized between nucleotides 150 and 180 (-55 to -25 relative to the primary RNA start site). Although this region also exhibited homologies to other transcriptional control elements, the simian virus 40 enhancer, and the adenovirus E1A enhancer, only the GC box and TATA box appear functional. These two motifs also play an essential role in vivo, although additional upstream sequences (between -139 and -55) are required for optimal transcription. DNase I footprinting, competitive gel retardation assays, and UV-photocrosslinking were used to identify Sp1-like proteins of 95 and 120 kilodaltons in A92L extracts that interact with the GC box of the minute virus of mice P4 promoter.
p53-plus-ras-transformed rat embryo fibroblasts express tumor-specific transplantation antigen activity which is shared by independently transformed cells
Authors: Frey A B , Levine A J
Published: January 2020

p53-plus-ras-transformed rat cell lines express a tumor-specific transplantation antigen that is common to a number (85%) of independently derived p53-plus-ras-transformed cell lines. This has been shown by immunizing rats with irradiated p53-plus-ras-transformed cells and demonstrating protection of these animals by subsequent live-cell tumor challenge. Several c-myc-plus-ras-transformed cell lines (54% of the lines tested) and one adenovirus E1a-plus-ras-transformed cell line (9% of those tested) were shown to share a common tumor-specific transplantation antigen by their ability to immunize a rat against a p53-plus-ras cell line challenge. Several experimental approaches have been used to fractionate and identify the antigen common to these cell lines. The experimental results reported here make it clear that the p53 protein common to most of these transformed cell lines is not likely to be the tumor-specific transplantation antigen.
A highly efficient retroviral vector allows detection of the transforming activity of the human c-fps/fes proto-oncogene
Authors: Feldman R A , Lowy D R , Vass W C , Velu T J
Published: January 2020

We have constructed an efficient new retroviral vector containing strong promoting elements derived from the Friend murine leukemia virus (F-MuLV) long terminal repeat (LTR) and have used the vector to demonstrate that overexpression of human c-fps/fes can transform established mouse cells. When a c-fps/fes cDNA was cloned into the vector, this viral DNA and the recovered virus induced very high levels of the c-fps/fes product NCP92 and tumorigenic transformation of NIH 3T3 cells. Compared with an isogenic vector under control of a Moloney MuLV-derived LTR, the vector driven by the F-MuLV LTR induced 3- to 10-times-higher levels of expression of c-fps/fes, a higher level of phosphotyrosine in cellular proteins, and a virus whose transforming activity was 2 orders of magnitude greater. We conclude (i) that normal c-fps/fes can induce morphologic transformation and that its transforming activity is a function of the level of expression of NCP92 and (ii) that the vector based on the F-MuLV LTR is more efficient than the vector driven by a Moloney MuLV LTR in inducing high levels of expression and measurable biological activity.
Release of pseudorabies virus from infected cells is controlled by several viral functions and is modulated by cellular components
Authors: Zsak L , Mettenleiter T C , Sugg N , Ben-Porat T
Published: January 2020

The role of the nonessential glycoproteins gI, gp63, and gIII in the release of pseudorabies virus from different cell lines was investigated. We show that these glycoproteins may have a beneficial or deleterious effect on virus release depending on the type of cell in which the virus is grown. Inactivation of the genes encoding either gI, gp63, or gIII has no detectable effect on virus release from rabbit kidney cells. Inactivation of gI or gp63 strongly promotes virus release from chicken embryo fibroblasts, whereas inactivation of gIII reduces virus release from these cells. A defect in both gI and gIII or in both gp63 and gIII diminishes virus release from rabbit kidney cells but improves release from chicken embryo fibroblasts. We demonstrate that all three nonessential glycoproteins contribute to one specific aspect of viral growth, namely, virus release, and that they affect virus release in conjunction with each other. Furthermore, our results show that the manifestation of the role of each of these viral functions in virus growth may differ in different cell types, i.e., that release is affected by these viral functions in conjunction with some unknown cellular function.
Constitutive expression of simian virus 40 large T antigen in monkey cells activates their capacity to support polyomavirus replication
Authors: Tang W J , Folk W R
Published: January 2020

Polyomavirus DNA replication is normally restricted to rodent cells, and simian virus 40 (SV40) DNA replication is restricted to primate cells. We demonstrate that DNAs containing the polyomavirus origin can be replicated in monkey cells which constitutively express SV40 large T antigen. Permissivity is most likely caused by SV40 T antigen modification of cellular protein(s) required to replicate the polyomavirus origin. A possible target for the T-antigen-induced modification is DNA polymerase alpha-DNA primase.
Biosynthesis of hepatitis B virus e antigen: directed mutagenesis of the putative aspartyl protease site
Authors: Jean-Jean O , Salhi S , Carlier D , Elie C , De Recondo A M , Rossignol J M
Published: January 2020

The C gene products of all mammalian hepadnaviruses contain a region with sequence similarities to the catalytic center of the aspartyl proteases. This region could have the capacity to cleave precore proteins, leading to the synthesis of e antigen. By site-directed mutagenesis on a plasmid containing the hepatitis B virus C gene, we have replaced either the Asp residue of the putative aspartyl protease catalytic center or an Asp residue located 3 amino acids upstream. Transient expression of the mutated hepatitis B virus C gene in human and mouse cells showed that none of these mutations prevented the secretion of an accurately processed HBe antigen. Thus, we demonstrated that the aspartyl protease responsible for e antigen precursor processing is not C gene encoded but is more likely to be a cellular enzyme. From these results, we suggest a model for the mechanism of e antigen synthesis.
The reduced virulence of the thymotropic Moloney murine leukemia virus derivative MoMuLV-TB is mapped to 11 mutations within the U3 region of the long terminal repeat
Authors: Yuen P H , Szurek P F
Published: January 2020

Chimeric constructs were generated by exchanging genomic fragments between the potent T-cell lymphoma inducer Moloney murine leukemia virus (MoMuLV) and its derivative MoMuLV-TB, which induces T-cell lymphoma after a relatively longer latent period. Analysis of the T-cell lymphoma-inducing potential of the hybrid viruses that were obtained localized the primary determinant critical to efficient T-cell lymphoma induction to the MoMuLV ClaI-XbaI fragment which comprises 48 nucleotides (nt) of p15E, p2E, the 3'-noncoding sequence, and 298 nt of U3. The 438-base-pair ClaI-XbaI fragments of MoMuLV and MoMuLV-TB differed in only 11 nt. Nine mutations were found within the enhancer. These mutations occurred within the two CORE, the two GRE-LVa, and two of the four NF1 nuclear factor-binding motifs. MoMuLV-TB replicated better than MoMuLV in thymus-bone marrow (TB) cells, a cultured cell line of lymphoid origin. In addition, MoMuLV-TB and NwtTB-2, a recombinant virus with the ClaI-SmaI fragment of MoMuLV-TB in a MoMuLV background, replicated in thymocytes as efficiently as did MoMuLV or TBNwt-2, the reciprocal recombinant virus, with the ClaI-SmaI fragment of MoMuLV in a MoMuLV-TB background. Like NwtTB-4, a recombinant virus with the ClaI-XbaI fragment of MoMuLV-TB in a MoMuLV background, NwtTB-2 induced lymphoma after a long latent period. The finding given above suggests that thymotropism is not the only factor that determines the T-cell lymphoma-inducing potential of MoMuLV. It appears likely that mutations in one or more of the MoMuLV-TB nuclear factor-binding motifs may have altered the interaction of the enhancer with specific nuclear factors; this, in turn, may affect the T-cell lymphoma-inducing potential of MoMuLV-TB.
Augmentation of retrovirus-induced lymphoid leukosis by Marek's disease herpesviruses in White Leghorn chickens
Authors: Bacon L D , Witter R L , Fadly A M
Published: January 2020

Our objective was to determine whether the cell-associated herpesvirus vaccines used in chickens to control Marek's disease tumors can augment development of lymphoid leukosis (LL) induced by exogenous avian leukosis virus (ALV). Various single or mixed Marek's disease vaccines were inoculated at day 1, and ALV was injected at 1 to 10 days, with chickens of several experimental or commercial strains. Development of LL was monitored at 16 to 48 weeks in various experiments. In several strains of chickens we repeatedly found that the widely used serotype 3 turkey herpesvirus vaccine did not augment LL in comparison with unvaccinated controls. However, LL development and incidence were prominently augmented in several chicken strains vaccinated with serotype 2 vaccines, used alone or as mixtures with other serotypes. In one chicken strain, augmentation was demonstrated after natural exposure to ALV or serotype 2 Marek's disease virus viremic shedder chickens. Augmentation of LL by virulent or attenuated Marek's disease viruses of serotype 1 was intermediate in effect. Serotype 2 Marek's disease virus augmentation of LL was prominent in three laboratory lines and one commercial strain of White Leghorns, but it was not observed in an LL-resistant laboratory line or four commercial strains susceptible to ALV infection. Chickens developed similar levels of viremia and neutralizing antibodies to ALV regardless of the presence of augmentation of LL, suggesting that the mechanism of enhanced LL did not result from differences in susceptibility or immune response to ALV. We postulate that the serotype 2 herpesviruses may augment LL through one of several possible influences on bursal cells that are subsequently transformed by exogenous ALV.
Linker insertion-deletion mutagenesis of the v-src gene: isolation of host- and temperature-dependent mutants
Authors: DeClue J E , Martin G S
Published: January 2020

The host cell regulators and substrates of the Rous sarcoma virus transforming protein pp60v-src remain largely unknown. Viral mutants which induce a host-dependent phenotype may result from mutations which affect the interaction of pp60v-src with host cell components. To isolate such mutants and to examine the role of different regions of src in regulating pp60v-src function, we generated 46 linker insertion and 5 deletion mutations within src. The mutant src genes were expressed in chicken embryo fibroblasts and in rat-2 cells by using retrovirus expression vectors. Most linker insertions within the kinase domain (residues 260 to 512) inactivated kinase activity and transforming capacity, while most insertions in the N-terminal domain and at the extreme C terminus were tolerated. A number of mutations generated a host-dependent phenotype. Insertions after residues 225 and 227, within the N-terminal regulatory domain (SH2), produced a fusiform transformation in chicken embryo fibroblasts and abolished transformation in rat-2 cells; a similar phenotype also resulted from two deletions affecting SH2 (residues 149 to 174 and residues 77 to 225). Insertions immediately C terminal to Lys-295, which is involved in ATP binding, also produced a conditional phenotype. Insertions after residues 299 and 300 produced a temperature-sensitive phenotype, while insertions after residues 304 and 306 produced a host cell-dependent phenotype. An insertion which removed the major tyrosine autophosphorylation site (Tyr-416) greatly reduced transformation of rat-2 cells, a property not previously observed with other mutations at this site. We conclude that mutations at certain sites within src result in conditional phenotypes. These sites may represent regions important in interactions with host cell components.
The Epstein-Barr virus (EBV) DR enhancer contains two functionally different domains: domain A is constitutive and cell specific, domain B is transactivated by the EBV early protein R
Authors: Chevallier-Greco A , Gruffat H , Manet E , Calender A , Sergeant A
Published: January 2020

The Epstein-Barr virus (EBV) DR promoter is located upstream of the PstI repeats, and besides the TATA box, it contains two cis-acting regulatory elements. One of them has enhancer properties. To define more precisely the functional region(s) in the DR enhancer, we generated 5' and 3' deletion mutants. These deletion mutants, which were transfected into various recipient cells of different origins, allowed us to identify two functionally distinct domains, A and B. Domain A was constitutively active in all cell lines tested, except in lymphoid B cells. Domain B was active in lymphoid B cells, and its activity required both EB1 (the BZLF1-encoded EBV trans-acting factor) and the presence of the EBV genome. This suggested that an EBV-encoded, EB1-inducible factor was activating the enhancer B domain. In effect, the B domain was trans-activated by R, an EBV early product encoded by the open reading frame BRLF1, and the activation by R occurred in epithelial, fibroblastic, and lymphoid cells. The R-responsive element has been reduced to 28 base pairs containing the double palindromic sequence TTGTCCCGTGGACAATGTCC. Both domains A and B act by increasing the initiation of specific RNAs.
Characterization of human immunodeficiency virus type 2 envelope glycoproteins: dimerization of the glycoprotein precursor during processing
Authors: Rey M A , Krust B , Laurent A G , Montagnier L , Hovanessian A G
Published: January 2020

Four glycoproteins with apparent molecular weights of 300,000, 140,000, 125,000, and 36,000 (gp300, gp140, gp125, and gp36) were detectable in human immunodeficiency virus type 2 (HIV-2)-infected cells. gp125 and gp36 are the external and transmembrane components, respectively, of the envelope glycoproteins of HIV-2 mature virions. gp300 and gp140 are only detectable in virus-infected cells. They have identical isoelectric points, suggesting that gp300 might be a dimeric form of the immature precursor, gp140. The purified gp300 can be dissociated in a slightly acidic buffer to give rise to monomers of 140,000 molecular weight. Such dissociated monomers and the purified gp140 showed identical patterns of polypeptides after partial proteolysis with Staphylococcus aureus V8 protease. Pulse-chase experiments indicated that gp300 is formed after synthesis of gp140 and before the detection of the mature external envelope glycoprotein, gp125. These results were confirmed by using various inhibitors of glycosylation and inhibitors of trimming enzymes. Dimer formation of the envelope glycoprotein precursor was also observed in cells infected with simian immunodeficiency virus (SIV), a virus closely related to HIV-2. On the other hand, the envelope glycoprotein precursor of HIV-1 did not form a dimer during its processing. Therefore, dimer formation seems to be a specific property of HIV-2 and SIV envelope gene expression. Such transient dimerization of the glycoprotein precursor might be required for its efficient transport to the Golgi apparatus and for its processing.
Fine structure mapping of five temperature-sensitive mutants in the 22- and 147-kilodalton subunits of vaccinia virus DNA-dependent RNA polymerase
Authors: Thompson C L , Hooda-Dhingra U , Condit R C
Published: January 2020

We have mapped the temperature-sensitive (ts) lesions of three mutants, ts51, ts53, and ts65, and two other mutants, ts7 and ts20, to regions on the vaccinia virus genome that encode the 147- and 22-kilodalton subunits of the viral DNA-dependent RNA polymerase, respectively. Plasmid and bacteriophage clones from the HindIII J region and the region spanning the HindIII J-H junction were used in marker rescue experiments to map the mutations. Sequence analysis of the region encoding the 22-kilodalton subunit in the wild-type, ts7, and ts20 viruses revealed a single base change in the mutants compared with that in the wild-type virus. The identification of these RNA polymerase mutants provides us with tools to understand transcription and its regulation in vaccinia virus.
Presentation of an immunodominant T-cell epitope of hepatitis B surface antigen by the HLA-DPw4 molecule
Authors: Celis E , Karr R W
Published: January 2020

Human T cells that recognize a major epitope of the hepatitis B surface antigen were studied for their ability to react with antigen when presented by mouse fibroblasts that express class II products of the human major histocompatibility gene complex after gene transfection. L cells expressing HLA-DPw4, but not those expressing HLA-DR4 or HLA-DR7, induced strong proliferative responses of antigen-specific T cells to either hepatitis B surface antigen or the synthetic peptide S1d, which bears the immunodominant T-cell epitope. These results identified a genetic restriction element of human helper T-lymphocyte responses to a major antigenic determinant of hepatitis B virus and might be important in the design of subunit vaccines to this pathogen. Peptides that induce T-cell responses that are restricted by a frequently encountered major histocompatibility complex molecule in the general population such as DPw4 would be ideal candidates as subunit vaccines.
Adeno-associated virus rep protein synthesis during productive infection
Authors: Redemann B E , Mendelson E , Carter B J
Published: January 2020

Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. We studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with [35S]methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.
Form-determining functions in Sindbis virus nucleocapsids: nucleosomelike organization of the nucleocapsid
Authors: Coombs K M , Brown D T
Published: January 2020

Purified intact Sindbis virus nucleocapsids were treated at different pH values or with various concentrations of divalent cations, cation chelators, salt, or formamide. The resulting structures were examined by velocity sedimentation, electron microscopy, and protein-protein cross-linking. Changes in each of the test conditions led to alterations in the sedimentation profile of treated nucleocapsids. Appropriate concentrations of formamide or divalent cations generated beaded strandlike structures similar in morphology to those generated from adenovirus cores and nucleosomes. The capsid protein and RNA remained associated with each other at NaCl concentrations less than or equal to 1 M or after treatment of the structures with alkaline pH up to and including pH 10.7. Protein and RNA were dissociated by salt concentrations of greater than 1 M, suggesting that the arginine-rich, amino-terminal portion of the capsid protein is responsible for binding the RNA. Protein-protein cross-linking also indicated that the capsid proteins remained associated in small aggregates under some of the conditions that caused dissociation of the nucleocapsid and suggested the presence of more than one type of protein-protein interaction in the nucleocapsids. Collectively, these data suggest that, like histones and adenovirus core proteins, the Sindbis virus capsid protein serves to package segments of the genome into nucleoprotein beads which are capable of interacting with each other to form the nucleocapsid structure.
Two related Epstein-Barr virus membrane proteins are encoded by separate genes
Authors: Sample J , Liebowitz D , Kieff E
Published: January 2020

The structures of the 2.3- and 2.0-kilobase Epstein-Barr virus (EBV) mRNAs, partially encoded within the EcoRI J fragment DNA of the viral genome, were determined by analysis of their cDNAs. Both mRNAs are transcribed across the fused terminal repeats of the EBV episome and consist of nine exons. The mRNAs are transcribed from different promoters and have a unique 5' exon from the U5 region of the genome but eight common exons from the U1 region. One principal open reading frame is present in each mRNA and is predicted to encode 54,000- and 40,000-dalton integral membrane proteins. This result was confirmed by in vitro translation of RNAs in the presence of canine pancreatic microsomes. The 2.3-kilobase mRNA is not expressed in Raji cells, owing to the deletion of the 5' regulatory and coding region of this gene, whereas neither mRNA is expressed in Namalwa cells, owing to inactivation as a result of integration of the EBV genome via the terminal repeats. Since these mRNAs are readily detected in largely latently infected cells and do not increase in abundance with EBV replication, these putative latent-infection membrane proteins are tentatively designated LMP-2A and LMP-2B, respectively.
Identification of proteins encoded by Epstein-Barr virus trans-activator genes
Authors: Marschall M , Leser U , Seibl R , Wolf H
Published: January 2020

Specific antisera were generated to characterize Epstein-Barr virus proteins reported to have trans-activating properties. Open reading frame BRLF1 was found to be expressed in two modifications in vivo, with molecular sizes ranging from 94 to 98 kilodaltons (kDa) depending on the cell line, whereas only one protein (Raji cells, 96 kDa) was detected by in vitro translation. Open reading frame BZLF1 encoded polypeptides of 38 and 35 kDa and additional smaller forms. A BZLF1-encoded 30-kDa protein could be detected under conditions in which expression was restricted to immediate early genes. Nuclear localization could be detected under conditions in which expression was restricted to immediate early genes. Nuclear localization could be shown for the proteins derived from reading frames BZLF1 and BMLF1. BMLF1 expression gave a heterogeneous protein pattern, with molecular sizes between 45 and 70 kDa, including a predominant 60-kDa protein detected in different B-cell lines.
During latency, herpes simplex virus type 1 DNA is associated with nucleosomes in a chromatin structure
Authors: Deshmane S L , Fraser N W
Published: January 2020

Latent herpes simplex virus type 1 DNA has a nucleosomal structure similar to that of cellular chromatin, as determined by micrococcal nuclease digestion. All of the major regions, including the transcriptionally active region of the genome, were found to be associated with nucleosomes. Such a chromatin structure is likely to be an important element in the control of herpes simplex virus type 1 gene expression during latency.
Mouse natural autoantibodies can interfere with murine alpha and beta interferons
Authors: Matsiota P , Saron M F , Guillon J C , Avrameas S
Published: January 2020

Natural polyspecific autoantibodies could impede the establishment of an antiviral state by mouse alpha and beta interferons (IFN) as determined by an IFN assay with L929 cells and with vesicular stomatitis virus as the challenge virus. This anti-IFN effect was due to interactions with cell surface constituents rather than to antibody activity against IFN. This observation supports the hypothesis that natural autoantibodies participate in specific immune regulation as well as in the regulation of nonspecific host defense.
Optimal replication of plasmids carrying polyomavirus origin regions requires two high-affinity binding sites for large T antigen
Authors: Weichselbraun I , Haider G , Wintersberger E
Published: January 2020

The efficiency of replication of plasmids containing the control region of polyomavirus DNA including one, two, or all three of the strong binding sites for large T antigen was measured in COP 8 cells which provide polyomavirus T antigen in trans. It was found that plasmids carrying only binding site A (the one closest to the origin core region) exhibited only 10% of the replication competence of plasmids with binding sites A and B or A and C. Plasmids containing all three binding sites, A, B, and C, did not replicate more efficiently than those with only two strong T-antigen-binding sites. We conclude, therefore, that optimal T-antigen-dependent replication of polyomavirus DNA requires two high-affinity T-antigen-binding sites.
Human papillomavirus type 16 transformation of primary human embryonic fibroblasts requires expression of open reading frames E6 and E7
Authors: Watanabe S , Kanda T , Yoshiike K
Published: January 2020

Human papillomavirus 16 (HPV-16) early genes were tested for their ability to transform primary human embryonic fibroblasts (WI38) or to extend the life span of the fibroblasts. The expression plasmids containing the transcriptional unit for the HPV-16 subgenomic DNA fragments controlled by the simian virus 40 promoter were introduced into WI38 cells with the aid of neomycin selection, and the drug-resistant cells that survived long-term culture were characterized. The fragments containing E6E7 open reading frames (ORFs) intact were found to be capable of extending the life span of WI38 cells. From the results obtained with frameshift mutations in either ORF and from those obtained by cotransfection with the separate plasmids containing E6 and E7 ORFs, it was concluded that HPV-16 transformation (extension of the life span) of human fibroblasts requires expression of genes E6 and E7.
Phosphorylation at serine 89 induces a shift in gel mobility but has little effect on the function of adenovirus type 5 E1A proteins
Authors: Dumont D J , Tremblay M L , Branton P E
Published: January 2020

Phosphorylation at serine 89 was shown to be the major cause of the shift in gel migration of the 289R and 243R early region 1A (E1A) proteins of human adenovirus type 5. However, conversion of Ser-89 to alanine by site-directed mutagenesis did not abolish E1A transactivating or transforming activities, suggesting that phosphorylation at this site is not necessary for these E1A functions.
Genetic variation occurring on the genome of an in vitro insertion mutant of poliovirus type 1
Authors: Kuge S , Kawamura N , Nomoto A
Published: January 2020

An insertion sequence of 72 nucleotides prepared from a polylinker sequence of plasmid pUC18 was introduced at nucleotide position 702 of the 5' noncoding sequence (742 nucleotides long) of the genome of the Sabin strain of poliovirus type 1 by using an infectious cDNA clone of the virus strain. The insertion mutant thus obtained showed a small-plaque phenotype compared with that of the parent virus. Apparent revertants (large-plaque variants) were easily generated from the insertion mutant. Nucleotide sequence analysis was performed on the revertant genomes to determine the mutation(s) by which the plaque size of the parent virus was regained. Some large-plaque variants lacked genomic sequences including all or a part of the insertion sequence. A computer-aided search for secondary structures with respect to the deletion sites detected possible supporting sequences which provided fairly stable secondary structures at the deletion sites. This result was consistent with our supporting sequence-loop model which had been proposed as a new copy-choice model for the generation of genetic rearrangements occurring on single-stranded RNA genomes (S. Kuge, I. Saito, and A. Nomoto, J. Mol. Biol. 192:473-487, 1986). The other large-plaque variants had point mutations at any one of three positions of an AUG existing in the insertion sequence. A small-plaque phenotype was observed when an AUG codon was inserted in frame or out of frame with regard to the initiation site of viral polyprotein synthesis. Our data strongly suggest that an AUG sequence in this genome region is deleterious for efficient poliovirus replication.
2'-5'-oligoadenylate synthetase gene expression in normal and murine sarcoma virus-transformed NIH 3T3 cells
Authors: David S , Nissim A , Chebath J , Salzberg S
Published: January 2020

Mouse fibroblasts transformed by murine sarcoma virus (MSV) are highly sensitive to the antiproliferative effect of interferon (IFN) (M. Bakhanashvili, D. H. Wreschner, and S. Salzberg, Cancer Res. 43:1289-1294, 1983). To elucidate the mechanism leading to this IFN sensitivity, the expression of the 2'-5'-oligoadenylate synthetase (2-5A synthetase) gene, the presence of the 2-5A synthetase protein, and the level of its enzymatic activity were determined in IFN-treated and untreated cultures. NIH 3T3 mouse fibroblasts were compared with two different NIH 3T3 clones transformed by MSV. Cultures were treated with 300 IU of beta IFN (IFN-beta) per ml for 16 to 24 h. While no detectable 2-5A synthetase-derived transcripts were seen in untreated NIH 3T3 cells, two size classes of RNA transcripts, i.e., 1.7 and 4.2 kilobases, were detected in IFN-treated cultures. Surprisingly, a similar amount of transcripts were present in untreated transformed cells. However, following IFN treatment, an eightfold increase in the level of RNA was readily detected in these cells, with no change in the size classes. Similar results were obtained with the 2-5A synthetase protein, for which three size classes of 42, 71, and 102 kilodaltons were demonstrated by immunoblotting, and with the enzymatic activity, for which again, the highest level was seen in IFN-treated MSV-transformed cultures. The basal level of 2-5A synthetase gene expression in the transformed cells has biological significance since these cells were more resistant to mengovirus infection than NIH 3T3 mouse fibroblasts. Medium collected from transformed cultures failed to induce 2-5A synthetase activity in NIH 3T3 cells. Furthermore, antibodies directed against mouse IFN-beta failed to inhibit 2-5A synthetase activity detected in transformed cultures. These results suggest that at least IFN-beta secretion is not involved in the elevated level of 2-5A synthetase gene expression in these cells.
Expression of Sindbis virus 26S cDNA in Spodoptera frugiperda (Sf9) cells, using a baculovirus expression vector
Authors: Oker-Blom C , Summers M D
Published: January 2020

To study protein processing in an insect Spodoptera frugiperda (fall armyworm; Sf9) cell line, a 26S cDNA encoding the sequence of Sindbis virus structural proteins (capsid protein, of 30 kilodaltons [kDa]; p62 [the precursor of E3 and E2], of 62 kDa; a 6-kDa peptide; and the E1 protein, of 56 kDa) was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) adjacent to the polyhedrin promoter. By immunoblot analysis with antisera directed against whole Sindbis virus and the individual structural proteins (capsid, E2, and E1), we have shown that polypeptides similar in size and antigenicity to those synthesized in Sindbis virus-infected BHK cells are expressed in Sf9 cells infected with the recombinant baculovirus Ac373-SV26. By pulse-chase labeling in the presence or absence of tunicamycin, by endo-beta-N-acetylglucosaminidase H (endo-H) treatment of the recombinant glycoproteins, and by N-terminal sequence analysis of the E1 envelope glycoprotein, we have further shown that the 26S transcription translation unit of Sindbis virus, although normally encoded by nonnuclear RNA, is expressed and proteolytically cleaved similarly, if not identically, in Sf9 cells as compared with BHK cells when a baculovirus expression vector is used.
Harvey murine sarcoma virus: influences of coding and noncoding sequences on cell transformation in vitro and oncogenicity in vivo
Authors: Velu T J , Vass W C , Lowy D R , Tambourin P E
Published: January 2020

The rat-derived Harvey murine sarcoma virus (Ha-MuSV) contains a transduced ras oncogene activated by two missense mutations and flanked by rat retroviruslike VL30 sequences. Ha-MuSV induces focal transformation of mouse NIH 3T3 cells in vitro and tumors (fibrosarcomas and splenic erythroleukemias) in newborn mice. We have used these two assays to study the contribution of coding and noncoding viral sequences to the biological activity of Ha-MuSV. A good correlation was found between the in vitro and in vivo assays. In several different isogenic Ha-MuSV variants, those with a rasH gene that had one or both of the Ha-MuSV missense mutations were much more active biologically than the corresponding proto-oncogene. A Ha-MuSV variant that encoded the proto-oncogene protein induced lymphoid leukemias (with thymomas), with a relatively long latent period, rather than the fibrosarcomas and erythroleukemias characteristic of Ha-MuSV with one or both missense mutations. A VL30-derived segment with enhancer activity was identified downstream from v-rasH. A mutant Ha-MuSV from which this 3' noncoding segment was deleted expressed lower levels of the wild-type viral protein, displayed impaired transforming activity in vitro, and induced lymphoid leukemias (with thymomas). 5' noncoding rat c-rasH sequences were found to increase the biological activity of the virus when substituted for the corresponding segment of v-rasH. We conclude that (i) the biological activity of Ha-MuSV can be influence significantly by noncoding sequences located outside the long terminal repeat as well as by coding sequences, (ii) VL30 sequences positively regulate the expression of v-rasH, (iii) relatively low biological levels of ras, whether resulting from low-level expression of wild type v-rasH or high-levels of ras proto-oncogene protein, induce a type of tumor that differs from tumors induced by high biological levels of ras, and (iv) the in vivo pathogenicity of the Ha-MuSV variants correlated with their transforming activity on NIH 3T3 cells.
Identification and some properties of a unique DNA polymerase from cells infected with human B-lymphotropic virus
Authors: Bapat A R , Bodner A J , Ting R C , Cheng Y C
Published: January 2020

A new DNA polymerase and DNase activity were identified from cells infected with human B-lymphotropic herpesvirus (HBLV). DNA polymerase associated with HBLV infection was similar in its sensitivity to inhibition by ppi analogs as other herpesvirus-specific DNA polymerases but was dissimilar in its inhibition by certain nucleoside triphosphates.
Adenovirus E1A represses transcription of the cellular JE gene
Authors: Timmers H T , van Dam H , Pronk G J , Bos J L , Van der Eb A J
Published: January 2020

The expression of the growth factor-inducible JE gene was severely reduced in cells expressing region E1A of human adenoviruses. This reduction was not caused by a decreased stability of JE mRNA but by a reduced transcription rate as determined by run-on transcription experiments. Nevertheless, JE mRNA could still be induced by growth factors, suggesting that intracellular signaling is not blocked but rather that the basal level of JE transcription is repressed by E1A.
In vitro mutagenesis of the human T-cell leukemia virus types I and II tax genes
Authors: Cann A J , Rosenblatt J D , Wachsman W , Chen I S
Published: January 2020

The tax gene of the human T-cell leukemia virus types I and II (HTLV-I and HTLV-II) is essential for viral replication and acts by increasing the level of RNA transcription. The tax genes of HTLV-I and HTLV-II encode proteins of 40 and 37 kilodaltons, respectively. By in vitro mutagenesis of the tax gene, we have investigated those regions of the protein which are essential for its function. Mutation of either the amino- or carboxy-terminal domain of the protein resulted in loss of trans-activation ability. In addition, specificity of its activity with regard to trans-activation of either the HTLV-I or HTLV-II long terminal repeats was conferred by the first 59 amino acids.
A neutralization-resistant Theiler's virus variant produces an altered disease pattern in the mouse central nervous system
Authors: Zurbriggen A , Fujinami R S
Published: January 2020

Theiler's murine encephalomyelitis virus infection of mice is an animal model for human demyelinating diseases. To further define the role of this virus in the disease process, we selected a virus variant resistant to neutralization by a monoclonal antibody to VP-1. This virus variant was then injected into SJL/J mice. Central nervous system tissue was compared between variant virus- and wild-type virus-infected mice. Within the brain, no large differences were observed between the two groups as to the distribution of inflammatory infiltrates around the injection site and the number of viral antigen-positive cells during the first weeks of the observation period. In contrast, in the spinal cord major differences were found between variant virus- and wild-type virus-infected mice regarding the number of inflammatory lesions, infected cells, and the size of the areas involved with time. By immunohistochemistry, equivalent numbers of infected cells could be found in the spinal cord 1 week postinfection (p.i.): however, after that time, the number of infected cells in the wild-type virus-infected mice continued to increase, whereas the virus-positive cells from the variant virus-infected mice gradually decreased. Thus, the number of viral antigen-containing cells peaked by 1 week p.i. in the variant virus-infected animals. Conversely, the number of infected cells in the spinal cords from mice inoculated with wild-type virus steadily increased until 8 weeks p.i. At this time (8 weeks p.i.), no more variant virus antigen-positive cells could be observed within the spinal cord. Plaque assay of central nervous system tissue confirmed these differences between the two groups observed by immunohistochemistry. No infectious variant virus could be isolated after 2 weeks p.i. from the brain and 4 weeks p.i. from the spinal cord, whereas infectious wild-type virus could be detected up to the end of the observation period (12 weeks p.i.). Virus which was isolated from variant virus-infected mice still retained the neutralization-resistant phenotype. These studies emphasize the important biological in vivo activity of Theiler's virus VP-1 in determining neurovirulence.