bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of Animal Models for Adeno-Associated Virus Site-Specific Integration
Authors: Rizzuto Gabriella , Gorgoni Barbara , Cappelletti Manuela , Lazzaro Domenico , Gloaguen Isabelle , Poli Valeria , Sgura Antonella , Cimini Daniela , Ciliberto Gennaro , Cortese Riccardo , Fattori Elena , La Monica Nicola
Published: December 2019

The adeno-associated virus (AAV) is unique in its ability to target viral DNA integration to a defined region of human chromosome 19 (AAVS1). Since AAVS1 sequences are not conserved in a rodent’s genome, no animal model is currently available to study AAV-mediated site-specific integration. We describe here the generation of transgenic rats and mice that carry the AAVS1 3.5-kb DNA fragment. To test the response of the transgenic animals to Rep-mediated targeting, primary cultures of mouse fibroblasts, rat hepatocytes, and fibroblasts were infected with wild-type wt AAV. PCR amplification of the inverted terminal repeat (ITR)-AAVS1 junction revealed that the AAV genome integrated into the AAVS1 site in fibroblasts and hepatocytes. Integration in rat fibroblasts was also observed upon transfection of a plasmid containing the
rep
gene under the control of the p5 and p19 promoters and a dicistronic cassette carrying the green fluorescent protein (GFP) and neomycin (
neo
) resistance gene between the ITRs of AAV. The localization of the GFP-Neo sequence in the AAVS1 region was determined by Southern blot and FISH analysis. Lastly, AAV genomic DNA integration into the AAVS1 site in vivo was assessed by virus injection into the quadriceps muscle of transgenic rats and mice. Rep-mediated targeting to the AAVS1 site was detected in several injected animals. These results indicate that the transgenic lines are proficient for Rep-mediated targeting. These animals should allow further characterization of the molecular aspects of site-specific integration and testing of the efficacy of targeted integration of AAV recombinant vectors designed for human gene therapy.
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

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

Thenefgene 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

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

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

Rhesus 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

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

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

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

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

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

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

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

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.
Role of CXCR4 in Cell-Cell Fusion and Infection of Monocyte-Derived Macrophages by Primary Human Immunodeficiency Virus Type 1 (HIV-1) Strains: Two Distinct Mechanisms of HIV-1 Dual Tropism
Authors: Yi Yanjie , Isaacs Stuart N. , Williams Darlisha A. , Frank Ian , Schols Dominique , De Clercq Erik , Kolson Dennis L. , Collman Ronald G.
Published: December 2019

Dual-tropic human immunodeficiency virus type 1 (HIV-1) strains infect both primary macrophages and transformed T-cell lines. Prototype T-cell line-tropic (T-tropic) strains use CXCR4 as their principal entry coreceptor (X4 strains), while macrophagetropic (M-tropic) strains use CCR5 (R5 strains). Prototype dual tropic strains use both coreceptors (R5X4 strains). Recently, CXCR4 expressed on macrophages was found to support infection by certain HIV-1 isolates, including the dual-tropic R5X4 strain 89.6, but not by T-tropic X4 prototypes like 3B. To better understand the cellular basis for dual tropism, we analyzed the macrophage coreceptors used for Env-mediated cell-cell fusion as well as infection by several dual-tropic HIV-1 isolates. Like 89.6, the R5X4 strain DH12 fused with and infected both wild-type and CCR5-negative macrophages. The CXCR4-specific inhibitor AMD3100 blocked DH12 fusion and infection in macrophages that lacked CCR5 but not in wild-type macrophages. This finding indicates two independent entry pathways in macrophages for DH12, CCR5 and CXCR4. Three primary isolates that use CXCR4 but not CCR5 (tybe, UG021, and UG024) replicated efficiently in macrophages regardless of whether CCR5 was present, and AMD3100 blocking of CXCR4 prevented infection in both CCR5 negative and wild-type macrophages. Fusion mediated by UG021 and UG024 Envs in both wild-type and CCR5-deficient macrophages was also blocked by AMD3100. Therefore, these isolates use CXCR4 exclusively for entry into macrophages. These results confirm that macrophage CXCR4 can be used for fusion and infection by primary HIV-1 isolates and indicate that CXCR4 may be the sole macrophage coreceptor for some strains. Thus, dual tropism can result from two distinct mechanisms: utilization of both CCR5 and CXCR4 on macrophages and T-cell lines, respectively (dual-tropic R5X4), or the ability to efficiently utilize CXCR4 on both macrophages and T-cell lines (dual-tropic X4).
Functionally Heterogeneous CD8+T-Cell Memory Is Induced by Sendai Virus Infection of Mice
Authors: Usherwood Edward J. , Hogan Robert J. , Crowther Graham , Surman Sherri L. , Hogg Twala L. , Altman John D. , Woodland David L.
Published: December 2019

It has recently been established that memory CD8+T cells induced by viral infection are maintained at unexpectedly high frequencies in the spleen. While it has been established that these memory cells are phenotypically heterogeneous, relatively little is known about the functional status of these cells. Here we investigated the proliferative potential of CD8+memory T cells induced by Sendai virus infection. High frequencies of CD8+T cells specific for both dominant and subdominant Sendai virus epitopes persisted for many weeks after primary infection, and these cells were heterogeneous with respect to CD62L expression (approximately 20% CD62Lhiand 80% CD62Llo). Reactivation of these cells with the antigenic peptide in vitro induced strong proliferation of antigen-specific CD8+T cells. However, approximately 20% of the cells failed to proliferate in vitro in response to a cognate peptide but nevertheless differentiated into effector cells and acquired full cytotoxic potential. These cells also expressed high levels of CD62L (in marked contrast to the CD62Llostatus of the proliferating cells in the culture). Direct isolation of CD62Lhiand CD62LloCD8+T cells from memory mice confirmed the correlation of this marker with proliferative potential. Taken together, these data demonstrate that Sendai virus infection induces high frequencies of memory CD8+T cells that are highly heterogeneous in terms of both their phenotype and their proliferative potential.
Identification of Amino Acid Residues of Influenza Virus Nucleoprotein Essential for RNA Binding
Authors: Elton Debra , Medcalf Liz , Bishop Konrad , Harrison Deborah , Digard Paul
Published: December 2019

The influenza virus nucleoprotein (NP) is a single-strand-RNA-binding protein associated with genome and antigenome RNA and is one of the four virus proteins necessary for transcription and replication of viral RNA. To better characterize the mechanism by which NP binds RNA, we undertook a physical and mutational analysis of the polypeptide, with the strategy of identifying first the regions in direct contact with RNA, then the classes of amino acids involved, and finally the crucial residues by mutagenesis. Chemical fragmentation and amino acid sequencing of NP that had been UV cross linked to radiolabelled RNA showed that protein-RNA contacts occur throughout the length of the polypeptide. Chemical modification experiments implicated arginine but not lysine residues as important for RNA binding, while RNA-dependent changes in the intrinsic fluorescence spectrum of NP suggested the involvement of tryptophan residues. Supporting these observations, single-codon mutagenesis identified five tryptophan, one phenylalanine, and two arginine residues as essential for high-affinity RNA binding at physiological temperature. In addition, mutants unable to bind RNA in vitro were unable to support virus gene expression in vivo. The mutationally sensitive residues are not localized to any particular region of NP but instead are distributed throughout the protein. Overall, these data are inconsistent with previous models suggesting that the NP-RNA interaction is mediated by a discrete N-terminal domain. Instead, we propose that high-affinity binding of RNA by NP requires the concerted interaction of multiple regions of the protein with RNA and that the individual protein-RNA contacts are mediated by a combination of electrostatic interactions between positively charged residues and the phosphate backbone and planar interactions between aromatic side chains and bases.
Cytoplasmic Polyadenylation in Development and Beyond
Authors: Richter Joel D.
Published: December 2019

Maternal mRNA translation is regulated in large part by cytoplasmic polyadenylation. This process, which occurs in both vertebrates and invertebrates, is essential for meiosis and body patterning. In spite of the evolutionary conservation of cytoplasmic polyadenylation, many of the cis elements and trans-acting factors appear to have some species specificity. With the recent isolation and cloning of factors involved in both poly(A) elongation and deadenylation, the underlying biochemistry of these reactions is beginning to be elucidated. In addition to early development, cytoplasmic polyadenylation is now known to occur in the adult brain, and there is circumstantial evidence that this process occurs at synapses, where it could mediate the long-lasting phase of long-term potentiation, which is probably the basis of learning and memory. Finally, there may be multiple mechanisms by which polyadenylation promotes translation. Important questions yet to be answered in the field of cytoplasmic polyadenylation are addressed.
PAS Domains: Internal Sensors of Oxygen, Redox Potential, and Light
Authors: Taylor Barry L. , Zhulin Igor B.
Published: December 2019

PAS domains are newly recognized signaling domains that are widely distributed in proteins from members of the Archaea and Bacteria and from fungi, plants, insects, and vertebrates. They function as input modules in proteins that sense oxygen, redox potential, light, and some other stimuli. Specificity in sensing arises, in part, from different cofactors that may be associated with the PAS fold. Transduction of redox signals may be a common mechanistic theme in many different PAS domains. PAS proteins are always located intracellularly but may monitor the external as well as the internal environment. One way in which prokaryotic PAS proteins sense the environment is by detecting changes in the electron transport system. This serves as an early warning system for any reduction in cellular energy levels. Human PAS proteins include hypoxia-inducible factors and voltage-sensitive ion channels; other PAS proteins are integral components of circadian clocks. Although PAS domains were only recently identified, the signaling functions with which they are associated have long been recognized as fundamental properties of living cells.
How a strong low-angle normal fault formed: The Whipple detachment, southeastern California
Authors: Axen Gary J.
Published: December 2019

Many low-angle normal faults (dip ≤30°) accommodate tens of kilometers of crustal extension, but their mechanics remain contentious. Most models for low-angle normal fault slip assume vertical maximum principal stress σ1, leading many authors to conclude that low-angle normal faults are poorly oriented in the stress field (≥60° from σ1) and weak (low friction). In contrast, models for low-angle normal fault formation in isotropic rocks typically assume Coulomb failure and require inclined σ1 (no misorientation). Here, a data-based, mechanical-tectonic model is presented for formation of the Whipple detachment fault, southeastern California. The model honors local and regional geologic and tectonic history and laboratory friction measurements. The Whipple detachment fault formed progressively in the brittle-plastic transition by linking of “minidetachments,” which are small-scale analogs (meters to kilometers in length) in the upper footwall.

Minidetachments followed mylonitic anisotropy along planes of maximum shear stress (45° from the maximum principal stress), not Coulomb fractures. They evolved from mylonitic flow to cataclasis and frictional slip at 300–400 °C and ∼9.5 km depth, while fluid pressure fell from lithostatic to hydrostatic levels. Minidetachment friction was presumably high (0.6–0.85), based upon formation of quartzofeldspathic cataclasite and pseudotachylyte. Similar mechanics are inferred for both the minidetachments and the Whipple detachment fault, driven by high differential stress (∼150–160 MPa). A Mohr construction is presented with the fault dip as the main free parameter. Using “Byerlee friction” (0.6–0.85) on the minidetachments and the Whipple detachment fault, and internal friction (1.0–1.7) on newly formed Reidel shears, the initial fault dips are calculated at 16°–26°, with σ1 plunging ∼61°–71° northeast. Linked minidetachments probably were not well aligned, and slip on the evolving Whipple detachment fault probably contributed to fault smoothing, by off-fault fracturing and cataclasis, and to formation of the fault core and fractured damage zone.

Stress rotation may have occurred only within the mylonitic shear zone, but asymmetric tectonic forces applied to the brittle crust probably caused gradual rotation of σ1 above it as a result of: (1) the upward force applied to the base of marginal North America by buoyant asthenosphere upwelling into an opening slab-free window and/or (2) basal, top-to-the-NE shear traction due to midcrustal mylonitic flow during tectonic exhumation of the Orocopia Schist. The mechanical-tectonic model probably applies directly to low-angle normal faults of the lower Colorado River extensional corridor, and aspects of the model (e.g., significance of anisotropy, stress rotation) likely apply to formation of other strong low-angle normal faults.
Heparan sulfate chains contribute to the anticoagulant milieu in malignant pleural effusion
Authors: Hardak Emilia , Peled Eli , Crispel Yonatan , Ghanem Shourouk , Attias Judith , Asayag Keren , Kogan Inna , Nadir Yona
Published: December 2019

Background

While malignant pleural effusion (MPE) is a common and significant cause of morbidity in patients with cancer, current treatment options are limited. Human heparanase, involved in angiogenesis and metastasis, cleaves heparan sulfate (HS) side chains on the cell surface.

Aims

To explore the coagulation milieu in MPE and infectious pleural effusion (IPE) focusing on the involvement of heparanase.

Methods

Samples of 30 patients with MPE and 44 patients with IPE were evaluated in comparison to those of 33 patients with transudate pleural effusions, using heparanase ELISA, heparanase procoagulant activity assay, thrombin and factor Xa chromogenic assays and thromboelastography. A cell proliferation assay was performed. EMT-6 breast cancer cells were injected to the pleural cavity of mice. A peptide inhibiting heparanase activity was administered subcutaneously.

Results

Levels of heparanase, factor Xa and thrombin were significantly higher in exudate than transudate. Thromboelastography detected almost no thrombus formation in the whole blood, mainly on MPE addition. This effect was completely reversed by bacterial heparinase. Direct measurement revealed high levels of HS chains in pleural effusions. Higher proliferation was observed in tumour cell lines incubated with exudate than with transudate and it was reduced when bacterial heparinase was added. The tumour size in the pleural cavity of mice treated with the heparanase inhibitor were significantly smaller compared with control (p=0.005).

Conclusions

HS chains released by heparanase form an anticoagulant milieu in MPE, preventing local thrombosis and enabling tumour cell proliferation. Inhibition of heparanase might provide a therapeutic option for patients with recurrent MPE.
Evaluation of Crack Formation and Propagation with Ultrasonic Root-End Preparation and Obturation Using a Digital Microscope and Optical Coherence Tomography
Authors: Rashed Bayan , Iino Yoshiko , Ebihara Arata , Okiji Takashi
Published: December 2019

Objective. This study is aimed at determining (1) the effect of root-end resection, ultrasonic root-end preparation, and root-end filling on the incidence of crack formation and propagation by using a digital microscope (DM) and optical coherence tomography (OCT) and (2) the performance of OCT on the detection of cracks by comparing with microcomputed tomography (micro-CT) as a reference standard. Methodology. Thirty extracted lower incisors were endodontically treated and subjected to root-end resection and ultrasonic root-end cavity preparation. Then, the teeth were divided into three groups (n=10, each), and the root-end cavity was either left unfilled or filled with mineral trioxide aggregate (MTA) or super-EBA. The resected surface was observed with OCT and DM after the root-end resection, ultrasonic root-end preparation, and root-end filling, and the frequency of incomplete and complete cracks were recorded. The observation was repeated after two weeks, one month, and two months, and micro-CT scans after two months were taken as the gold standard. Results. The DM results show dentinal crack formation in 47% of the samples following root-end resection and in 87% following ultrasonic preparation. After the ultrasonic preparation, no existing crack propagated to a complete crack, but new cracks were formed. MTA and super-EBA had no effect on crack formation. The Spearman correlation coefficient between OCT and DM was 0.186 (very weak correlation; p=0.015). Sensitivity and specificity in comparison to micro-CT were 0.50 and 0.55 in OCT and 1.00 and 0.35 in DM, respectively. McNemar’s test showed a significant difference between OCT and DM (p<0.05). Conclusion. Apical resection and ultrasonic preparation could form dentinal cracks. OCT and DM showed different detection frequencies of cracks with very weak correlation. DM showed superior sensitivity compared with OCT.
Nanolayer Film on Poly(Styrene/Ethylene Glycol Dimethacrylate) High Internal Phase Emulsion Porous Polymer Surface as a Scaffold for Tissue Engineering Application
Authors: Preechawong Jitima , Noulta Kornkanok , Dubas Stephan T. , Nithitanakul Manit , Sapsrithong Pornsri
Published: December 2019

Highly open, porous polymer or poly(high internal phase emulsion) (polyHIPE) obtained from polymerized high internal phase emulsions (HIPE) have recently attracted much attention and increasing interest in tissue engineering applications because of their excellent properties. This research is aimed at preparing and developing a process for producing polyHIPE for use as a scaffold in tissue engineering applications. Poly(styrene/ethylene glycol dimethacrylate) (poly(S/EGDMA)) loaded with hydroxyapatite was used to prepare the polyHIPE. Further improvement on hydrophilicity and biological response to tissue fluids of the polyHIPE porous polymer was carried out using a nanolayer coating via the layer-by-layer polyelectrolyte multilayer (PEM) technique. Three types of chemicals were used for coating on the surface of the polyHIPE porous polymer such as poly(sodium 4-styrene sulfonate) (PSS), gelatin (GEL), and alginic acid (ALG). The change in surface properties of the modified poly(S/EGDMA)HIPE was characterized by UV-visible spectroscopy and contact angle measurement. Further assessments consisted of cytotoxicity testing, cell attachment, and proliferation of L929, fibroblast-like cells that were seeded on the surface of the polyHIPE porous polymer. A three-dimensional structure poly(S/EGDMA)HIPE porous polymer with high porosity was successfully prepared. Moreover, it was found that surface modification encouraged poly(S/EGDMA)HIPE with a hydrophilic nanolayer, as observed by the decrease in contact angle degree and cell adhesion of L929 fibroblast-like cells, indicating that the poly(S/EGDMA)HIPE porous polymer was effectively improved by using the layer-by-layer technique. It was revealed by MTT assay that the poly(S/EGDMA)HIPE porous polymer coated with a nanolayer of a polyelectrolyte multilayer led to an enhancement in the amount of cell adhesion and proliferation on the modified poly(S/EGDMA)HIPE porous polymer. Additionally, the most effective polyelectrolyte solution for improving cell adhesion of the poly(S/EGDMA)HIPE was PSS.
Epigenetic Targeting of TERT-Associated Gene Expression Signature in Human Neuroblastoma with TERT Overexpression
Authors: Huang Min , Zeki Jasmine , Sumarsono Nathan , Coles Garry L. , Taylor Jordan S. , Danzer Enrico , Bruzoni Matias , Hazard Florette K. , Lacayo Norman J. , Sakamoto Kathleen M. , Dunn James C.Y. , Spunt Sheri L. , Chiu Bill
Published: January 2020

Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Particularly, the pathophysiology of tumors without MYCN amplification remains poorly defined. Utilizing an unbiased approach, we performed gene set enrichment analysis of RNA-sequencing data from 498 patients with neuroblastoma and revealed a differentially overexpressed gene signature in MYCN nonamplified neuroblastomas with telomerase reverse transcriptase (TERT) gene overexpression and coordinated activation of oncogenic signaling pathways, including E2Fs, Wnt, Myc, and the DNA repair pathway. Promoter rearrangement of the TERT gene juxtaposes the coding sequence to strong enhancer elements, leading to TERT overexpression and poor prognosis in neuroblastoma, but TERT-associated oncogenic signaling remains unclear. ChIP-seq analysis of the human CLB-GA neuroblastoma cells harboring TERT rearrangement uncovered genome-wide chromatin co-occupancy of Brd4 and H3K27Ac and robust enrichment of H3K36me3 in TERT and multiple TERT-associated genes. Brd4 and cyclin-dependent kinases (CDK) had critical regulatory roles in the expression and chromatin activation of TERT and multiple TERT-associated genes. Epigenetically targeting Brd4 or CDKs with their respective inhibitors suppressed the expression of TERT and multiple TERT-associated genes in neuroblastoma with TERT overexpression or MYCN amplification. ChIP-seq and ChIP-qPCR provided evidence that the CDK inhibitor directly inhibited Brd4 recruitment to activate chromatin globally. Therefore, inhibiting Brd4 and CDK concurrently with AZD5153 and dinaciclib would be most effective in tumor growth suppression, which we demonstrated in neuroblastoma cell lines, primary human cells, and xenografts. In summary, we describe a unique mechanism in neuroblastoma with TERT overexpression and an epigenetically targeted novel therapeutic strategy.

Significance:

Epigenetically cotargeting Brd4 and Cdks suppresses human neuroblastoma with TERT overexpression by inhibiting the TERT-associated gene expression networks.
Long Noncoding RNA DRAIC Inhibits Prostate Cancer Progression by Interacting with IKK to Inhibit NF-κB Activation
Authors: Saha Shekhar , Kiran Manjari , Kuscu Canan , Chatrath Ajay , Wotton David , Mayo Marty W. , Dutta Anindya
Published: January 2020

DRAIC is a 1.7 kb spliced long noncoding RNA downregulated in castration-resistant advanced prostate cancer. Decreased DRAIC expression predicts poor patient outcome in prostate and seven other cancers, while increased DRAIC represses growth of xenografted tumors. Here, we show that cancers with decreased DRAIC expression have increased NF-κB target gene expression. DRAIC downregulation increased cell invasion and soft agar colony formation; this was dependent on NF-κB activation. DRAIC interacted with subunits of the IκB kinase (IKK) complex to inhibit their interaction with each other, the phosphorylation of IκBα, and the activation of NF-κB. These functions of DRAIC mapped to the same fragment containing bases 701–905. Thus, DRAIC lncRNA inhibits prostate cancer progression through suppression of NF-κB activation by interfering with IKK activity.

Significance:

A cytoplasmic tumor-suppressive lncRNA interacts with and inhibits a major kinase that activates an oncogenic transcription factor in prostate cancer.
A Phase I Study of ASTX660, an Antagonist of Inhibitors of Apoptosis Proteins, in Adults with Advanced Cancers or Lymphoma
Authors: Mita Monica M. , LoRusso Patricia M. , Papadopoulos Kyriakos P. , Gordon Michael S. , Mita Alain C. , Ferraldeschi Roberta , Keer Harold , Oganesian Aram , Su Xiang Yao , Jueliger Simone , Tolcher Anthony W.
Published: January 2020

Purpose:

This first-in-human, phase I study evaluated ASTX660, an oral, small-molecule antagonist of cellular/X-linked inhibitors of apoptosis proteins in patients with advanced solid tumors or lymphoma.

Patients and Methods:

ASTX660 was administered orally once daily on a 7-day-on/7-day-off schedule in a 28-day cycle. Dose escalation followed a standard 3+3 design to determine the MTD and recommended phase II dose (RP2D). Dose expansion was conducted at the RP2D.

Results:

Forty-five patients received ASTX660 (range 15–270 mg/day). Dose-limiting toxicity of grade 3 increased lipase with or without increased amylase occurred in 3 patients at 270 mg/day and 1 patient at 210 mg/day. The MTD was determined to be 210 mg/day and the RP2D 180 mg/day. Common treatment-related adverse events included fatigue (33%), vomiting (31%), and nausea (27%). Grade ≥3 treatment-related adverse events occurred in 7 patients, most commonly anemia (13%), increased lipase (11%), and lymphopenia (9%). ASTX660 was rapidly absorbed, with maximum concentration achieved at approximately 0.5–1.0 hour. An approximately 2-fold accumulation in AUC exposures was observed on day 7 versus 1. ASTX660 suppressed cellular inhibitor of apoptosis protein-1 in peripheral blood mononuclear cells, which was maintained into the second cycle beyond the off-therapy week at the 180-mg/day RP2D and above. Clinical activity was seen in a patient with cutaneous T-cell lymphoma.

Conclusions:

ASTX660 demonstrated a manageable safety profile and exhibited evidence of pharmacodynamic and preliminary clinical activity at the 180-mg/day RP2D. The phase II part of the study is ongoing.