bluuebunny/crossref_metadata_2025 huggingface dataset:
Construction and application of plasmids containing bidirectional promoters of vaccinia virus
Authors: Tsao H , Liu G Q , Ruan L , Chu C M
Published: January 2020

A bidirectional expression vector containing both the 11KD late promoter (p11) and the presumptive 25KD early promoter (p25) was constructed. These bidirectional vectors have been applied to the expression of hepatitis B surface antigen by using one of the promoters for beta-galactosidase as the marker gene and the other one for hepatitis B surface antigen as the target gene.
trans-activation of viral enhancers by the hepatitis B virus X protein
Authors: Spandau D F , Lee C H
Published: January 2020

We have used the chloramphenicol acetyltransferase (cat) gene expression system to study the effect of the X protein of hepatitis B virus (HBV) on viral enhancers. Plasmids containing the HBV enhancer and the core gene promoter linked to the cat gene were cotransfected with a plasmid containing the X gene into the human hepatoma cell line PLC/PRF/5. Our results indicate that the transfected X gene caused a trans-activation of the HBV enhancer. If a frameshift mutation or a deletion in the X structural gene was created, this trans-activation function was abolished. This result and the observation that the frameshift mutation did not alter the transcription of X mRNA suggest that the X protein is the trans-activating factor. Using similar techniques, we found that the X protein was also capable of trans-activating the simian virus 40 (SV40) and Rous sarcoma virus enhancers (pSV2cat and pRSVcat) in CV-1 cells. However, trans-activation of the SV40 enhancer by the X protein was not observed in COS-1 cells. By cotransfecting pSV2cat and the X gene with a plasmid containing either the intact SV40 genome, the SV40 genome devoid of the T-antigen (T-ag) gene, or only the T-ag gene, we demonstrated that SV40 T-ag can suppress trans-activation by the X protein. SV40 T-ag did not inhibit expression of the X gene or inactivate the X protein. The most probable mechanism of this inhibition is that T-ag competes with the X protein for a common target.
A C-terminal domain in the avian sarcoma-leukosis virus pol gene product is not essential for viral replication
Authors: Katz R A , Skalka A M
Published: January 2020

The virion proteins encoded by the avian retroviral pol gene (reverse transcriptase and endonuclease) are formed by the proteolytic processing of a gag-pol fusion protein precursor. Recent studies have predicted that the avian sarcoma-leukosis virus pol precursor protein undergoes a previously undetected processing event resulting in the formation of common C termini for the endonuclease (pp32) and the beta subunit of reverse transcriptase (F. Alexander, J. Leis, D. A. Soltis, R. M. Crowl, W. Danho, M. S. Poonian, Y.-C. E. Pan, and A. M. Skalka, J. Virol. 61:534-542, 1987; D. Grandgenett, T. Quinn, P. J. Hippenmeyer, and S. Oroszlan, J. Biol. Chem. 260:8243-8249, 1985). This processing event removes 37 amino acids, thus defining a new pol domain. In this report, we present evidence that this C-terminal domain is translated as part of the gag-pol precursor but is not required for replication of the virus in tissue culture cells.
Gamma interferon regulates long terminal repeat-controlled oncogene expression in transformed mouse fibroblasts at the level of mRNA transcription
Authors: Seliger B , Kruppa G , Schäfer R , Redmond S M , Pfizenmaier K
Published: January 2020

In transformed NIH 3T3 cells, murine gamma interferon reduces the expression of the long terminal repeat-controlled oncogenes v-mos, c-myc, and v-Ha-ras by a direct effect on the activity of retroviral promoters, as revealed by analyses of RNA half-life and transcriptional activity of retroviral genes as well as by analyses of chloramphenicol acetyltransferase activity in cells transformed with the cat gene under the control of long terminal repeats.
trans-activation of the simian virus 40 enhancer by a pX product of human T-cell leukemia virus type I
Authors: Saito S , Nakamura M , Ohtani K , Ichijo M , Sugamura K , Hinuma Y
Published: January 2020

A trans-acting factor, p40, of human T-cell leukemia virus type I profoundly potentiated the function of the enhancer from simian virus 40 but not polyomavirus and Rous sarcoma and murine sarcoma viruses. This trans-activation was seen in a limited repertoire of cells, in contrast to trans-activation of the human T-cell leukemia virus type I enhancer by p40.
Development of avian sarcoma and leukosis virus-based vector-packaging cell lines
Authors: Stoker A W , Bissell M J
Published: January 2020

We have constructed an avian leukosis virus derivative with a 5' deletion extending from within the tRNA primer binding site to a SacI site in the leader region. Our aim was to remove cis-acting replicative and/or encapsidation sequences and to use this derivative, RAV-1 psi-, to develop vector-packaging cell lines. We show that RAV-1 psi- can be stably expressed in the quail cell line QT6 and chicken embryo fibroblasts and that it is completely replication deficient in both cell types. Moreover, we have demonstrated that QT6-derived lines expressing RAV-1 psi- can efficiently package four structurally different replication-defective v-src expression vectors into infectious virus, with very low or undetectable helper virus release. These RAV-1 psi--expressing cell lines comprise the first prototype avian sarcoma and leukosis virus-based vector-packaging system. The construction of our vectors has also shown us that a sequence present within gag, thought to facilitate virus packaging, is not necessary for efficient vector expression and high virus production. We show that quantitation and characterization of replication-defective viruses can be achieved with a sensitive immunocytochemical procedure, presenting an alternative to internal selectable vector markers.
Human immune responses to major human cytomegalovirus glycoprotein complexes
Authors: Liu Y N , Kari B , Gehrz R C
Published: January 2020

Sera from both human cytomegalovirus (HCMV)-seropositive adults and infants with congenital HCMV infection recognized two major HCMV glycoprotein complexes. However, proliferative responses of peripheral blood mononuclear cells to these complexes varied among seropositive adults and were not detected in any of the infants. Thus, these glycoproteins alone may not be sufficient to develop a subviral HCMV vaccine.
Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen
Authors: Hoshino Y , Saif L J , Sereno M M , Chanock R M , Kapikian A Z
Published: January 2020

A single-gene substitution reassortant 11-1 was generated from two porcine rotaviruses, OSU (serotype 5) and Gottfried (serotype 4). This reassortant derived 10 genes, including gene 4 encoding VP3, from the OSU strain and only gene 9, encoding a major neutralization glycoprotein (VP7), from the Gottfried strain and was thus designated VP3:5; VP7:4. Oral administration of this reassortant to colostrum-deprived gnotobiotic newborn pigs induced a high level of neutralizing antibodies not only to Gottfried VP7 but also to OSU VP3, thus demonstrating that VP3 is as potent an immunogen as VP7 in inducing neutralizing antibodies during experimental oral infection. Gnotobiotic piglets infected previously with the reassortant were completely resistant to oral challenge with the virulent Gottfried strain (VP3:4; VP7:4), as indicated by failure of symptoms to develop and lack of virus shedding. Similarly, prior infection with the reassortant induced almost complete protection against diarrhea and significant restriction of virus replication after oral challenge with the virulent OSU strain (VP3:5; VP7:5). Thus, it appears that (i) the immune system of the piglet responds equally well to two rotavirus outer capsid proteins, VP3 and VP7, during primary enteric rotavirus infection; (ii) antibody to VP3 and antibody to VP7 are each associated with resistance to diarrhea; and (iii) infection with a reassortant rotavirus bearing VP3 and VP7 neutralization antigens derived from two viruses of different serotype induces immunity to both parental viruses. The relevance of these findings to the development of effective reassortant rotavirus vaccines is discussed.
Expression of the M gene of vesicular stomatitis virus cloned in various vaccinia virus vectors
Authors: Li Y , Luo L Z , Snyder R M , Wagner R R
Published: January 2020

Initial attempts to clone the matrix (M) gene of vesicular stomatitis virus (VSV) in a vaccinia virus expression vector failed, apparently because the expressed M protein, and particularly a carboxy-terminus-distal two-thirds fragment, was lethal for the virus recombinant. Therefore, a transient eucaryotic expression system was used in which a cDNA clone of the VSV M protein mRNA was inserted into a region of plasmid pTF7 flanked by the promoter and terminator sequences for the T7 bacteriophage RNA polymerase. When CV-1 cells infected with recombinant vaccinia virus vTF1-6,2 expressing the T7 RNA polymerase were transfected with pTF7-M3, the cells produced considerable amounts of M protein reactive by Western blot (immunoblot) analysis with monoclonal antibodies directed to VSV M protein. Evidence for biological activity of the plasmid-expressed wild-type M protein was provided by marker rescue of the M gene temperature-sensitive mutant tsO23(III) at the restrictive temperature. Somewhat higher levels of M protein expression were obtained in CV-1 cells coinfected with a vaccinia virus-M gene recombinant under control of the T7 polymerase promoter along with T7 polymerase-expressing vaccinia virus vTF1-6,2.
Encapsidation of Sendai virus genome RNAs by purified NP protein during in vitro replication
Authors: Baker S C , Moyer S A
Published: January 2020

The ability of the Sendai virus major nucleocapsid protein, NP, to support the in vitro synthesis and encapsidation of viral genome RNA during Sendai virus RNA replication was studied. NP protein was purified from viral nucleocapsids isolated from Sendai virus-infected BHK cells and shown to be a soluble monomer under the reaction conditions used for RNA synthesis. The purified NP protein alone was necessary and sufficient for in vitro genome RNA synthesis and encapsidation from preinitiated intracellular Sendai virus defective interfering particle (DI-H) nucleocapsid templates. The amount of DI-H RNA replication increased linearly with the addition of increasing amounts of NP protein. With purified detergent-disrupted DI-H virions as the template, however, there was no genome RNA synthesis in either the absence or presence of the NP protein. Furthermore, addition of the soluble protein fraction of uninfected cells alone or in the presence of purified NP protein also did not support DI-H genome RNA synthesis from purified DI-H. Another viral component in addition to the NP protein appears to be required for the initiation of encapsidation, since the soluble protein fraction of infected but not uninfected cells did support DI-H genome replication from purified DI-H.
Attachment of human polymorphonuclear leukocytes to herpes simplex virus-infected fibroblasts mediated by antibody-independent complement activation
Authors: van Strijp J A , van Kessel K P , Miltenburg L A , Fluit A C , Verhoef J
Published: January 2020

Herpes simplex virus (HSV)-infected cells can activate the human complement system without interference of specific anti-HSV antibodies. Analysis by flow cytometry showed that C3-like molecules were deposited on the membrane of the infected cell when incubated with human serum without specific antibodies. Depletion of calcium to block the classical pathway of the complement system had no effect on fluorescence intensity. The complement activation could be blocked by chelating both calcium and magnesium or by heating the serum. Furthermore, in the fluid phase C3 was converted to C3b by infected cells and not by uninfected cells. The antibody-independent activation did not lead to lysis of the virus-infected fibroblasts, indicating that the complement cascade is abrogated before formation of the membrane attack complex. This was also confirmed by measurement of the 50% hemolytic complement activities for total and alternative pathways. Polymorphonuclear leukocytes attached to infected fibroblasts after incubation of these fibroblasts with intact complement. This is most probably mediated by complement receptor binding of C3b and C3bi which is deposited on the membrane of the HSV-infected cell. Both type 1 and type 2 HSVs showed the same characteristics in complement activation and thereby mediated polymorphonuclear leukocyte adherence.
Tissue tropism and target cells of bluetongue virus in the chicken embryo
Authors: Wang L , Kemp M C , Roy P , Collisson E W
Published: January 2020

In situ cytohybridization was used to determine the tissue tropism and target cells for replication of bluetongue virus (BTV) in the developing chicken embryo. Hybridization with a biotinylated probe specific for segment 3 of BTV serotype 17 detected viral replication in embryos inoculated with U.S. serotypes 2, 10, 11, 13, and 17 or sheep blood containing a BTV field strain. At the final stages of infection, when the embryos were hemorrhagic, viral infection could consistently be detected in the brain, kidney, spinal cord, heart, lung, and liver, with the brain and kidney most severely affected. Other tissues, such as the retina, skin, tongue, and intestinal villi, also supported viral replication in some embryos. Greater concentrations of virus tended to be localized within epithelial cells, such as those lining the kidney tubules and tertiary bronchi of the lungs. Kinetics studies with BTV serotypes 11 and 17 and a field strain indicated that within 24 h after inoculation, viral replication occurred initially in the brain and kidney. By 48 h, viral replication was also detected in the lungs, heart, and spinal cord, with the liver being severely infected by 72 h. Low levels of hybridization could be detected in embryos infected with epizootic hemorrhagic disease virus, which is antigenically related to BTV.
B-cell lymphoproliferation and lymphomagenesis are associated with clonotypic intracellular terminal regions of the Epstein-Barr virus
Authors: Brown N A , Liu C R , Wang Y F , Garcia C R
Published: January 2020

We analyzed 17 B-cell lineages cloned from two patients with infectious mononucleosis and found that different B-cell lineages exhibited notable variation in the length of the fused Epstein-Barr virus (EBV) terminal region on intracellular EBV episomes. EBV termini in different B-cell clones from the same person differed by as many as 15 to 20 reiterations of the ca. 500-base-pair terminal repeat sequence. In contrast, analysis of seven B-cell lineages cloned from a patient with a fatal, oligoclonal lymphoma revealed that three of the cell clones had the same-sized EBV terminal region. These three clones had previously been shown, by immunoglobulin gene analysis, to be metastatic daughter cells descended from a common progenitor. Similarity of the EBV terminal regions in the three daughter clones suggested that EBV infected the progenitor cell before proliferation and metastasis. Individual, EBV-infected cells from a single individual showed sufficient heterogeneity in their EBV termini to allow use of terminal fragment size as a clonal marker in studies addressing the contribution of EBV to the clonal pathogenesis of tumors with which this virus has been associated.
Transforming viruses spontaneously arise from nontransforming reticuloendotheliosis virus strain T-derived viruses as a result of increased accumulation of spliced viral RNA
Authors: Miller C K , Embretson J E , Temin H M
Published: January 2020

The highly oncogenic avian retrovirus reticuloendotheliosis virus strain T (Rev-T) contains a substitution of the oncogene v-rel for much of env and a deletion of gag and pol relative to the helper virus Rev-A. Replacement of gag and pol sequences in Rev-T suppresses transformation by reducing the accumulation of spliced viral mRNA and v-rel protein in infected cells (C. K. Miller and H. M. Temin, J. Virol 58:75-80, 1986). After infection of spleen cells with viruses containing gag and pol sequences, revertant viruses that are strongly transforming were found. Approximately three-fourths of the revertant viruses appeared structurally the same as the parental virus, and approximately one-fourth of the revertant viruses had large deletions (similar in size and location to the deletion in Rev-T). Two revertant viruses that appeared structurally the same as the parental virus were molecularly cloned. The regions sufficient to change the parental virus to a strongly transforming virus were determined by construction of recombinant viruses. In one revertant virus, the region sufficient for transformation contained a 327-base-pair insertion 5' of the 3' splice site used by Rev-T. In the other revertant virus, the region sufficient for transformation contained a 1-base-pair transition and a deletion of one copy of a 9-base-pair direct repeat, both 3' of the 3' splice site used by Rev-T. These differences resulted in the accumulation of increased levels of subgenomic v-rel mRNA and protein, ultimately leading to transformation.
Structural and functional analysis of long terminal repeats of Suncus murinus mammary tumor virus
Authors: Yanagawa S , Murakami A , Hoshino M , Tanaka H
Published: January 2020

A 2.7-kilobase (kb) cDNA sequence complementary to Suncus murinus mammary tumor virus (Sm-MTV) genomic RNA [corrected] was prepared using purified virions produced by the Sm-MT cell line, which had been established from a spontaneous mammary tumor of S. murinus. It was found, by using this cDNA in Southern hybridization experiments, that Sm-MTV was endogenous to this animal and that some 50 copies of endogenous provirus were present per haploid cellular genome. In addition, a proviral Sm-MTV DNA sequence, 9.4 kb long (Sm-P-MTV10), was cloned from a Sm-MT cell genomic library, and its long terminal repeat was found to be 720 base pairs (bp) long, with the U3.R and U5 regions 574 and 146 bp long, respectively. The boundary between U3 and R was not determined with certainty, though in the cDNA, the U3 and R regions were 462 and 105 bp long, respectively. The overall homology between the U3.R regions in the cDNA and Sm-P-MTV was 75%. These two DNAs differed in such transcription regulatory signals as CCAAT and TATAA, the first being missing from the cDNA. Nevertheless, chloramphenicol acetyltransferase assays showed that the long terminal repeats of the cDNA and the Sm-P-MTV were transcriptionally active but not steroid hormone responsive. Like Mason-Pfizer monkey virus, Sm-MTV used tRNA(1,2Lys) as a primer for reverse transcription. In addition, the immunosuppressive peptide sequence common to many retroviruses was found in the env region of Sm-MTV. In these two points, Sm-MTV differed from mouse MTV.
Purification and partial characterization of poliovirus protease 2A by means of a functional assay
Authors: König H , Rosenwirth B
Published: January 2020

The purification of poliovirus protease 2A from infected cells by a functional assay is described. A small synthetic peptide was cleaved specifically by an esterase present in poliovirus-infected cells. Since the enzyme proved extremely unstable in crude extracts a rapid and efficient purification procedure had to be developed. By treatment with different detergents followed by high-speed centrifugation, the esterase activity was separated from inactivating cellular enzymes and was solubilized. Purification to more than 90% homogeneity could be achieved by a single chromatography step, namely, by gel filtration through Superose 12 under fast-protein liquid chromatography conditions. The esterase activity was associated with a protein of 17,000 daltons and copurified with poliovirus protein 2A. Furthermore, antibodies to 2A specifically precipitated the esterase activity. Thus, the esterase was identified as poliovirus protease 2A. Inhibition studies with known protease inhibitors revealed that 2A is probably a sulfhydryl protease. Of the metal ions tested, only zinc exerted significant inhibitory effects. The esterase activity was optimal near neutral pH and had an extremely short half-life at physiological temperatures.
Regulatory elements within the murine leukemia virus enhancer regions mediate glucocorticoid responsiveness
Authors: Celander D , Hsu B L , Haseltine W A
Published: January 2020

Enhancer elements within nonleukemogenic (Akv) and T-cell leukemogenic (SL3-3) murine leukemia viruses demonstrate strong cell type preference in transcriptional activity. These transcription elements are additionally regulated by the synthetic glucocorticoid dexamethasone, and this pattern of regulation varies according to cell type. The sequences required for dexamethasone regulation for both Akv and SL3-3 are shown to include a 17-nucleotide consensus sequence previously termed the glucocorticoid response element (GRE). Although the GREs are identical for both viral enhancers, the sequences surrounding these elements differ, as does the spatial arrangement of the GRE sequences with respect to one another. It is proposed that the spatial arrangement of the GREs, as well as their precise sequence context, determines the difference in the response to dexamethasone of the enhancers in different cell types.
Delayed formation of defective interfering particles in vesicular stomatitis virus-infected cells: kinetic studies of viral protein and RNA synthesis during autointerference
Authors: Von Laer D M , Mack D , Kruppa J
Published: January 2020

The time course of defective interfering (DI) particle and B particle release from vesicular stomatitis virus-infected BHK-21 cells was studied at different multiplicities of defective and infective particles. Particle release was progressively delayed in cells infected with an increasing DI-to-B particle ratio. The delayed particle release during interference was found to be connected with a reduced but prolonged synthesis of viral proteins, a slower accumulation of viral proteins, and a delayed shutoff of cellular protein synthesis. The relative synthesis of M and G proteins was reduced during interference, whereas the relative synthesis of N and NS proteins was increased. On the level of genomic RNA replication, we found that DI RNA was replicated more slowly during interference than the standard genomic RNA was during acute infection. The ratio of DI particles to B particles which were released increased throughout the infectious cycle. At a given time in the infectious cycle, this ratio was independent of the multiplicity of infecting DI and B particles. On the basis of the kinetic studies, we argue that cells infected with higher amounts of DI particles compared with B particles synthesize a higher DI-to-B particle ratio and release these progeny particles later than cells infected with a low DI-to-B particle ratio.
Specificity of cleavage in replicative-form DNA of bovine herpesvirus 1
Authors: Hammerschmidt W , Ludwig H , Buhk H J
Published: January 2020

The linear double-stranded DNA genome of herpesvirus as it is present in infectious virions needs to be circularized after infection of host cells and before DNA replication. Replicative-form genomes have to be cleaved into linear unit-length molecules during virion maturation and are most probably the substrate for inversion of the short segment relative to the long segment of the bovine herpesvirus 1 (BHV-1) genome. Those regions of the BHV-1 genome which are functionally involved in these processes have been analyzed at the molecular level by cloning and sequencing the genomic termini, the fusion of both termini from replicative-form molecules, and the junction between the short and the long genome segment. On the basis of the simple genome arrangement of BHV-1, it was inferable that the cleavage of replicative-form genomes by a hypothetical BHV-1 terminase activity may be specified by a sequence at the left end of UL (An element), which is located proximal to a reiterated beta element that makes up the cleavage site itself. The relationship of those elements in BHV-1 and the comparison to similar regions of other herpesviruses indicate consensus sequence elements which are functionally important for cleavage and isomerization of viral DNA during maturation of virions.
Mapping Pieris rapae granulosis virus transcripts and their in vitro translation products
Authors: Dwyer K G , Granados R R
Published: January 2020

Poly(A)+ RNA was isolated from Pieris rapae granulosis virus (PrGV)-infected P. rapae larvae at times early (40 h postinoculation) and late (88 h postinoculation) in larval infection. Using Northern (RNA) blot analysis, we determined the sizes, relative abundances, and map locations of over 100 PrGV transcripts. Differences were found in the transcripts which had accumulated at the two time points. Splicing of these transcripts was not detected. Evidence for the expression of overlapping RNAs in PrGV was obtained. A minimum of 35 PrGV translation products were detected via hybrid selection of poly(A)+ RNA with specific PrGV restriction endonuclease fragments, followed by in vitro translation.
Efficient expression in insect cells of a soluble, active human insulin receptor protein-tyrosine kinase domain by use of a baculovirus vector
Authors: Ellis L , Levitan A , Cobb M H , Ramos P
Published: January 2020

The human insulin receptor (IR) is a transmembrane glycoprotein, whose cytoplasmic domain contains an insulin-activated protein-tyrosine kinase (EC 2.7.1.112). By the use of an appropriately engineered baculovirus expression vector, a soluble cytoplasmic derivative of this domain was expressed in the insect cell line Spodoptera frugiperda (Sf9). At 24 to 48 h after Sf9 cells were infected with recombinant virus, a protein of the size expected for this domain (approximately 48 kilodaltons) constituted a major band when total cell lysates of metabolically labeled cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. This protein (designated AchIRPTK) was immunoprecipitated by three monoclonal antibodies, each of which recognizes a distinct antigenic site of the IR cytoplasmic domain and requires the native structure of the protein for recognition and one of which binds at or near the physiologically relevant site(s) of IR autophosphorylation. In vivo, AchIRPTK was phosphorylated on both tyrosine and serine residues. When affinity purified, the kinase was active in vitro; it autophosphorylated exclusively on tyrosine residues, and phosphorylated the exogenous substrates histone H2b and poly(Glu-Tyr). The expression of an active IR protein-tyrosine kinase molecule in this heterologous cell system provides an efficient experimental method for producing this domain in quantity for enzymatic and structural studies.
Production of infectious duck hepatitis B virus in a human hepatoma cell line
Authors: Galle P R , Schlicht H J , Fischer M , Schaller H
Published: January 2020

The differentiated human hepatoma cell line Hep-G2 was transfected with cloned duck hepatitis B virus (DHBV) DNA. Introduction of closed circular DNA into the human liver cells resulted in the production of viral proteins: core antigen was detected in the cytoplasm, and e antigen, a related product, was secreted into the medium. Moreover, viral particles were released into the tissue culture medium which were indistinguishable from authentic DHBV by density, antigenicity, DNA polymerase activity, and morphology. Intravenous injection of tissue culture-derived DHBV particles into Pekin ducks established DHBV infection. In conclusion, transfection of human hepatoma cells with cloned DHBV DNA results in the production of infectious virus, as occurs with cloned human hepatitis B virus DNA. Human liver cells are therefore competent to support production of the avian and mammalian hepadnaviruses, indicating that liver-specific viral gene expression is controlled by evolutionarily conserved mechanisms. This new DHBV transfection system offers the opportunity to rapidly produce mutated DHBV which then can be further investigated in Pekin ducks.
Genetic control and fine specificity of the immune response to a synthetic peptide of influenza virus hemagglutinin
Authors: Brown L E , Murray J M , Anders E M , Tang X L , White D O , Tregear G W , Jackson D C
Published: January 2020

The immune response to a synthetic peptide, H3 HA1(305-328), representing the C'-terminal 24 amino acid residues of the HA1 chain of the hemagglutinin of the H3 subtype of influenza virus is controlled by genes in the I region of the major histocompatibility complex. Mice of the H-2d haplotype are high responders and produce antibody for several months after a single injection of peptide without carrier. Mice of the H-2b, H-2k, and H-2q haplotypes are low antibody responders. Investigation of recombinant and congenic mouse strains revealed that high responsiveness requires the genes that encode the I-Ed molecule. Immunoassays, involving direct binding to analogs of this peptide and inhibition by both these analogs and synthetic epitopes, were used to analyze the specificity of the polyclonal response. In BALB/c mice, the primary antibody response is directed principally against the antigenic site 314-LKLAT-318, whereas the secondary response after a boost is predominantly directed to a distinct site, 320-MRNVPEKQT-328. The T-cell response to the peptide H3 HA1(305-328), as measured by antigen-induced proliferation of primed T cells in vitro, is also I-Ed restricted in high-responder H-2d mice and is directed against an antigenic site that does not require the four C-terminal residues unique to the H3 influenza subtype. A different epitope appears to be recognized by T cells from CBA (H-2k) mice, which proliferate to a moderate extent on exposure to the peptide but, nevertheless, do not provide help for an antibody response.
The cytoplasmic domain of herpes simplex virus type 1 glycoprotein C is required for membrane anchoring
Authors: Holland T C , Lerch R J , Earhart K
Published: January 2020

The herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) gene was altered so that it encoded a truncated glycoprotein lacking a cytoplasmic domain but retaining 20 of 23 amino acids of the transmembrane domain. No additional amino acid residues were introduced into the glycoprotein encoded by the altered gene. The gene was recombined into the HSV-1 genome by marker transfer. Two recombinant viruses, dl1 and dl2, that expressed the mutant gene were isolated. Characterization of these viruses showed that a substantial fraction of the mutant glycoprotein was secreted from infected cells. Pulse-chase experiments showed that the kinetics of posttranslational modification of the mutant glycoprotein were similar to those of the wild type. However, comparison of the kinetics of secretion of gC by dl2 and gC-3, a gC mutant lacking both the transmembrane and cytoplasmic domains, showed that dl2 gC was secreted much more slowly than gC-3 gC. Iodination of plasma membrane glycoproteins showed that dl2 gC was initially expressed on the cell surface as a membrane protein and subsequently was slowly released from the membrane into the medium. These data indicate that a major function of the cytoplasmic domain of gC is to ensure the stable anchoring of the glycoprotein in plasma membranes. In contrast to these major changes in the membrane-anchoring properties of gC, characterization of the virions produced by dl1 and dl2 showed that they contain significant amounts of gC. Thus the cytoplasmic domain does not appear to be essential for incorporation of this glycoprotein into virions.
In vivo RNA-RNA recombination of coronavirus in mouse brain
Authors: Keck J G , Matsushima G K , Makino S , Fleming J O , Vannier D M , Stohlman S A , Lai M M
Published: January 2020

RNA-RNA recombination between different strains of the murine coronavirus mouse hepatitis virus (MHV) occurs at a very high frequency in tissue culture. To demonstrate that RNA recombination may play a role in the evolution and pathogenesis of coronaviruses, we sought to determine whether MHV recombination could occur during replication in the animal host of the virus. By using two selectable markers, i.e., temperature sensitivity and monoclonal antibody neutralization, we isolated several recombinant viruses from the brains of mice infected with two different strains of MHV. The recombination frequency was very high, and recombination occurred at multiple sites on the viral RNA genome. This finding suggests that RNA-RNA recombination may play a significant role in natural evolution and neuropathogenesis of coronaviruses.
Prediction of similar transforming regions in simian virus 40 large T, adenovirus E1A, and myc oncoproteins
Authors: Figge J , Webster T , Smith T F , Paucha E
Published: January 2020

Regions containing similar elements of primary and predicted secondary structure were identified in simian virus 40 large T, adenovirus E1A, c-myc and v-myc proteins by a computer program with a set of highly specific, complex pattern descriptors. In all cases these regions were localized in domains of the respective proteins known to be required for transforming activity. We suggest that these apparently structurally similar regions may mediate a common biological function.
Minus-strand RNA synthesis in the spinal cords of mice persistently infected with Theiler's virus
Authors: Cash E , Chamorro M , Brahic M
Published: January 2020

Theiler's virus, a murine picornavirus, causes a chronic neurological disease characterized by primary demyelination in SJL/J mice. The lesions are very reminiscent of those of multiple sclerosis. Theiler's virus persists in oligodendrocytes and to a lesser extent in astrocytes and macrophages throughout the disease. Viral RNA and capsid protein syntheses are minimal in these cells. This restriction could play a central role in the mechanism of virus persistence. By quantitating plus- and minus-strand RNAs in infected central nervous system cells, we showed that RNA replication was blocked at the level of minus-strand RNA synthesis.
Analysis of neutralizing epitopes on foot-and-mouth disease virus
Authors: Pfaff E , Thiel H J , Beck E , Strohmaier K , Schaller H
Published: January 2020

For the investigation of the antigenic determinant structure of foot-and-mouth disease virus (FMDV), neutralizing monoclonal antibodies (MAbs) against complete virus were characterized by Western blot (immunoblot), enzyme immunoassay, and competition experiments with a synthetic peptide, isolated coat protein VP1, and viral particles as antigens. Two of the four MAbs reacted with each of these antigens, while the other two MAbs recognized only complete viral particles and reacted only very poorly with the peptide. The four MAbs showed different neutralization patterns with a panel of 11 different FMDV strains. cDNA-derived VP1 protein sequences of the different strains were compared to find correlations between the primary structure of the protein and the ability of virus to be neutralized. Based on this analysis, it appears that the first two MAbs recognized overlapping sequential epitopes in the known antigenic site represented by the peptide, whereas the two other MAbs recognized conformational epitopes. These conclusions were supported and extended by structural analyses of FMDV mutants resistant to neutralization by an MAb specific for a conformational epitope. These results demonstrate that no amino acid exchanges had occurred in the primary antigenic site of VP1 but instead in the other coat proteins VP2 and VP3, which by themselves do not induce neutralizing antibodies.
Togavirus-associated pathologic changes in the midgut of a natural mosquito vector
Authors: Weaver S C , Scott T W , Lorenz L H , Lerdthusnee K , Romoser W S
Published: January 2020

Arthropod-borne viruses were not previously believed to cause discernible pathologic changes in their natural mosquito vectors. We report cytopathologic lesions in the midgut of the mosquito, Culiseta melanura, 2 to 5 days after oral infection with eastern equine encephalomyelitis virus. Sloughing of densely staining, heavily infected epithelial cells into the midgut lumen was observed by light and transmission electron microscopy, along with degeneration of cells within the epithelium. Pathological changes in midgut epithelial cells sometimes included loss of brush border and basal lamina integrity. Disruption of the midgut basal lamina could result in bypassing of barriers to virus dissemination within the mosquito and allow rapid transmission to occur. Alternatively, luminal sloughing of heavily infected midgut epithelial cells may serve to modulate mosquito infections. These findings challenge previous beliefs regarding the benign nature of arbovirus-invertebrate host relationships.
Functional and topographical analyses of epitopes on the hemagglutinin (VP4) of the simian rotavirus SA11
Authors: Burns J W , Greenberg H B , Shaw R D , Estes M K
Published: January 2020

An immunochemical analysis of the hemagglutinin (VP4) of the simian rotavirus SA11 was performed to better understand the structure and function of this molecule. Following immunization of mice with double-shelled virus particles and VP4-enriched fractions from CsCl gradients, a battery of anti-SA11 hybridomas was generated. A total of 13 clones secreting high levels of anti-VP4 monoclonal antibody (MAb) was characterized and compared with two cross-reactive anti-VP4 MAbs generated against heterologous rhesus (RRV) and porcine (OSU) rotavirus strains. These cross-reactive MAbs effectively neutralized SA11 infectivity in vitro. The epitopes recognized by these 15 MAbs were grouped into six antigenic sites on the SA11 hemagglutinin. These sites were identified following analysis of the MAbs by using a simple competitive binding enzyme-linked immunosorbent assay (ELISA) and biological assays. Three of the antigenic sites were involved in neutralization of virus infectivity in vitro. All the MAbs with neutralization activity and two nonneutralizing MAbs were able to inhibit viral hemagglutination of human erythrocytes. Competitive binding ELISA data showed a positive cooperative binding effect with some pairs of the anti-VP4 MAbs, apparently due to a conformational change induced by the binding of the first MAb. Some of the MAbs also bound better to trypsin-treated virus than to non-trypsin-treated virus. A topographic map for VP4 is proposed on the basis of the observed properties of each antigenic site.
Simian virus 40 (SV40) T antigen binds specifically to double-stranded DNA but not to single-stranded DNA or DNA/RNA hybrids containing the SV40 regulatory sequences
Authors: Auborn K J , Markowitz R B , Wang E , Yu Y T , Prives C
Published: January 2020

Simian virus 40 T antigen has been shown previously to bind specifically with high affinity to sites within the regulatory region of double-stranded simian virus 40 DNA. Using competition filter binding and the DNA-binding immunoassay, we show that T antigen did not bind specifically to either early or late single-stranded DNA containing these binding sites. Moreover, T antigen did not bind these sequences present in single-stranded RNA, RNA/RNA duplexes, or RNA/DNA hybrids. T antigen did, however, bind as efficiently to single-stranded DNA-cellulose as to double-stranded DNA-cellulose. This binding was nonspecific because it was independent of the presence of T-antigen-binding sites. The implications of these observations are discussed.
A sequence-specific single-strand-binding protein for the late-coding strand of the simian virus 40 control region
Authors: Gaillard C , Weber M , Strauss F
Published: January 2020

We have purified a protein from uninfected monkey CV1 cells that binds specifically in vitro to the late-coding simian virus 40 DNA strand in the region of transcription control without any detectable binding to the complementary single strand. Nuclease protection experiments detected two binding sites in the 21-base-pair repeat region. The protein did not bind to this region in the double-stranded form, nor did it bind to RNA synthesized in vitro by using either DNA strand as a template. This protein, and perhaps other DNA single-strand-sequence-specific proteins, may play a role in the control of gene expression in higher organisms.
A family of interferon-induced Mx-related mRNAs encodes cytoplasmic and nuclear proteins in rat cells
Authors: Meier E , Fäh J , Grob M S , End R , Staeheli P , Haller O
Published: January 2020

Mouse Mx protein, an interferon (IFN)-induced nuclear protein, confers selective resistance to influenza virus. We show here that, as with influenza virus-resistant Mx+ mouse embryo cells, influenza virus mRNA accumulation and protein synthesis are strongly inhibited in rat embryo cells treated with IFN-alpha/beta. IFN-alpha/beta induced in rat cells the synthesis of Mx-related mRNAs migrating on Northern (RNA) gels as two bands of about 3.5 and 2.5 kilobases which directed the synthesis of three electrophoretically distinct proteins called rat Mx proteins 1, 2, and 3. The three rat proteins were antigenically related to the mouse Mx protein but differed in molecular weight and intracellular location. Rat Mx protein 1 was found predominantly in the nucleus and, on the basis of several criteria, resembled the nuclear mouse Mx protein. It was induced by IFN-alpha/beta in all 28 inbred rat strains tested. Rat Mx proteins 2 and 3 differed from protein 1 at the carboxy terminus and were predominantly cytoplasmic like the human Mx homolog. Sequence data of partial cDNA clones indicate that three Mx-related genes, rather than one, exist in the rat.
Identification of cross-reactive and serotype 2-specific neutralization epitopes on VP3 of human rotavirus
Authors: Taniguchi K , Maloy W L , Nishikawa K , Green K Y , Hoshino Y , Urasawa S , Kapikian A Z , Chanock R M , Gorziglia M
Published: January 2020

The group A rotaviruses are composed of at least seven serotypes. Serotype specificity is defined mainly by an outer capsid protein, VP7. In contrast, the other surface protein, VP3 (775 amino acids), appears to be associated with both serotype-specific and heterotypic immunity. To identify the cross-reactive and serotype-specific neutralization epitopes on VP3 of human rotavirus, we sequenced the VP3 gene of antigenic mutants resistant to each of seven anti-VP3 neutralizing monoclonal antibodies (N-MAbs) which exhibited heterotypic or serotype 2-specific reactivity, and we defined three distinct neutralization epitopes on VP3. The mutants sustained single amino acid substitutions at position 305, 392, 433, or 439. Amino acid position 305 was critical to epitope I, whereas amino acid position 433 was critical to epitope III. In contrast, epitope II appeared to be more dependent upon conformation and protein folding because both amino acid positions 392 and 439 appeared to be critical. These four positions clustered in a relatively limited area of VP5, the larger of the two cleavage products of VP3. At the positions where amino acid substitutions occurred, there was a correlation between amino acid sequence homology among different serotypes and the reactivity patterns of various viruses with the N-MAbs used for selection of mutants. A synthetic peptide (amino acids 296 to 313) which included the sequence of epitope I reacted with its corresponding N-MAb, suggesting that the region contains a sequential antigenic determinant. These data may prove useful in current efforts to develop vaccines against human rotavirus infection.
Model for studying virus attachment: identification and quantitation of Epstein-Barr virus-binding cells by using biotinylated virus in flow cytometry
Authors: Inghirami G , Nakamura M , Balow J E , Notkins A L , Casali P
Published: January 2020

Epstein-Barr virus (EBV) was purified and biotinylated without significant loss of its cell-transforming activity. The use of biotinylated virus in conjunction with antibodies specific for selected cell surface molecules and flow cytometric analysis allowed for the positive identification of the virus-binding lymphocytes among a heterogeneous mononuclear cell population. Biotinylated EBV efficiently bound to all B lymphocytes, including those bearing surface mu, delta, gamma, and alpha immunoglobulin heavy chains or the surface CD5 (Leu-1) marker, but not to T lymphocytes, natural killer cells, or monocytes. By using biotinylated EBV and specific monoclonal antibodies in competitive inhibition experiments, it was also found that the virus attaches to an epitope on the CR2 molecule (the receptor for C3d and EBV), which is close to or identical with the one recognized by OKB7 monoclonal antibody, and that cell surface structures other than CR2 cannot mediate attachment of EBV. Moreover, studies on the binding of the virus to induced B lymphocytes (cells in S through G2 phase), and this was associated with the disappearance of the surface CR2 molecule and the inability of the virus to attach to these cells. The approach described here should be useful in studying the attachment of other viruses, identifying the specific cell types involved, and analyzing the effect of the cell cycle on virus binding.
Mapping of the fibrotropic and lymphotropic host range determinants of the parvovirus minute virus of mice
Authors: Gardiner E M , Tattersall P
Published: January 2020

The fibrotropic and lymphotropic strains of minute virus of mice are each unable to grow lytically in the differentiated host cell type of the other strain. To map the viral sequence responsible for the target cell specificities of the two strains, we constructed chimeric viral genomes in vitro from infectious genomic clones. The phenotypes of viral progeny derived from the chimeric genomes were tested by transfecting the plasmids into fibroblast monolayers and assaying plaque formation and by testing stocks of the recombinant viruses for cytotoxicity in fibroblast and lymphocyte cultures. Both the fibrotropic and lymphotropic determinants mapped to the same 237-nucleotide sequence within the coding region of the virus structural gene. A second sequence, near the viral promoter at map unit 38, was also shown to affect viral growth in fibroblast host cells profoundly.
A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation
Authors: Jang S K , Kräusslich H G , Nicklin M J , Duke G M , Palmenberg A C , Wimmer E
Published: January 2020

Picornavirus RNAs are uncapped messengers and have unusually long 5' nontranslated regions (5'NTRs) which contain many noninitiating AUG triplets. The translational efficiency of different picornavirus RNAs varies between different cell-free extracts and even in the same extract, such as micrococcal nuclease-treated rabbit reticulocyte lysates. The effect of the poliovirus 5'NTR on in vitro translation was compared with that of the 5'NTR of encephalomyocarditis virus by the use of synthetic mRNAs, micrococcal nuclease-treated HeLa cell extracts, and rabbit reticulocyte lysates. Artificial mono- and dicistronic mRNAs synthesized with T7 RNA polymerase were used to investigate whether the 5'NTR of encephalomyocarditis virus RNA contains a potential internal ribosomal entry site. The sequence between nucleotides 260 and 484 in the 5'NTR of encephalomyocarditis RNA was found to play a critical role in the efficient translation in both mono- and dicistronic mRNAs. Our data suggest that an internal ribosomal entry site resides in this region.