bluuebunny/crossref_metadata_2025_split huggingface dataset:
Vaccinia Virus 15-Kilodalton (A14L) Protein Is Essential for Assembly and Attachment of Viral Crescents to Virosomes
Authors: Rodríguez Juan Ramón , Risco Cristina , Carrascosa José L. , Esteban Mariano , Rodríguez Dolores 
Published: December 2019

ABSTRACT

Early stages in vaccinia virus (VV) assembly involve the recruitment of cellular membranes from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) to virus factories (or virosomes). The key viral factors involved in this process are not yet known. We have previously identified and characterized two viral proteins, of 21 kDa (A17L gene) and 15 kDa (A14L gene), that associate with tubulovesicular elements related to the ERGIC and are localized in viral membranes at all stages of virion assembly. We showed that the 21-kDa protein is not responsible for the recruitment of membranes from the ERGIC to viral factories. However, it appears to be essential for the organization of viral membranes. In this investigation we have generated a VV recombinant, VVindA14L, in which the expression of the A14L gene is inducibly regulated by the
Escherichia coli lacI
operator-repressor system. Repression of 15-kDa protein synthesis has a dramatic effect on virus yields and severely impairs plaque formation. Compared to wild-type VV, reduced amounts of 15-kDa protein are produced in VVindA14L-infected cells in the presence of IPTG (isopropyl-β-
d
-thiogalactoside), and this correlates with a small-plaque phenotype and reduced VVindA14L yields under these conditions. In the absence of the 15-kDa protein, early and late viral protein syntheses proceed normally; however, proteolytic cleavage of the major core precursors is inhibited. Electron microscopic examination of cells infected with VVindA14L under nonpermissive conditions reveals the presence of numerous membranous elements that look like unfinished or disassembled crescents interespersed between electron-dense masses. These abnormal membrane elements are usually well separated from the surfaces of the dense structures. These findings show that the 15-kDa protein is essential for VV morphogenesis and indicate that this polypeptide is necessary both for the correct assembly of viral crescents and for their stable attachment to the surfaces of viral factories.
STP and Tip Are Essential for Herpesvirus Saimiri Oncogenicity
Authors: Duboise S. Monroe , Guo Jie , Czajak Sue , Desrosiers Ronald C. , Jung Jae U. 
Published: December 2019

ABSTRACTMutant forms of herpesvirus saimiri (HVS) subgroup C strain 488 with deletions in either STP-C488 or Tip were constructed. The transforming potentials of the HVS mutants were tested in cell culture and in common marmosets. Parental HVS subgroup C strain 488 immortalized common marmoset T lymphocytes in vitro to interleukin-2-independent growth, but neither of the deletion mutants produced such growth transformation. Wild-type HVS produced fatal lymphoma within 19 to 20 days of experimental infection of common marmosets, while HVS ΔSTP-C488 and HVS ΔTip were nononcogenic. Virus was repeatedly isolated from the peripheral blood of marmosets infected with mutant virus for more than 5 months. These results demonstrate that STP-C488 and Tip are not required for replication or persistence, but each is essential for transformation in cell culture and for lymphoma induction in common marmosets.
Entry of Amphotropic and 10A1 Pseudotyped Murine Retroviruses Is Restricted in Hematopoietic Stem Cell Lines
Authors: von Laer Dorothee , Thomsen Silke , Vogt Birgit , Donath Martina , Kruppa Joachim , Rein Alan , Ostertag Wolfram , Stocking Carol 
Published: December 2019

ABSTRACT

Although transduction with amphotropic murine leukemia virus (MLV) vectors has been optimized successfully for hematopoietic differentiated progenitors, gene transfer to early hematopoietic cells (stem cells) is still highly restricted. A similar restriction to gene transfer was observed in the mouse stem cell line FDC-Pmix compared with transfer in the more mature myeloid precursor cell line FDC-P1 and the human erythroleukemia cell line K562. Gene transfer was not improved when the vector was pseudotyped with gp70
SU
of the 10A1 strain of MLV, which uses the receptor of the gibbon ape leukemia virus (Pit1), in addition to the amphotropic receptor (Pit2). Although 10A1 and amphotropic gp70
SU
bound to FDC-P1, K562, and fibroblasts, no binding to FDC-Pmix cells was detected. This indicates that FDC-Pmix cells lack functional Pit2 and Pit1 receptors. Pseudotyping with the vesicular stomatitis virus G protein improved transduction efficiency in FDC-Pmix stem cells by 2 orders of magnitude, to fibroblast levels, confirming a block to retroviral infection at the receptor level.
Genetic Determinants Responsible for Acquisition of Dengue Type 2 Virus Mouse Neurovirulence
Authors: Bray Michael , Men Ruhe , Tokimatsu Issei , Lai Ching-Juh 
Published: December 2019

ABSTRACTStudies conducted some 50 years ago showed that serial intracerebral passage of dengue viruses in mice selected for neurovirulent mutants that also exhibited significant attenuation for humans. We investigated the genetic basis of mouse neurovirulence of dengue virus because it might be directly or indirectly associated with attenuation for humans. Analysis of the sequence in the C-PreM-E-NS1 region of the parental dengue type 2 virus (DEN2) New Guinea C (NGC) strain and its mouse-adapted, neurovirulent mutant revealed that 10 nucleotide changes occurred during serial passage in mice. Seven of these changes resulted in amino acid substitutions, i.e., Leu55-Phe and Arg57-Lys in PreM, Glu71-Asp, Glu126-Lys, Phe402-Ile, and Thr454-Ile in E, and Arg105-Gln in NS1. The sequence of C was fully conserved between the parental and mutant DEN2. We constructed intertypic chimeric dengue viruses that contained the PreM-E genes or only the NS1 gene of neurovirulent DEN2 NGC substituting for the corresponding genes of DEN4. The DEN2 (PreM-E)/DEN4 chimera was neurovirulent for mice, whereas DEN2 (NS1)/DEN4 was not. The mutations present in the neurovirulent DEN2 PreM-E genes were then substituted singly or in combination into the sequence of the nonneurovirulent, parental DEN2. Intracerebral titration of the various mutant chimeras so produced identified two amino acid changes, namely, Glu71-Asp and Glu126-Lys, in DEN2 E as being responsible for mouse neurovirulence. The conservative amino acid change of Glu71-Asp probably had a minor effect, if any. The Glu126-Lys substitution in DEN2 E, representing a change from a negatively charged amino acid to a positively charged amino acid, most likely plays an important role in conferring mouse neurovirulence.
A Determinant for Central Nervous System Persistence Localized in the Capsid of Theiler’s Murine Encephalomyelitis Virus by Using Recombinant Viruses
Authors: Adami Cecilia , Pritchard Arthur E. , Knauf Todd , Luo Ming , Lipton Howard L. 
Published: December 2019

ABSTRACT
The demyelinating process in Theiler’s murine encephalomyelitis virus (TMEV) infection in mice requires virus persistence in the central nervous system. Using recombinant TMEV assembled between the virulent GDVII and less virulent BeAn virus cDNAs, we now provide additional evidence supporting the localization of a persistence determinant to the leader P1 (capsid) sequences. Further, recombinant viruses in which BeAn sequences progressively replaced those of GDVII within the capsid starting at the leader NH2 terminus suggest that a conformational determinant requiring homologous sequences in both the VP2 puff and VP1 loop regions, which are in close contact on the virion surface, might underlie persistence.
Human Immunodeficiency Virus Type 1 Matrix Protein Interacts with Cellular Protein HO3
Authors: Lama Juan , Trono Didier 
Published: December 2019

ABSTRACT

The matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) plays a critical role in virion morphogenesis and fulfills important functions during the early steps of infection. In an effort to identify cellular partners of MA, a
Saccharomyces cerevisiae
two-hybrid screen was utilized. A specific interaction between MA and HO3, a putative histidyl-tRNA synthetase, was demonstrated in this system. HO3-specific mRNA was detected in several tissues relevant for HIV infection, such as spleen, thymus, and peripheral blood lymphocytes, as well as in a number of T-lymphoid-cell lines. The binding of MA to HO3 was confirmed in transfected cells by coimmunoprecipitation. This interaction was abrogated by replacing two lysine residues at positions 26 and 27 of MA by threonine (MA

KK

27
TT

). HO3 localized both to the cytoplasm and to the nucleus of acutely transfected 293T cells. When overexpressed in HIV-1-producing cells, HO3 was incorporated into wild-type virions but not in ones containing the dilysine-mutated variant of MA. Correspondingly, overexpression of HO3 in virus producer cells enhanced the infectivity of wild-type but not MA

KK

27
AA

HIV-1 particles. The stimulating effect of HO3 was independent from the presence of Envelope, Vpr, or Vpu. Taken together, these results suggest that HO3, through its recognition of MA, plays a role in the life cycle of HIV-1.
Construction and Characterization of a Hybrid Mouse Mammary Tumor Virus/Murine Leukemia Virus-Based Retroviral Vector
Authors: Saller Robert M. , Öztürk Feride , Salmons Brian , Günzburg Walter H. 
Published: December 2019

ABSTRACT
Mouse mammary tumor virus (MMTV)-based vectors are characterized by low titers. In an effort to transfer MMTV-specific regulation of gene expression to a more efficient murine leukemia virus (MLV) vector, we have replaced the complete 3′ U3 region of MLV with the complete U3 region of MMTV. Virus titers were not significantly affected by this modification, there was no impairment of reverse transcription and integration, and after infection of cells, the MMTV promoter is duplicated and translocated to the 5′ long terminal repeat, resulting in glucocorticoid-regulatable RNA expression.
Both the Polycythemia- and Anemia-Inducing Strains of Friend Spleen Focus-Forming Virus Induce Constitutive Activation of the Raf-1/Mitogen-Activated Protein Kinase Signal Transduction Pathway
Authors: Muszynski Karen W. , Ohashi Takashi , Hanson Charlotte , Ruscetti Sandra K. 
Published: December 2019

ABSTRACT
The erythroleukemia-inducing Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein which allows erythroid cells to proliferate and differentiate in the absence of erythropoietin (Epo). In an attempt to understand how the virus causes Epo independence, we have been studying signal transduction pathways activated by Epo to determine if SFFV exerts its biological effects by constitutively activating any of these pathways in the absence of Epo. We previously demonstrated that Stat proteins, the downstream components of the Epo-induced Jak-Stat pathway, are constitutively activated in SFFV-infected cells. In this study, we demonstrate that SFFV also activates Raf-1, MEK and mitogen-activated protein (MAP) kinase, the downstream components of the Raf-1/MAP kinase pathway. This pathway was activated in cells infected with the polycythemia-inducing strain of SFFV, which induces both proliferation and differentiation of erythroid cells in the absence of Epo, as well as in cells infected with the anemia-inducing strain of the virus, which still require Epo for differentiation. Inhibition of Raf-1 by using antisense oligonucleotides led to a partial inhibition of the Epo-independent proliferation of SFFV-infected cells. Expression of the transcription factors c-Jun and JunB, but not c-Fos, was induced in SFFV-infected cells in the absence of Epo, suggesting that constitutive activation of the Raf-1/MAP kinase pathway by the virus may result in deregulation of AP-1 activity. We conclude from our studies that infection of erythroid cells with SFFV leads to the constitutive activation of signal transduction molecules in both the Jak-Stat and Raf-1/MAP kinase pathways and that both of these pathways must be activated to achieve maximum proliferation and differentiation of erythroid cells in the absence of Epo.
Disruption of the G
1
/S Transition in Human Papillomavirus Type 16 E7-Expressing Human Cells Is Associated with Altered Regulation of Cyclin E
Authors: Martin Larry G. , Demers G. William , Galloway Denise A. 
Published: December 2019

ABSTRACT

The development of neoplasia frequently involves inactivation of the p53 and retinoblastoma (Rb) tumor suppressor pathways and disruption of cell cycle checkpoints that monitor the integrity of replication and cell division. The human papillomavirus type 16 (HPV-16) oncoproteins, E6 and E7, have been shown to bind p53 and Rb, respectively. To further delineate the mechanisms by which E6 and E7 affect cell cycle control, we examined various aspects of the cell cycle machinery. The low-risk HPV-6 E6 and E7 proteins did not cause any significant change in the levels of cell cycle proteins analyzed. HPV-16 E6 resulted in very low levels of p53 and p21 and globally elevated cyclin-dependent kinase (CDK) activity. In contrast, HPV-16 E7 had a profound effect on several aspects of the cell cycle machinery. A number of cyclins and CDKs were elevated, and despite the elevation of the levels of at least two CDK inhibitors, p21 and p16, CDK activity was globally increased. Most strikingly, cyclin E expression was deregulated both transcriptionally and posttranscriptionally and persisted at high levels in S and G
2
/M. Transit through G
1
was shortened by the premature activation of cyclin E-associated kinase activity. Elevation of cyclin E levels required both the CR1 and CR2 domains of E7. These data suggest that cyclin E may be a critical target of HPV-16 E7 in the disruption of G
1
/S cell cycle progression and that the ability of E7 to regulate cyclin E involves activities in addition to the release of E2F.
Delayed Human Immunodeficiency Virus Type 1-Induced Apoptosis in Cells Expressing Truncated Forms of CD4
Authors: Guillerm Claire , Coudronnière Nolwenn , Robert-Hebmann Véronique , Devaux Christian 
Published: December 2019

ABSTRACTIt has been reported previously that cells expressing a truncated form of CD4 which lacks the cytoplasmic tail of the molecule (truncation at position 402) were not sensitive to human immunodeficiency virus type 1 (HIV-1)-induced apoptosis in an acute-phase model of infection (J. Corbeil, M. Tremblay, and D. D. Richman, J. Exp. Med. 183:39–48, 1996). The role played by the cytoplasmic domain of CD4 in HIV-1-induced apoptosis was reexamined here with clones of A2.01 cells expressing different forms of CD4 and the DNA intercalant YOPRO-1 assay. Six days after virus exposure, we found evidence of apoptosis in A2.01 cells expressing the wild-type CD4 (A2.01/CD4), whereas enhanced apoptosis remained absent in cultures of A2.01/CD4.401 and A2.01/CD4.403 cells (A2.01 cells which express CD4.401 and CD4.403 molecules with truncations at positions 401 and 403, respectively). However, cell death by apoptosis measured with YOPRO-1 was found in cultures of A2.01/CD4.401 and A2.01/CD4.403 cells 15 days after virus exposure. This result was confirmed with a terminal dUTP nick end-labeling assay and propidium iodide staining. The long lag time postinfection required for apoptosis to be observed in cultures of infected cells expressing truncated forms of CD4 was due to the delayed viral replication in these cells, as shown by monitoring of the viral reverse transcriptase activity and HIV-1 p24gagantigen expression. These results emphasize the relationship between virus replication and cell death by apoptosis.
Three Amino Acid Substitutions in the L Protein of the Human Parainfluenza Virus Type 3
cp
45 Live Attenuated Vaccine Candidate Contribute to Its Temperature-Sensitive and Attenuation Phenotypes
Authors: Skiadopoulos Mario H. , Durbin Anna P. , Tatem Joanne M. , Wu Shin-Lu , Paschalis Maribel , Tao Tao , Collins Peter L. , Murphy Brian R. 
Published: December 2019

ABSTRACT

Studies were initiated to define the genetic basis of the temperature-sensitive (
ts
), cold adaptation (
ca
), and attenuation (
att
) phenotypes of the human parainfluenza virus type 3 (PIV3)
cp
45 live attenuated vaccine candidate. Genetic data had previously suggested that the L polymerase protein of
cp
45, which contains three amino acid substitutions at positions 942, 992, and 1558, contributed to its temperature sensitivity (R. Ray, M. S. Galinski, B. R. Heminway, K. Meyer, F. K. Newman, and R. B. Belshe, J. Virol. 70:580–584, 1996; A. Stokes, E. L. Tierney, C. M. Sarris, B. R. Murphy, and S. L. Hall, Virus Res. 30:43–52, 1993). To study the individual and aggregate contributions that these amino acid substitutions make to the
ts
,
att
, and
ca
phenotypes of
cp
45, seven PIV3 recombinant viruses (three single, three double, and one triple mutant) representing all possible combinations of the three amino acid substitutions were recovered from full-length antigenomic cDNA and analyzed for their
ts
,
att
, and
ca
phenotypes. None of the seven mutant recombinant PIVs was cold adapted. The substitutions at L protein amino acid positions 992 and 1558 each specified a 10
5
-fold reduction in plaque formation in cell culture at 40°C, whereas the substitution at position 942 specified a 300-fold reduction. Thus, each of the three mutations contributes individually to the
ts
phenotype. The triple recombinant which possesses an L protein with all three mutations was almost as temperature sensitive as
cp
45, indicating that these mutations are the major contributors to the
ts
phenotype of
cp
45. The three individual mutations in the L protein each contributed to restricted replication in the upper or lower respiratory tract of hamsters, and this likely contributes to the observed stability of the
ts
and
att
phenotypes of
cp
45 during replication in vivo. Importantly, the recombinant virus possessing L protein with all three mutations was as restricted in replication as was the
cp
45 mutant in both the upper and lower respiratory tracts of hamsters, indicating that the L gene of the
cp
45 virus is a major attenuating component of this candidate vaccine.
Phosphatidylinositol-Dependent Membrane Fusion Induced by a Putative Fusogenic Sequence of Ebola Virus
Authors: Ruiz-Argüello M. Begoña , Goñi Félix M. , Pereira Francisca B. , Nieva José L. 
Published: December 2019

ABSTRACT

The membrane-interacting abilities of three sequences representing the putative fusogenic subdomain of the Ebola virus transmembrane protein have been investigated. In the presence of calcium, the sequence EBO
GE
(GAAIGLAWIPYFGPAAE) efficiently fused unilamellar vesicles composed of phosphatidylcholine, phosphatidylethanolamine, cholesterol, and phosphatidylinositol (molar ratio, 2:1:1:0.5), a mixture that roughly resembles the lipid composition of the hepatocyte plasma membrane. Analysis of the lipid dependence of the process demonstrated that the fusion activity of EBO
GE
was promoted by phosphatidylinositol but not by other acidic phospholipids. In comparison, EBO
EA
(EGAAIGLAWIPYFGPAA) and EBO
EE
(EGAAIGLAWIPYFGPAAE) sequences, which are similar to EBO
GE
except that they bear the negatively charged glutamate residue at the N terminus and at both the N and C termini, respectively, induced fusion to a lesser extent. As revealed by binding experiments, the glutamate residue at the N terminus severely impaired peptide-vesicle interaction. In addition, the fusion-competent EBO
GE
sequence did not associate significantly with vesicles lacking phosphatidylinositol. Tryptophan fluorescence quenching by vesicles containing brominated phospholipids indicated that the EBO
GE
peptide penetrated to the acyl chain level only when the membranes contained phosphatidylinositol. We conclude that binding and further penetration of the Ebola virus putative fusion peptide into membranes might be governed by the nature of the N-terminal residue and by the presence of phosphatidylinositol in the target membrane. Moreover, since insertion of such a peptide leads to membrane destabilization and fusion, the present data would be compatible with the involvement of this sequence in Ebola virus fusion.
Receptor-Binding Properties of a Soluble Form of Human Cytomegalovirus Glycoprotein B
Authors: Boyle Kathleen A. , Compton Teresa 
Published: December 2019

ABSTRACT

The human cytomegalovirus (HCMV) glycoprotein B (gB) (also known as gpUL55) homolog is an important mediator of virus entry and cell-to-cell dissemination of infection. To examine the potential ligand-binding properties of gB, a soluble form of gB (gB-S) was radiolabeled, purified, and tested in cell-binding experiments. Binding of gB-S to human fibroblast cells was found to occur in a dose-dependent, saturable, and specific manner. Scatchard analysis demonstrated a biphasic plot with the following estimated dissociation constants (

K
d

):
K

d
1

, 4.96 × 10
−6
M;
K

d
2

, 3.07 × 10
−7
M. Cell surface heparan sulfate proteoglycans (HSPGs) were determined to serve as one class of receptors able to facilitate gB-S binding. Both HSPG-deficient Chinese hamster ovary (CHO) cells and fibroblast cells with enzymatically removed HSPGs had 40% reductions in gB-S binding, whereas removal of chondroitin sulfate had no effect. However, a significant proportion of gB-S was able to associate with the cell surface in the absence of HSPGs via an undefined nonheparin component. Binding affinity analysis of gB-S binding to wild-type CHO-K1 cells demonstrated biphasic binding kinetics (
K

d
1

, 9.85 × 10
−6
M;

K
d2

, 4.03 × 10
−8
M), whereas gB-S binding to HSPG-deficient CHO-677 cells exhibited single-component binding kinetics (

K
d

, 7.46 × 10
−6
M). Together, these data suggest that gB-S associates with two classes of cellular receptors. The interaction of gB with its receptors is physiologically relevant, as evidenced by an inhibitory effect on HCMV entry when cells were pretreated with purified gB-S. This inhibition was determined to be manifested at the level of virus attachment. We conclude that gB is a ligand for HCMV that mediates an interaction with a cellular receptor(s) during HCMV infection.
Multiple Functions within the Epstein-Barr Virus EBNA-3A Protein
Authors: Cludts Isabelle , Farrell Paul J. 
Published: December 2019

ABSTRACT
Two regions of the EBNA-3A protein of Epstein-Barr virus were shown to be capable of binding to the cell protein RBP-Jk (also known as CBF-1), a component of the Notch signaling pathway. Consistent with this binding, EBNA-3A inhibited reporter gene expression from plasmids containing RBP-Jk DNA binding sites within their promoters, including the Cp promoter. When EBNA-3A was linked to a GAL4 DNA binding domain, it repressed the activity of a promoter containing GAL4 binding sites at all plasmid concentrations tested. However, a deletion mutant of EBNA-3A lacking amino acids 100 to 364 showed a biphasic response in the GAL4 assay: it inhibited transcription at low DNA concentrations but activated it at high DNA concentrations. There appears to be a gene activation function within EBNA-3A that is masked in the full-length protein in this assay. Current models for EBNA-3 function have stressed transcription repression through binding to RBP-Jk, but we consider an alternative scheme in which the role of the binding of EBNA-3A, -3B, and -3C to RBP-Jk is to buffer the levels of active EBNA-3 protein. We have also found that the behavior of EBNA-3A in a cell fractionation procedure that distinguishes insoluble matrix from soluble cell fractions is modified by EBNA-LP, indicating a further novel level of interplay between the EBNA proteins.