bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunogenicity and Protective Efficacy in Mice of Influenza B Virus Vaccines Grown in Mammalian Cells or Embryonated Chicken Eggs
Authors: Alymova I. V. , Kodihalli S. , Govorkova E. A. , Fanget B. , Gerdil C. , Webster R. G. 
Published: December 2019

ABSTRACTThe immunogenicity and protective efficacy of formalin-inactivated influenza B/Memphis/1/93 virus vaccines propagated exclusively in Vero cells, MDCK cells, or embryonated chicken eggs (hereafter referred to as eggs) were investigated. Mammalian cell-grown viruses differ from the egg-grown variant at amino acid position 198 (Pro/Thr) in the hemagglutinin gene. The level of neuraminidase activity was highest in egg-grown virus, while MDCK and Vero cell-derived viruses possessed 70 and 90% less activity, respectively. After boosting, each of the vaccines induced high levels of hemagglutinin-inhibiting, neuraminidase-inhibiting, and neutralizing antibodies that provided complete protection from MDCK-grown virus challenge. Mammalian cell-derived virus vaccines induced serum antibodies that were more cross-reactive, while those induced by egg-grown virus vaccines were more specific to the homologous antigen. Enzyme-linked immunospot analysis indicated that cell-grown virus vaccines induced high frequencies of immunoglobulin G (IgG)-producing cells directed against both cell- and egg-grown virus antigens, whereas egg-grown virus vaccine induced higher frequencies of IgG- and IgM-producing cells reacting with homologous antigen and low levels of IgG-producing cells reactive with cell-grown viruses. These studies indicate that influenza B virus variants selected in different host systems can elicit different immune responses, but these alterations had no detectable influence on the protective efficacy of the vaccines with the immunization protocol used in this study.
Respiratory Syncytial Virus Infection of Human Alveolar Epithelial Cells Enhances Interferon Regulatory Factor 1 and Interleukin-1β-Converting Enzyme Gene Expression but Does Not Cause Apoptosis
Authors: Takeuchi Ryoh , Tsutsumi Hiroyuki , Osaki Masaya , Haseyama Keiji , Mizue Nobuo , Chiba Shunzo 
Published: December 2019

ABSTRACT
The induction kinetics of the transcriptional activities of interferon regulatory factor 1 (IRF-1), interleukin-1β-converting enzyme (ICE), and CPP32 by respiratory syncytial virus (RSV) infection of human type II alveolar epithelial cells (A549 cells) were analyzed semiquantitatively by reverse transcriptase PCR. The appearance of ICE and CPP32 protein in cell lysate was examined by Western blotting analysis. The induction of apoptosis by RSV infection was examined by the appearance of DNA fragmentation detected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling. RSV moderately enhanced IRF-1 mRNA as early as 4 h after infection, and this enhancement lasted several hours. Following induction of the IRF-1 gene, ICE gene expression increased significantly, and an increase of ICE protein was observed in the RSV-infected cell lysate. These increments were observed in cells treated with live RSV but not in cells treated with inactivated RSV or control antigen. However, no infection-specific increase of CPP32 gene expression or the protein was observed. No nucleosomal fragmentation was observed in RSV-infected cells during the whole course of infection, despite the appearance of extensive cytopathic change and cell death. These observations suggest that RSV infection of human alveolar epithelial cells induces the ICE gene and its protein as a result of increased IRF-1 induction but that the increased ICE was insufficient to cause apoptosis in the RSV-infected cells. ICE might not be able to activate CPP32, which is thought to be the more important protease for apoptosis.
Processing of the Borna Disease Virus Glycoprotein gp94 by the Subtilisin-Like Endoprotease Furin
Authors: Richt Jürgen A. , Fürbringer Thomas , Koch Andreas , Pfeuffer Isolde , Herden Christiane , Bause-Niedrig Ingrid , Garten Wolfgang 
Published: December 2019

ABSTRACT
Open reading frame IV (ORF-IV) of Borna disease virus (BDV) encodes a protein with a calculated molecular mass of ca. 57 kDa (p57), which increases after N glycosylation to 94 kDa (gp94). The unglycosylated and glycosylated proteins are proteolytically cleaved by the subtilisin-like protease furin. Furin most likely recognizes one of three potential cleavage sites, namely, an arginine at position 249 of the ORF-IV gene product. The furin inhibitor decRVKRcmk decreases the production of infectious BDV significantly, indicating that proteolytic cleavage of the gp94 precursor molecule is necessary for the full biological activity of the BDV glycoprotein.
Characterization of Determinants for Envelope Binding and Infection in Tva, the Subgroup A Avian Sarcoma and Leukosis Virus Receptor
Authors: Rong Lijun , Gendron Kristin , Strohl Barbara , Shenoy Rajeev , Wool-Lewis Rouven J. , Bates Paul 
Published: December 2019

ABSTRACT
Tva is the cellular receptor for subgroup A avian leukosis and sarcoma virus (ALSV-A). The viral interaction domain of Tva is determined by a 40-residue, cysteine-rich module closely related to the ligand binding domain of the human low-density lipoprotein receptor (LDLR). In this report, we examined the role of the LDLR-like module of Tva in envelope binding and viral infection by mutational analysis. We found that the entire LDLR module in Tva is essential for efficient binding to the viral envelope protein. However, the 17 N-terminal residues of this module can be deleted without affecting receptor function, suggesting that the major determinants for viral entry are located at the C terminus of the module. The effect on viral infection of many amino acid substitutions and deletions in the LDLR module is context dependent, suggesting that the residues important for viral entry are dispersed throughout the LDLR module. In addition, we found that all 27 mutations at residues D46, E47, and W48 greatly reduced envelope binding. These results are discussed in relation to a recently elucidated structure for an LDLR module.
Rep*: a Viral Element That Can Partially Replace the Origin of Plasmid DNA Synthesis of Epstein-Barr Virus
Authors: Kirchmaier Ann L. , Sugden Bill 
Published: December 2019

ABSTRACT

Replication of the Epstein-Barr viral (EBV) genome occurs once per cell cycle during latent infection. Similarly, plasmids containing EBV’s plasmid origin of replication,
oriP
, are replicated once per cell cycle. Replication from
oriP
requires EBV nuclear antigen 1 (EBNA-1) in
trans
; however, its contributions to this replication are unknown.
oriP
contains 24 EBNA-1 binding sites; 20 are located within the family of repeats, and 4 are found within the dyad symmetry element. The site of initiation of DNA replication within
oriP
is at or near the dyad symmetry element. We have identified a plasmid that contains the family of repeats but lacks the dyad symmetry element whose replication can be detected for a limited number of cell cycles. The detection of short-term replication of this plasmid requires EBNA-1 and can be inhibited by a dominant-negative inhibitor of EBNA-1. We have identified two regions within this plasmid which can independently contribute to this replication in the absence of the dyad symmetry element of
oriP
. One region contains native EBV sequences within the
Bam
HI C fragment of the B95-8 genome of EBV; the other contains sequences within the simian virus 40 genome. We have mapped the region contributing to replication within the EBV sequences to a 298-bp fragment, Rep*. Plasmids which contain three copies of Rep* plus the family of repeats support replication more efficiently than those with one copy, consistent with a stochastic model for the initiation of DNA synthesis. Plasmids with three copies of Rep* also support long-term replication in the presence of EBNA-1. These observations together indicate that the latent origin of replication of EBV is more complex than formerly appreciated; it is a multicomponent origin of which the dyad symmetry element is one efficient component. The experimental approach described here could be used to identify eukaryotic sequences which mediate DNA synthesis, albeit inefficiently.
N-Terminal Extension of Human Immunodeficiency Virus Capsid Protein Converts the In Vitro Assembly Phenotype from Tubular to Spherical Particles
Authors: Gross Ingolf , Hohenberg Heinz , Huckhagel Carola , Kräusslich Hans-Georg 
Published: December 2019

ABSTRACT

Expression of retroviral Gag polyproteins is sufficient for morphogenesis of virus-like particles with a spherical immature protein shell. Proteolytic cleavage of Gag into the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains (in the case of human immunodeficiency virus [HIV]) leads to condensation to the mature cone-shaped core. We have analyzed the formation of spherical or cylindrical particles on in vitro assembly of purified HIV proteins or inside
Escherichia coli
cells. CA protein alone yielded cylindrical particles, while all N-terminal extensions of CA abolished cylinder formation. Spherical particles with heterogeneous diameters or amorphous protein aggregates were observed instead. Extending CA by 5 amino acids was sufficient to convert the assembly phenotype to spherical particles. Sequences C-terminal of CA were not required for sphere formation. Proteolytic cleavage of N-terminally extended CA proteins prior to in vitro assembly led to the formation of cylindrical particles, while proteolysis of in vitro assembly products caused disruption of spheres but not formation of cylinders. In vitro assembly of CA and extended CA proteins in the presence of cyclophilin A (CypA) at a CA-to-CypA molar ratio of 10:1 yielded significantly longer cylinders and heterogeneous spheres, while higher concentrations of CypA completely disrupted particle formation. We conclude that the spherical shape of immature HIV particles is determined by the presence of an N-terminal extension on the CA domain and that core condensation during virion maturation requires the liberation of the N terminus of CA.
Rescue and Autonomous Replication of Adeno-Associated Virus Type 2 Genomes Containing Rep-Binding Site Mutations in the Viral p5 Promoter
Authors: Wang Xu-Shan , Srivastava Arun 
Published: December 2019

ABSTRACT
The Rep proteins encoded by the adeno-associated virus type 2 (AAV) play a crucial role in the rescue, replication, and integration of the viral genome. In the absence of a helper virus, little expression of the AAV Rep proteins occurs, and the AAV genome fails to undergo DNA replication. Since previous studies have established that expression of the Rep78 and Rep68 proteins from the viral p5 promoter is controlled by the Rep-binding site (RBS) and the YY1 factor-binding site (YBS), we constructed a number of recombinant AAV plasmids containing mutations and/or deletions of the RBS and the YBS in the p5 promoter. These plasmids were transfected in HeLa or 293 cells and analyzed for the potential to undergo AAV DNA rescue and replication. Our studies revealed that (i) a low-level rescue and autonomous replication of the wild-type AAV genome occurred in 293 but not in HeLa cells; (ii) mutations in the RBS resulted in augmented expression from the p5 promoter, leading to more efficient rescue and/or replication of the AAV genome in 293 but not in HeLa cells; (iii) little rescue and/or replication occurred from plasmids containing mutations in the YBS alone in the absence of coinfection with adenovirus; (iv) expression of the adenovirus E1A gene products was insufficient to mediate rescue and/or replication of the AAV genome in HeLa cells; (v) autonomously replicated AAV genomes in 293 cells were successfully encapsidated in mature progeny virions that were biologically active in secondary infection of HeLa cells in the presence of adenovirus; and (vi) stable transfection of recombinant AAV plasmids containing a gene for resistance to neomycin significantly affected stable integration only in 293 cells, presumably because rescue and autonomous replication of the AAV genome from these plasmids occurred in 293 cells but not in HeLa or KB cells. These data suggest that in the absence of adenovirus, the AAV Rep protein-RBS interaction plays a dominant role in down-regulating viral gene expression from the p5 promoter and that perturbation in this interaction is sufficient to confer autonomous replication competence to AAV in 293 cells.
Structure-Based Mutational Analysis of the C-Terminal DNA-Binding Domain of Human Immunodeficiency Virus Type 1 Integrase: Critical Residues for Protein Oligomerization and DNA Binding
Authors: Lutzke Ramon A. Puras , Plasterk Ronald H. A. 
Published: December 2019

ABSTRACT

The C-terminal domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a dimer that binds to DNA in a nonspecific manner. The structure of the minimal region required for DNA binding (IN
220–270
) has been solved by nuclear magnetic resonance spectroscopy. The overall fold of the C-terminal domain of HIV-1 IN is similar to those of Src homology region 3 domains. Based on the structure of IN
220–270
, we studied the role of 15 amino acid residues potentially involved in DNA binding and oligomerization by mutational analysis. We found that two amino acid residues, arginine 262 and leucine 234, contribute to DNA binding in the context of IN
220–270
, as indicated by protein-DNA UV cross-link analysis. We also analyzed mutant proteins representing portions of the full-length IN protein. Amino acid substitution of residues located in the hydrophobic dimer interface, such as L241A and L242A, results in the loss of oligomerization of IN; consequently, the levels of 3′ processing, DNA strand transfer, and intramolecular disintegration are strongly reduced. These results suggest that dimerization of the C-terminal domain of IN is important for correct multimerization of IN.
The Rep52 Gene Product of Adeno-Associated Virus Is a DNA Helicase with 3′-to-5′ Polarity
Authors: Smith Richard H. , Kotin Robert M. 
Published: December 2019

ABSTRACT

The
rep
gene of adeno-associated virus type 2 encodes four overlapping proteins from two separate promoters, termed P
5
and P
19
. The P
5
-promoted Rep proteins, Rep78 and Rep68, are essential for viral DNA replication, and a wealth of data concerning the biochemical activities of these proteins has been reported. In contrast, data concerning the biochemical functions of the P
19
-promoted Rep proteins, Rep52 and Rep40, are lacking. Here, we describe enzymatic activities associated with a bacterially expressed maltose-binding protein (MBP)-Rep52 fusion protein. Purified MBP-Rep52 possesses 3′-to-5′ DNA helicase activity that is strictly dependent upon the presence of nucleoside triphosphate and divalent cation cofactors. In addition, MBP-Rep52 demonstrates a constitutive ATPase activity that is active in the absence of DNA effector molecules. An MBP-Rep52 chimera bearing a lysine-to-histidine substitution at position 116 (K116H) within a consensus helicase- and ATPase-associated motif (motif I or Walker A site) was deficient for both DNA helicase and ATPase activities. In contrast to a Rep78 A-site mutant protein bearing a corresponding amino acid substitution at position 340 (K340H), the MBP-Rep52 A-site mutant protein failed to exhibit a
trans
-dominant negative effect when it was mixed with wild-type MBP-Rep52 or MBP-Rep78 in vitro. This lack of
trans
dominance, coupled with the results of coimmunoprecipitation and gel filtration chromatography experiments reported here, suggests that the ability of Rep52 to engage in multimeric interactions may differ from that of Rep78 or -68.
Long-Term Evolution of the Hypervariable Region of Hepatitis C Virus in a Common-Source-Infected Cohort
Authors: McAllister Jane , Casino Carmela , Davidson Fiona , Power Joan , Lawlor Emer , Yap Peng Lee , Simmonds Peter , Smith Donald B. 
Published: December 2019

ABSTRACTThe long-term evolution of the hepatitis C virus hypervariable region (HVR) and flanking regions of the E1 and E2 envelope proteins have been studied in a cohort of women infected from a common source of anti-D immunoglobulin. Whereas virus sequences in the infectious source were relatively homogeneous, distinct HVR variants were observed in each anti-D recipient, indicating that this region can evolve in multiple directions from the same point. Where HVR variants with dissimilar sequences were present in a single individual, the frequency of synonymous substitution in the flanking regions suggested that the lineages diverged more than a decade previously. Even where a single major HVR variant was present in an infected individual, this lineage was usually several years old. Multiple lineages can therefore coexist during long periods of chronic infection without replacement. The characteristics of amino acid substitution in the HVR were not consistent with the random accumulation of mutations and imply that amino acid replacement in the HVR was strongly constrained. Another variable region of E2 centered on codon 60 shows similar constraints, while HVR2 was relatively unconstrained. Several of these features are difficult to explain if a neutralizing immune response against the HVR is the only selective force operating on E2. The impact of PCR artifacts such as nucleotide misincorporation and the shuffling of dissimilar templates is discussed.
Permissive Cytomegalovirus Infection of Primary Villous Term and First Trimester Trophoblasts
Authors: Hemmings D. G. , Kilani R. , Nykiforuk C. , Preiksaitis J. , Guilbert L. J. 
Published: December 2019

ABSTRACT
Forty percent of women with primary cytomegalovirus (CMV) infections during pregnancy infect their fetuses with complications for the baby varying from mild to severe. How CMV crosses the syncytiotrophoblast, the barrier between maternal blood and fetal tissue in the villous placenta, is unknown. Virus may cross by infection of maternal cells that pass through physical breaches in the syncytiotrophoblast or by direct infection of the syncytiotrophoblast, with subsequent transmission to underlying fetal placental cells. In this study, we show that pure (>99.99%), long-term and healthy (>3 weeks) cultures of syncytiotrophoblasts are permissively infected with CMV. Greater than 99% of infectious progeny virus remained cell associated throughout culture periods up to 3 weeks. Infection of term trophoblasts required a higher virus inoculum, was less efficient, and progressed more slowly than parallel infections of placental and human embryonic lung fibroblasts. Three laboratory strains (AD169, Towne, and Davis) and a clinical isolate from a congenitally infected infant all permissively infected trophoblasts, although infection efficiencies varied. The infection of first trimester syncytiotrophoblasts with strain AD169 occurred at higher frequency and progressed more rapidly than infection of term cells but less efficiently and rapidly than infection of fibroblasts. These results show that villous syncytiotrophoblasts can be permissively infected by CMV but that the infection requires high virus titers and proceeds slowly and that progeny virus remains predominantly cell associated.
Mapping of Homologous Interaction Sites in the Hepatitis B Virus Core Protein
Authors: König Sabine , Beterams Gertrud , Nassal Michael 
Published: December 2019

ABSTRACT
Hepatitis B virus consists of an outer envelope and an inner capsid, or core, that wraps around the small genome plus the viral replication enzyme. The icosahedrally symmetric nucleocapsid is assembled from multiple dimeric subunits of a single 183-residue capsid protein, which must therefore contain interfaces for monomer dimerization and for dimer multimerization. The atomic structure of the protein is not known, but electron microscopy-based image reconstructions suggested a hammerhead shape for the dimer and, very recently, led to a tentative model for the main chain trace. Here we used a combination of interaction screening techniques and functional analyses of core protein variants to define, at the primary sequence level, the regions that mediate capsid assembly. Both the two-hybrid system and the pepscan technique identified a strongly interacting region I between amino acids (aa) 78 and 117 that probably forms part of the dimer interface. Surprisingly, mutations in this region, in the context of a C-terminally truncated but assembly-competent core protein variant, had no detectable effect on assembly. By contrast, mutations in a second region, bordered by aa 113 and 143, markedly influenced capsid stability, strongly suggesting that this region II is the main contributor to dimer multimerization. Based on the electron microscopic data, it must therefore be located at the basal tips of the dimer, experimentally supporting the proposed main chain trace.
Limited Transmission of Kaposi’s Sarcoma-Associated Herpesvirus in Cultured Cells
Authors: Renne Rolf , Blackbourn David , Whitby Denise , Levy Jay , Ganem Don 
Published: December 2019

ABSTRACT
Kaposi’s sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) is a novel gammaherpesvirus strongly implicated in the pathogenesis of Kaposi’s sarcoma. Although virions can be produced in high yield from latently infected B-cell lines treated with phorbol esters, little is known about the infectivity of such virus, and efficient serial propagation of KSHV has been problematic. Here we report on the infectivity of KSHV produced from phorbol-induced BCBL-1 cells, employing an assay based on the detection of a spliced late mRNA by a sensitive reverse transcriptase PCR (RT-PCR) method. The results of this study confirm previous observations that 293 cells are susceptible to viral infection; however, infection with BCBL-1-derived virus is inefficient and the pattern of viral gene expression in infected cells may not fully reproduce that of authentic lytic infection. In keeping with this finding, serial propagation of BCBL-1-derived virus could not be demonstrated on 293 cells. Eleven of 38 other cell lines tested also supported KSHV infection, as judged by this RT-PCR assay, including cells of B-cell, endothelial, epithelial, and fibroblastic origin; however, in all cases, infection proceeded at or below the levels observed in 293 cells.
Relative Rates of Retroviral Reverse Transcriptase Template Switching during RNA- and DNA-Dependent DNA Synthesis
Authors: Bowman Robert R. , Hu Wei-Shau , Pathak Vinay K. 
Published: December 2019

ABSTRACT

Retroviral reverse transcriptases (RTs) frequently switch templates during DNA synthesis, which can result in mutations and recombination. The relative rates of in vivo RT template switching during RNA- and DNA-dependent DNA synthesis are unknown. To determine the relative rates of RT template switching during copying of RNA and DNA templates, we constructed spleen necrosis virus-based retroviral vectors containing a 400-bp direct repeat. The directly repeated sequences were upstream of the polypurine tract (PPT) in the RB-LLP vector; the same direct repeats flanked the PPT and attachment site (
att
) in the RB-LPL vector. RT template switching events could occur during either RNA- or DNA-dependent DNA synthesis and delete one copy of the direct repeat plus the intervening sequences. RB-LLP vectors that underwent direct repeat deletions during RNA- and DNA-dependent DNA synthesis generated viral DNA that could integrate into the host genome. However, any deletion of the direct repeats in the RB-LPL vector that occurred during RNA-dependent DNA synthesis resulted in deletion of the essential PPT and
att
site and generated a dead-end viral DNA product. Thus, only RB-LPL vectors that underwent direct repeat deletions during DNA-dependent DNA synthesis could integrate to form proviruses. The RB-LLP and RB-LPL vectors were permitted to undergo a single replication cycle, and the frequencies of direct repeat deletions were determined by PCR and Southern analysis of the resulting proviruses. A comparison of the frequency of direct repeat deletions in the RB-LLP and RB-LPL vectors indicated that the in vivo rates of RT template switching during RNA- and DNA-dependent DNA synthesis are nearly identical.