bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activated Mouse Notch1 Transactivates Epstein-Barr Virus Nuclear Antigen 2-Regulated Viral Promoters
Authors: Höfelmayr Heike , Strobl Lothar J. , Stein Charlotte , Laux Gerhard , Marschall Gabriele , Bornkamm Georg W. , Zimber-Strobl Ursula 
Published: December 2019

ABSTRACT

Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for B-cell immortalization by EBV, most probably by its ability to transactivate a number of cellular and viral genes. EBNA2-responsive elements (EBNA2REs) have been identified in several EBNA2-regulated viral promoters, each of them carrying at least one RBP-Jκ recognition site. RBP-Jκ recruits EBNA2 to the EBNA2RE and, once complexed to EBNA2, is converted from a repressor into an activator. An activated form of the cellular receptor Notch also interacts with RBP-Jκ, providing a link between EBNA2 and Notch signalling. To determine whether activated Notch is able to transactivate EBNA2-responsive viral promoters, we performed cotransfection experiments with activated mouse Notch1 (mNotch1-IC) and luciferase constructs of the
Bam
HI C, LMP1, and LMP2A promoters. We present here evidence that mNotch1-IC transactivates viral promoters known to be regulated by EBNA2. As shown for EBNA2, mutations or deletions of the RBP-Jκ sites diminish or eliminate mNotch1-IC-mediated transactivation of the promoters, pointing to an essential role for Notch–RBP-Jκ interaction. In addition to RBP-Jκ, other cellular factors may bind within the EBNA2REs of viral promoters. While some factors appear to play an important role in both EBNA2- and mNotch1-IC-mediated transactivation, others are only important for the activity of either EBNA2 or mNotch1-IC. We could observe specific mNotch1-IC-responsive regions, thereby throwing more light upon which cofactors interact with EBNA2 and mNotch1-IC, thus enabling them to become functionally transactivators in vivo.
Nef Enhances Human Immunodeficiency Virus Replication and Responsiveness to Interleukin-2 in Human Lymphoid Tissue Ex Vivo
Authors: Glushakova Svetlana , Grivel Jean-Charles , Suryanarayana Kalachar , Meylan Pascal , Lifson Jeffrey D. , Desrosiers Ronald , Margolis Leonid 
Published: December 2019

ABSTRACTThenefgene is important for the pathogenicity associated with simian immunodeficiency virus infection in rhesus monkeys and with human immunodeficiency virus type 1 (HIV-1) infection in humans. The mechanisms by whichnefcontributes to pathogenesis in vivo remain unclear. We investigated the contribution ofnefto HIV-1 replication in human lymphoid tissue ex vivo by studying infection with parental HIV-1 strain NL4-3 and with anefmutant (ΔnefNL4-3). In human tonsillar histocultures, NL4-3 replicated to higher levels than ΔnefNL4-3 did. Increased virus production with NL4-3 infection was associated with increased numbers of productively infected cells and greater loss of CD4+T cells over time. While the numbers of productively infected T cells were increased in the presence ofnef, the levels of viral expression and production per infected T cell were similar whether thenefgene was present or not. Exogenous interleukin-2 (IL-2) increased HIV-1 production in NL4-3-infected tissue in a dose-dependent manner. In contrast, ΔnefNL4-3 production was enhanced only marginally by IL-2. Thus, Nef can facilitate HIV-1 replication in human lymphoid tissue ex vivo by increasing the numbers of productively infected cells and by increasing the responsiveness to IL-2 stimulation.
In Vitro Infection of Human Peripheral Blood Mononuclear Cells by GB Virus C/Hepatitis G Virus
Authors: Fogeda Marta , Navas Sonia , Martín Julio , Casqueiro Mercedes , Rodríguez Elena , Arocena Carlos , Carreño Vicente 
Published: December 2019

ABSTRACT
GB virus C (GBV-C), also known as hepatitis G virus, is a recently discovered flavivirus-like RNA agent with unclear pathogenic implications. To investigate whether human peripheral blood mononuclear cells (PBMC) are susceptible to in vitro GBV-C infection, we have incubated PBMC from four healthy blood donors with a human GBV-C RNA-positive serum. By means of (i) strand-specific reverse transcription-PCR, cloning, and sequencing; (ii) sucrose ultracentrifugation and RNase sensitivity assays; (iii) fluorescent in situ hybridization; and (iv) Western blot analysis, it has been demonstrated that GBV-C is able to infect in vitro cells and replicate for as long as 30 days under the conditions developed in our cell culture system. The concentration of GBV-C RNA increased during the second and third weeks of culture. The titers of the genomic strand were 10 times higher than the titers of the antigenomic strand. In addition, the same predominant GBV-C sequence was found in all PBMC cultures and in the in vivo-GBV-C-infected PBMC isolated from the donor of the inoculum. GBV-C-specific fluorescent in situ hybridization signals were confined to the cytoplasm of cells at different times during the culture period. Finally, evidence obtained by sucrose ultracentrifugation, RNase sensitivity assays, and Western blot analysis of the culture supernatants suggests that viral particles are released from in vitro-GBV-C-infected PBMC. In conclusion, our study has demonstrated, for the first time, GBV-C replication in human lymphoid cells under experimental in vitro infection conditions.
A Recombinant Measles Virus Expressing Hepatitis B Virus Surface Antigen Induces Humoral Immune Responses in Genetically Modified Mice
Authors: Singh Mahender , Cattaneo Roberto , Billeter Martin A. 
Published: December 2019

ABSTRACT
It has been shown previously that measles virus (MV) can be successfully used to express foreign proteins (M. Singh and M. A. Billeter, J. Gen. Virol. 80:101–106, 1998). To develop an inexpensive MV-based vaccine, we generated recombinant MVs that produce structural proteins of hepatitis B virus (HBV). A recombinant virus that expressed the HBV small surface antigen (HBsAg) was analyzed in terms of its replication characteristics, its genetic stability in cell culture, and its immunogenic potential in genetically modified mice. Although this virus showed a progression of replication slightly slower than that of the parental MV, it appeared to stably maintain the added genetic information; it uniformly expressed the appropriately glycosylated HBsAg after 10 serial passages. Genetically modified mice inoculated with this recombinant MV produced humoral immune responses against both HBsAg and MV proteins.
Identification of the R1 Oncogene and Its Protein Product from the Rhadinovirus of Rhesus Monkeys
Authors: Damania Blossom , Li Mengtao , Choi Joong-Kook , Alexander Louis , Jung Jae U. , Desrosiers Ronald C. 
Published: December 2019

ABSTRACTRhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus that is most closely related to the human Kaposi’s sarcoma-associated herpesvirus (KSHV). We have identified a distinct open reading frame at the left end of RRV and designated it R1. The position of the R1 gene is equivalent to that of the saimiri transforming protein (STP) of herpesvirus saimiri (HVS) and of K1 of KSHV, other members of the gamma-2 or rhadinovirus subgroup of herpesviruses. The R1 sequence revealed an open reading frame encoding a product of 423 amino acids that was predicted to contain an extracellular domain, a transmembrane domain, and a C-terminal cytoplasmic tail reflective of a type I membrane-bound protein. The predicted structural motifs of R1, including the presence of immunoreceptor tyrosine-based activation motifs, resembled those in K1 of KSHV but were distinct from those of STP. R1 sequences from four independent isolates from three different macaque species revealed 0.95 to 7.3% divergence over the 423 amino acids. Variation was located predominantly within the predicted extracellular domain. The R1 protein migrated at 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was extensively glycosylated. Tagged R1 protein was localized to the cytoplasmic and plasma membranes of transfected cells. Expression of the R1 gene in Rat-1 fibroblasts induced morphologic changes and focus formation, and injection of R1-expressing cells into nude mice induced the formation of multifocal tumors. A recombinant herpesvirus in which the STP oncogene of HVS was replaced by R1 immortalized T lymphocytes to interleukin-2-independent growth. These results indicate that R1 is an oncogene of RRV.
Induction of AIDS in Rhesus Monkeys by a Recombinant Simian Immunodeficiency Virus Expressing
nef
of Human Immunodeficiency Virus Type 1
Authors: Alexander Louis , Du Zhenjian , Howe Anita Y. M. , Czajak Susan , Desrosiers Ronald C. 
Published: December 2019

ABSTRACT

A
nef
gene is present in all primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus of macaque monkeys (SIVmac). However, the
nef
genes of HIV-1 and SIVmac exhibit minimal sequence identity, and not all properties are shared by the two. Nef sequences of SIVmac239 were replaced by four independent
nef
alleles of HIV-1 in a context that was optimal for expression. The sources of the HIV-1
nef
sequences included NL 4-3, a variant NL 4-3 gene derived from a recombinant-infected rhesus monkey, a patient
nef
allele, and a
nef
consensus sequence. Of 16 rhesus monkeys infected with these SHIVnef chimeras, 9 maintained high viral loads for prolonged periods, as observed with the parental SIVmac239, and 6 have died with AIDS 52 to 110 weeks postinfection. Persistent high loads were observed at similar frequencies with the four different SIV recombinants that expressed these independent HIV-1
nef
alleles. Infection with other recombinant SHIVnef constructions resulted in sequence changes in infected monkeys that either created an open
nef
reading frame or optimized the HIV-1
nef
translational context. The HIV-1
nef
gene was uniformly retained in all SHIVnef-infected monkeys. These results demonstrate that HIV-1
nef
can substitute for SIVmac
nef
in vivo to produce a pathogenic infection. However, the model suffers from an inability to consistently obtain persisting high viral loads in 100% of the infected animals, as is observed with the parental SIVmac239.
Evidence of Avian Leukosis Virus Subgroup E and Endogenous Avian Virus in Measles and Mumps Vaccines Derived from Chicken Cells: Investigation of Transmission to Vaccine Recipients
Authors: Tsang Shirley X. , Switzer William M. , Shanmugam Vedapuri , Johnson Jeffrey A. , Goldsmith Cynthia , Wright Anthony , Fadly Aly , Thea Donald , Jaffe Harold , Folks Thomas M. , Heneine Walid 
Published: December 2019

ABSTRACT

Reverse transcriptase (RT) activity has been detected recently in all chicken cell-derived measles and mumps vaccines. A study of a vaccine manufactured in Europe indicated that the RT is associated with particles containing endogenous avian retrovirus (EAV-0) RNA and originates from the chicken embryonic fibroblasts (CEF) used as a substrate for propagation of the vaccine. We investigated the origin of RT in measles and mumps vaccines from a U.S. manufacturer and confirm the presence of RT and EAV RNA. Additionally, we provide new evidence for the presence of avian leukosis virus (ALV) in both CEF supernatants and vaccines. ALV
pol
sequences were first identified in particle-associated RNA by amplification with degenerate retroviral
pol
primers. ALV RNA sequences from both the
gag
and
env
regions were also detected. Analysis of hypervariable region 2 of
env
revealed a subgroup E sequence, an endogenous-type ALV. Both CEF- and vaccine-derived RT activity could be blocked by antibodies to ALV RT. Release of ALV-like virus particles from uninoculated CEF was also documented by electron microscopy. Nonetheless, infectivity studies on susceptible 15B
1
chicken cells gave no evidence of infectious ALV, which is consistent with the phenotypes of the
ev
loci identified in the CEF. PCR analysis of ALV and EAV proviral sequences in peripheral blood mononuclear cells from 33 children after measles and mumps vaccination yielded negative results. Our data indicate that the sources of RT activity in all RT-positive measles and mumps vaccines may not be similar and depend on the particular endogenous retroviral loci present in the chicken cell substrate used. The present data do not support transmission of either ALV or EAV to recipients of the U.S.-made vaccine and provide reassurance for current immunization policies.
Hepatitis A Virus Capsid Protein VP1 Has a Heterogeneous C Terminus
Authors: Graff Judith , Richards Oliver C. , Swiderek Kristine M. , Davis Michael T. , Rusnak Felicia , Harmon Shirley A. , Jia Xi-Yu , Summers Donald F. , Ehrenfeld Ellie 
Published: December 2019

ABSTRACT

Hepatitis A virus (HAV) encodes a single polyprotein which is posttranslationally processed into the functional structural and nonstructural proteins. Only one protease, viral protease 3C, has been implicated in the nine protein scissions. Processing of the capsid protein precursor region generates a unique intermediate, PX (VP1-2A), which accumulates in infected cells and is assumed to serve as precursor to VP1 found in virions, although the details of this reaction have not been determined. Coexpression in transfected cells of a variety of P1 precursor proteins with viral protease 3C demonstrated efficient production of PX, as well as VP0 and VP3; however, no mature VP1 protein was detected. To identify the C-terminal amino acid residue of HAV VP1, we performed peptide sequence analysis by protease-catalyzed [
18
O]H
2
O incorporation followed by liquid chromatography ion-trap microspray tandem mass spectrometry of HAV VP1 isolated from purified virions. Two different cell culture-adapted isolates of HAV, strains HM175pE and HM175p35, were used for these analyses. VP1 preparations from both virus isolates contained heterogeneous C termini. The predominant C-terminal amino acid in both virus preparations was VP1-Ser274, which is located N terminal to a methionine residue in VP1-2A. In addition, the analysis of HM175pE recovered smaller amounts of amino acids VP1-Glu273 and VP1-Thr272. In the case of HM175p35, which contains valine at amino acid position VP1-273, VP1-Thr272 was found in addition to VP1-Ser274. The data suggest that HAV 3C is not the protease responsible for generation of the VP1 C terminus. We propose the involvement of host cell protease(s) in the production of HAV VP1.
Numerous Length Polymorphisms at Short Tandem Repeats in Human Cytomegalovirus
Authors: Davis Clara L. , Field Dawn , Metzgar David , Saiz Robert , Morin Phillip A. , Smith Irene L. , Spector Stephen A. , Wills Christopher 
Published: December 2019

ABSTRACT
We show the presence of numerous short tandem repeats in the human cytomegalovirus (HCMV) genome and assess their usefulness as molecular markers. The genome is shown to contain at least 24 microsatellite regions that exhibit length polymorphisms. Insertion-deletion polymorphisms at these short tandem repeats are common (80% of repeats examined are polymorphic among two laboratory strains and 10 clinical isolates). This is the first report of widespread microsatellite length polymorphism in a viral genome. Some regions are highly polymorphic: one was revealed by DNA sequencing to contain length variants at five closely linked sites, which combined resulted in 10 variants for this region among the 12 strains and isolates examined. This study not only provides a new molecular marker system for this virus but also extends our understanding of microsatellite polymorphism in two important ways. First, variable-length repeats in HCMV can be considerably shorter than polymorphic repeats previously found in other organisms. Second, highly variable microsatellite repeats are not confined to prokaryotes and eukaryotes, as previously assumed. This variation provides a useful marker system for distinguishing viral isolates, and similar markers are also likely to be found in other large-genome DNA viruses.
The Furin Protease Cleavage Recognition Sequence of Sindbis Virus PE2 Can Mediate Virion Attachment to Cell Surface Heparan Sulfate
Authors: Klimstra William B. , Heidner Hans W. , Johnston Robert E. 
Published: December 2019

ABSTRACT
Cell culture-adapted Sindbis virus strains attach to heparan sulfate (HS) receptors during infection of cultured cells (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357–7366, 1998). At least three E2 glycoprotein mutations (E2 Arg 1, E2 Lys 70, and E2 Arg 114) can independently confer HS attachment in the background of the consensus sequence Sindbis virus (TR339). In the studies reported here, we have investigated the mechanism by which the E2 Arg 1 mutation confers HS-dependent binding. Substitution of Arg for Ser at E2 1 resulted in a significant reduction in the efficiency of PE2 cleavage, yielding virus particles containing a mixture of PE2 and mature E2. Presence of PE2 was associated with an increase in HS-dependent attachment to cells and efficient attachment to heparin-agarose beads, presumably because the furin recognition site for PE2 cleavage also represents a candidate HS binding sequence. A comparison of mutants with partially or completely inhibited PE2 cleavage demonstrated that efficiency of cell binding was correlated with the amount of PE2 in virus particles. Viruses rendered cleavage defective due to deletions of portions or all of the furin cleavage sequence attached very poorly to cells, indicating that an intact furin cleavage sequence was specifically required for PE2-mediated attachment to cells. In contrast, a virus containing a partial deletion was capable of efficient binding to heparin-agarose beads, suggesting different requirements for heparin bead and cell surface HS binding. Furthermore, virus produced in C6/36 mosquito cells, which cleave PE2 more efficiently than BHK cells, exhibited a reduction in cell attachment efficiency correlated with reduced content of PE2 in particles. Taken together, these results strongly argue that the XBXBBX (B, basic; X, hydrophobic) furin protease recognition sequence of PE2 can mediate the binding of PE2-containing Sindbis viruses to HS. This sequence is very similar to an XBBXBX heparin-HS interaction consensus sequence. The attachment of furin protease cleavage sequences to HS may have relevance to other viruses whose attachment proteins are cleaved during maturation at positively charged recognition sequences.
Dissection of Individual Functions of the Sendai Virus Phosphoprotein in Transcription
Authors: Bowman Mary Catherine , Smallwood Sherin , Moyer Sue A. 
Published: December 2019

ABSTRACT
The Sendai virus P protein is an essential component of the viral RNA polymerase (P-L complex) required for RNA synthesis. To identify amino acids important for P-L binding, site-directed mutagenesis of the P gene changed 17 charged amino acids, singly or in groups, and two serines to alanine within the L binding domain from amino acids 408 to 479. Each of the 10 mutants was wild type for P-L and P-P protein interactions and for binding of the P-L complex to the nucleocapsid template, yet six showed a significant inhibition of in vitro mRNA and leader RNA synthesis. To determine if binding was instead hydrophobic in nature, five conserved hydrophobic amino acids in this region were also mutated. Each of these P mutants also retained the ability to bind to L, to itself, and to the template, but two gave a severe decrease in mRNA and leader RNA synthesis. Since all of the mutants still bound L, the data suggest that L binding occurs on a surface of P with a complex tertiary structure. Wild-type biological activity could be restored for defective polymerase complexes containing two P mutants by the addition of wild-type P protein alone, while the activity of two others could not be rescued. Gradient sedimentation analyses showed that rescue was not due to exchange of the wild-type and mutant P proteins within the P-L complex. Mutants which gave a defective RNA synthesis phenotype and could not be rescued by P establish an as-yet-unknown role for P within the polymerase complex, while the mutants which could be rescued define regions required for a P protein function independent of polymerase function.
An Avian Sarcoma/Leukosis Virus-Based Gene Trap Vector for Mammalian Cells
Authors: Zheng Xiangqun H. , Hughes Stephen H. 
Published: December 2019

ABSTRACT
RCASBP-M2C is a retroviral vector derived from an avian sarcoma/leukosis virus which has been modified so that it uses the envelope gene from an amphotropic murine leukemia virus (E. V. Barsov and S. H. Hughes, J. Virol. 70:3922–3929, 1996). The vector replicates efficiently in avian cells and infects, but does not replicate in, mammalian cells. This makes the vector useful for gene delivery, mutagenesis, and other applications in mammalian systems. Here we describe the development of a derivative of RCASBP-M2C, pGT-GFP, that can be used in gene trap experiments in mammalian cells. The gene trap vector pGT-GFP contains a green fluorescent protein (GFP) reporter gene. Appropriate insertion of the vector into genes causes GFP expression; this facilitates the rapid enrichment and cloning of the trapped cells and provides an opportunity to select subpopulations of trapped cells based on the subcellular localization of GFP. With this vector, we have generated about 90 gene-trapped lines using D17 and NIH 3T3 cells. Five trapped NIH 3T3 lines were selected based on the distribution of GFP in cells. The cellular genes disrupted by viral integration have been identified in four of these lines by using a 5′ rapid amplification of cDNA ends protocol.
Human Immunodeficiency Virus Type 1 Tat Protein Activates Transcription Factor NF-κB through the Cellular Interferon-Inducible, Double-Stranded RNA-Dependent Protein Kinase, PKR
Authors: Demarchi Francesca , Gutierrez Maria Ines , Giacca Mauro 
Published: December 2019

ABSTRACT
The transactivator protein of human immunodeficiency virus type 1 (HIV-1) (Tat) is a powerful activator of nuclear factor-κB (NF-κB), acting through degradation of the inhibitor IκB-α (F. Demarchi, F. d’Adda di Fagagna, A. Falaschi, and M. Giacca, J. Virol. 70:4427–4437, 1996). Here, we show that this activity of Tat requires the function of the cellular interferon-inducible protein kinase PKR. Tat-mediated NF-κB activation and transcriptional induction of the HIV-1 long terminal repeat were impaired in murine cells in which the PKR gene was knocked out. Both functions were restored by cotransfection of Tat with the cDNA for PKR. Expression of a dominant-negative mutant of PKR specifically reduced the levels of Tat transactivation in different human cell types. Activation of NF-κB by Tat required integrity of the basic domain of Tat; previous studies have indicated that this domain is necessary for specific Tat-PKR interaction.