bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel Role for E4 Region Genes in Protection of Adenovirus Vectors from Lysis by Cytotoxic T Lymphocytes
Authors: Kaplan Johanne M. , Armentano Donna , Scaria Abraham , Woodworth Lisa A. , Pennington Sarah E. , Wadsworth Samuel C. , Smith Alan E. , Gregory Richard J. 
Published: December 2019

ABSTRACTTarget cells infected with adenovirus (Ad) vectors containing intact E3 and E4 regions were found to be relatively resistant to lysis by Ad-specific cytotoxic T lymphocytes. Elements from both the E3 and the E4 regions were required for this effect, leading to the identification of a previously undescribed role for E4 gene products in resistance to cytolysis.
An Arginine-Faced Amphipathic Alpha Helix Is Required for Adenovirus Type 5 E4orf6 Protein Function
Authors: Orlando Joseph S. , Ornelles David A. 
Published: December 2019

ABSTRACT

A region in the carboxy terminus of the protein encoded by open reading frame 6 in early region 4 (E4orf6) of adenovirus type 5 was determined to be required for directing nuclear localization of the E1B 55-kDa protein and for efficient virus replication. A peptide encompassing this region, corresponding to amino acids 239 through 255 of the E4orf6 protein, was analyzed by circular dichroism spectroscopy. The peptide showed evidence of self-interaction and displayed the characteristic spectra of an amphipathic α helix in the helix-stabilizing solvent trifluoroethanol. Disrupting the integrity of this α helix in the E4orf6 protein by proline substitutions or by removing amino acids 241 through 250 abolished its ability to direct the E1B 55-kDa protein to the nucleus when both proteins were transiently expressed in HeLa cells. Expression of E4orf6 variants that failed to direct nuclear localization of the E1B 55-kDa protein failed to enhance replication of the E4 mutant virus,
dl
1014, whereas expression of the wild-type E4orf6 protein restored growth of
dl
1014 to near-wild-type levels. These results suggest that the E4orf6 protein contains an arginine-faced, amphipathic α helix that is critical for a functional interaction with the E1B 55-kDa protein in the cell and for the function of the E4orf6 protein during a lytic infection.
Mutational Analysis, Using a Full-Length Rubella Virus cDNA Clone, of Rubella Virus E1 Transmembrane and Cytoplasmic Domains Required for Virus Release
Authors: Yao Jiansheng , Gillam Shirley 
Published: December 2019

ABSTRACT
We report on the construction of a full-length cDNA clone, pBRM33, derived from wild-type rubella virus M33 strain. The RNA transcripts synthesized in vitro from pBRM33 are highly infectious, and the viruses produced retain the phenotypic characteristics of the parental M33 virus in growth rate and plaque size. This cDNA clone was used to study the role of E1 transmembrane and cytoplasmic domains in virus assembly by site-directed mutagenesis. Three different alanine substitutions were introduced in the transmembrane domain of E1. These included substitution of leucine 464, cysteine 466, cysteine 467, and both cysteines 466 and 467 to alanine. In the E1 cytoplasmic domain, cysteine 470 and leucine 471 were altered to alanine. We found that these mutations did not significantly affect viral RNA replication, viral structural protein synthesis and transport, or E2/E1 heterodimer formation. Except for the substitution of cysteine 470, these mutations did, however, lead to a reduction in virus release. Substitution of cysteine 467 in the transmembrane region and of leucine 471 in the cytoplasmic domain dramatically reduced virus yield, resulting in the production of only 1 and 10% of the parental virus yield, respectively, in a parallel infection. These data show that E1 transmembrane and cytoplasmic domains play an important role in late stages of virus assembly, possibly during virus budding, consistent with earlier studies indicating that the E1 cytoplasmic domain may interact with nucleocapsids and that this interaction drives virus budding.
Novel Endogenous Type D Retroviral Particles Expressed at High Levels in a SCID Mouse Thymic Lymphoma
Authors: Ristevski Sika , Purcell Damian F. J. , Marshall John , Campagna Daniella , Nouri Sara , Fenton Simon P. , McPhee Dale A. , Kannourakis George 
Published: December 2019

ABSTRACTA xenograft model of the human disease Langerhans cell histiocytosis (LCH) was investigated with severe combined immunodeficiency (SCID) mice. Transplantation of human LCH biopsy material into SCID mice resulted in the generation of mouse tumors resembling lymphomas. A thymoma cell line (ThyE1M6) was generated from one of these mice and found to display significant levels of Mg2+-dependent reverse transcriptase activity. Electron microscopy revealed particles with type D retroviral morphology budding from ThyE1M6 cells at a high frequency, whereas control cultures were negative. Reverse transcription-PCR of virion RNA with degenerate primers for conserved regions of various mouse, human, and primate retroviruses amplified novel sequences related to primate type D retroviruses, murine intracisternal A particles, Jaagsiekte sheep retrovirus, and murine long interspersed nuclear elements but not other retroviral classes. We demonstrate that these sequences represent a novel group of endogenous retroviruses expressed at low levels in mice but expressed at high levels in the ThyE1M6 cell line. Furthermore, we propose that the activation of endogenous retroviral elements may be associated with a high incidence of thymomas in SCID mice.
Proline Residues in Human Immunodeficiency Virus Type 1 p6
Gag
Exert a Cell Type-Dependent Effect on Viral Replication and Virion Incorporation of Pol Proteins
Authors: Dettenhofer Markus , Yu Xiao-Fang 
Published: December 2019

ABSTRACT

The C terminus of the HIV-1 Gag protein contains a proline-rich domain termed p6
Gag
. This domain has been shown to play a role in efficient virus release and incorporation of Vpr into virions. In a previous study (X. F. Yu, L. Dawson, C. J. Tian, C. Flexner, and M. Dettenhofer, J. Virol. 72:3412–3417, 1998), we observed that the removal of the p6 domain of Gag as well as drastic mutations in the PTAP motif resulted in reduced virion-associated Pol proteins from transfected COS cells. In the present study, amino acid substitutions at residues 5 and 7 of p6
Gag
resulted in a cell type-dependent replication of the mutant virus in CD4
+
T cells; the virus was replication competent in Jurkat cells but restricted in H9 cells and primary blood-derived monocytes. Established Jurkat and H9 cell lines expressing p6
Gag
mutant and parental virus were used to further understand this defect. Mutant virions produced from H9 cells, which displayed no defect in extracellular virion production, showed an ∼16-fold reduction in Pol protein levels, whereas the levels of Pol proteins were only marginally reduced in mutant virions produced from Jurkat cells. The reduction in the virion-associated Pol proteins could not be accounted for by differences in the levels of intracellular p160
Gag-Pol
or in the interaction between p55
Gag
and p160
Gag-Pol
precursors. Electron microscopic analysis of the p6
Gag
mutant virions showed a predominately immature morphology in the absence of significant defects in Gag proteolytic cleavage. Taken together, these data suggest that the proline-rich motif of p6
Gag
is involved in the late stages of virus maturation, which include the packaging of cleaved Pol proteins in viral particles, a process which may involve cell-type-specific factors.
Phenotypic Consequences of Rearranging the P, M, and G Genes of Vesicular Stomatitis Virus
Authors: Ball L. Andrew , Pringle Craig R. , Flanagan Brian , Perepelitsa Victoria P. , Wertz Gail W. 
Published: December 2019

ABSTRACT

The nonsegmented negative-strand RNA viruses (order
Mononegavirales
) include many important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene expression, since genes that are close to the single promoter site at the 3′ end of the viral genome are transcribed at higher levels than those that occupy more distal positions. We manipulated an infectious cDNA clone of the prototypic vesicular stomatitis virus (VSV) to rearrange three of the five viral genes, using an approach which left the viral nucleotide sequence otherwise unaltered. The central three genes in the gene order, which encode the phosphoprotein P, the matrix protein M, and the glycoprotein G, were rearranged into all six possible orders. Viable viruses were recovered from each of the rearranged cDNAs. The recovered viruses were examined for their levels of gene expression, growth potential in cell culture, and virulence in mice. Gene rearrangement changed the expression levels of the encoded proteins in concordance with their distance from the 3′ promoter. Some of the viruses with rearranged genomes replicated as well or slightly better than wild-type virus in cultured cells, while others showed decreased replication. All of the viruses were lethal for mice, although the time to symptoms and death following inoculation varied. These data show that despite the highly conserved gene order of the
Mononegavirales
, gene rearrangement is not lethal or necessarily even detrimental to the virus. These findings suggest that the conservation of the gene order observed among the
Mononegavirales
may result from immobilization of the ancestral gene order due to the lack of a mechanism for homologous recombination in this group of viruses. As a consequence, gene rearrangement should be irreversible and provide an approach for constructing viruses with novel phenotypes.
Hepatitis C Virus Core Protein Inhibits Fas- and Tumor Necrosis Factor Alpha-Mediated Apoptosis via NF-κB Activation
Authors: Marusawa Hiroyuki , Hijikata Makoto , Chiba Tsutomu , Shimotohno Kunitada 
Published: December 2019

ABSTRACT
The effects of hepatitis C virus (HCV) proteins on anti-Fas (CD95/APO-1) antibody- and tumor necrosis factor alpha (TNF-α)-mediated apoptosis in different human cell lines were investigated by magnetic concentration of cells which transiently produced the exogenous protein. HepG2 cells, which produced whole HCV proteins, became resistant to anti-Fas-induced apoptotic cell death. Furthermore, the core protein among HCV proteins had a key role in protecting the various cells from apoptosis mediated by not only anti-Fas but also TNF-α. We also found that the core functioned in the activation of nuclear factor κB (NF-κB) in all cells examined. Deletion analysis of the core revealed that the region required for NF-κB activation was closely correlated with that for its antiapoptotic function. In addition, we revealed in some cases that the antiapoptotic effect of the core was restrained by coproduction of the inhibitor of NF-κB, IκB-α protein. These results demonstrated that the core inhibits Fas- and TNF-α-mediated apoptotic cell death via a mechanism dependent on the activation of NF-κB in particular cell lines.
Role of the Individual Interferon Systems and Specific Immunity in Mice in Controlling Systemic Dissemination of Attenuated Pseudorabies Virus Infection
Authors: Grob Philipp , Schijns Virgil E. C. J. , van den Broek Maries F. , Cox Silvie P. J. , Ackermann Mathias , Suter Mark 
Published: December 2019

ABSTRACTThe importance of each of the two interferon (IFN) systems in impeding herpesvirus replication and in stimulating virus-specific lymphocytes to control an acute systemic infection is not completely understood. To further our knowledge, pseudorabies virus, attenuated by deletion of the glycoprotein E gene to impair its neurovirulence and by deletion of the thymidine kinase gene (gE−TK−PRV), was used to infect wild-type 129Sv/Ev and congenic mice with immune system-associated genetic deficiencies. Mice with mature B and T lymphocytes but lacking either one or both functional receptors for members of each of the two IFN families were infected with gE−TK−PRV. At 3 and 7 but not 14 days after infection, replicating gE−TK−PRV could be isolated only from livers or spleens of mice lacking the receptors for both IFN families, and these mice survived the infection. Therefore, functional IFN receptors were not required to induce a protective immune response against an acute infection with gE−TK−PRV. Furthermore, PRV-specific antibodies of all immunoglobulin G isotypes were produced in these mice. Mice without mature B and T lymphocytes and lacking either one or both functional receptors for members of each of the two IFN families were also infected with gE−TK−PRV. Three days after infection, replicating virus could be isolated only from mice lacking both mature B and T lymphocytes and functional IFN receptors, and these mice were not able to clear the virus. We present evidence that mice with an intact gamma IFN system but without mature B and T cells were able to prevent systemic dissemination of gE−TK−PRV.
The 5′ RNA Terminus of Spleen Necrosis Virus Contains a Novel Posttranscriptional Control Element That Facilitates Human Immunodeficiency Virus Rev/RRE-Independent Gag Production
Authors: Butsch Melinda , Hull Stacey , Wang Yalai , Roberts Tiffiney M. , Boris-Lawrie Kathleen 
Published: December 2019

ABSTRACT

Previous work has shown that spleen necrosis virus (SNV) long terminal repeats (LTRs) are associated with Rex/Rex-responsive element-independent expression of bovine leukemia virus RNA and supports the hypothesis that SNV RNA contains a
cis
-acting element that interacts with cellular Rex-like proteins. To test this hypothesis, the human immunodeficiency virus type 1 (HIV) Rev/RRE-dependent
gag
gene was used as a reporter to analyze various SNV sequences. Gag enzyme-linked immunosorbent assay and Western blot analyses reveal that HIV Gag production is enhanced at least 20,000-fold by the 5′ SNV LTR in COS, D17, and 293 cells. Furthermore, SNV RU5 in the sense but not the antisense orientation is sufficient to confer Rev/RRE-independent expression onto a cytomegalovirus-
gag
plasmid. In contrast, the SNV 3′ LTR and 3′ untranslated sequence between
env
and the LTR did not support Rev-independent
gag
expression. Quantitative RNase protection assays indicate that the SNV 5′ RNA terminus enhances cytoplasmic accumulation and polysome association of HIV unspliced and spliced transcripts. However, comparison of the absolute amounts of polysomal RNA indicates that polysome association is not sufficient to account for the significant increase in Gag production by the SNV sequences. Our analysis reveals that the SNV 5′ RNA terminus contains a unique
cis
-acting posttranscriptional control element that interacts with hypothetical cellular Rev-like proteins to facilitate HIV RNA transport and efficient translation.
Immortalization of CD4+and CD8+T Lymphocytes by Human T-Cell Leukemia Virus Type 1 Tax Mutants Expressed in a Functional Molecular Clone
Authors: Robek Michael D. , Ratner Lee 
Published: December 2019

ABSTRACTThe human T-cell leukemia virus type 1 (HTLV-1) transcriptionaltrans-activator Tax has been demonstrated to have transforming activity in multiple cell culture and transgenic-mouse models. In addition to activating transcription from the viral long terminal repeat (LTR) through the cyclic AMP response element binding protein/activating transcription factor (CREB/ATF) family of transcription factors, Tax activates the expression of multiple cellular promoters through the NF-κB pathway of transcriptional activation. The Tax mutants M22 and M47 have previously been demonstrated to selectively abrogate the ability of Tax to activate transcription through the NF-κB or CREB/ATF pathway, respectively. These mutations were introduced in thetaxgene of the ACH functional molecular clone of HTLV-1, and virus produced from the mutant ACH clones was examined for the ability to replicate and immortalize primary human lymphocytes. While virus derived from the clone containing the M47 mutation retained the ability to immortalize T lymphocytes, the M22 mutant lost the ability to immortalize infected cells. These results indicate that activation of the CREB/ATF pathway by Tax is dispensable for the immortalization of T cells by HTLV-1, whereas activation of the NF-κB pathway may be critical.
Identification of the Murine
Mx2
Gene: Interferon-Induced Expression of the Mx2 Protein from the Feral Mouse Gene Confers Resistance to Vesicular Stomatitis Virus
Authors: Jin Hee Kyung , Takada Ayato , Kon Yasuhiro , Haller Otto , Watanabe Tomomasa 
Published: December 2019

ABSTRACT

The mouse genome contains two related interferon-regulated genes,
Mx1
and
Mx2
. Whereas
Mx1
codes for the nuclear 72-kDa protein that interferes with influenza virus replication after interferon treatment, the
Mx2
gene is nonfunctional in all laboratory mouse strains examined, since its open reading frame (ORF) is interrupted by an insertional mutation and a subsequent frameshift mutation. In the present study, we demonstrate that
Mx2
mRNA of cells from feral mouse strains NJL (
Mus musculus musculus
) and SPR (
Mus spretus
) differs from that of the laboratory mouse strains tested. The
Mx2
mRNA of the feral strains contains a single long ORF consisting of 656 amino acids. We further show that Mx2 protein in the feral strains is expressed upon interferon treatment and localizes to the cytoplasm much like the rat Mx2 protein, which inhibits vesicular stomatitis virus replication. Furthermore, transfected 3T3 cell lines of laboratory mouse origin expressing
Mx2
from feral strains acquire slight resistance to vesicular stomatitis virus.
Natural Variation in Translational Activities of the 5′ Nontranslated RNAs of Hepatitis C Virus Genotypes 1a and 1b: Evidence for a Long-Range RNA-RNA Interaction outside of the Internal Ribosomal Entry Site
Authors: Honda Masao , Rijnbrand Rene , Abell Geoff , Kim Desok , Lemon Stanley M. 
Published: December 2019

ABSTRACT

The 5′ nontranslated RNA (5′NTR) of a genotype 1b hepatitis C virus (HCV-N) directs cap-independent translation of the HCV-N polyprotein with about twofold less efficiency than the 5′NTR of a genotype 1a virus under physiologic conditions (Hutchinson strain, or HCV-H) (M. Honda et al., Virology 222:31–42, 1996). Here, we show by mutational analysis that substitution of the AG dinucleotide sequence at nucleotides (nt) 34 and 35 of HCV-N with GA (present in HCV-H) restores the translational activity to that of the HCV-H 5′NTR both in vitro and in vivo. These nucleotides are located upstream of the minimal essential internal ribosome entry site (IRES), as a 6-nt deletion spanning nt 32 to 37 also increased the translational activity of the HCV-N 5′NTR to that of HCV-H. Thus, the upstream AG dinucleotide sequence has an inhibitory effect on IRES-directed translation. Surprisingly, however, this inhibitory effect was observed only when the translated, downstream RNA sequence contained nt 408 to 929 of HCV (capsid-coding RNA). Further analysis of RNA transcripts containing frameshift mutations demonstrated that the nucleotide sequence of the transcript, and not the amino acid sequence of the expressed capsid protein, determines this difference in translation efficiency. The difference between the translational activities of the HCV-N and HCV-H transcripts was increased when translation was carried out in reticulocyte lysates containing high K
+
concentrations, with a sevenfold difference evident at 130 to 150 mM K
+
. These results suggest that there is an RNA-RNA interaction involving 5′NTR and capsid-coding sequences flanking the IRES and that this is responsible for the reduced IRES activity of the genotype 1b virus, HCV-N.
Oligomerization within Virions and Subcellular Localization of Human Immunodeficiency Virus Type 1 Integrase
Authors: Petit Caroline , Schwartz Olivier , Mammano Fabrizio 
Published: December 2019

ABSTRACT
Previous biochemical and genetic evidence indicated that the functional form of retroviral integrase protein (IN) is a multimer. A direct demonstration of IN oligomerization during the infectious cycle was, however, missing, due to the absence of a sensitive detection method. We describe here the generation of infectious human immunodeficiency virus type 1 (HIV-1) viral clones carrying IN protein tagged with highly antigenic epitopes. In this setting, we could readily visualize IN both in producer cells and in viral particles. More interestingly, we detected IN oligomers, the formation of which was dependent on disulfide bridges and took place inside virions. Additionally, expression of a tagged HIV-1 IN in the absence of other viral components resulted in almost exclusive nuclear accumulation of the protein. Mutation of a conserved cysteine in the proposed dimer interface determined the loss of viral infectivity, associated with a reduction of IN oligomer formation and the redistribution of the mutated protein in the nucleus and cytoplasm. Epitope tagging of HIV-1 IN expressed alone or in the context of a replication-competent viral clone provides powerful tools to validate debated issues on the implication of this enzyme in different steps of the viral cycle.
Inhibition of Tumor Necrosis Factor Alpha by an Adenovirus-Encoded Soluble Fusion Protein Extends Transgene Expression in the Liver and Lung
Authors: Peng YuFeng , Trevejo Jose , Zhou JunLiang , Marino Michael W. , Crystal Ronald G. , Falck-Pedersen Erik , Elkon Keith B. 
Published: December 2019

ABSTRACTThe cellular and humoral immune responses to adenovirus (Ad) remain a major barrier to Ad-mediated gene therapy. We recently reported that mice deficient in tumor necrosis factor alpha (TNF-α) or Fas (APO-1, CD95) have prolonged expression of an Ad transgene expressing a foreign protein in the liver. To determine whether blockade of TNF-α or Fas would have the same effect in normal mice, we created transgenes that expressed soluble murine CD8 or CD8 fused to the extracellular regions of TNF receptor 1 (TNFR) or Fas and inserted into the left-end region of first-generation (E1/E3−) Ad vectors. Consistent with the results observed in TNF-deficient mice, expression of the TNFR-CD8 fusion protein was prolonged in vivo compared to that of control proteins. Not only did expression of TNFR-CD8 persist in the liver and the lung, but when coadministered with another first-generation vector, the protein provided “transprotection” for the companion vector and transgene. In addition, TNFR-CD8 attenuated the humoral immune response to the Ad. Together, these findings demonstrate that blockade of TNF-α is likely to be useful in extending the expression of an Ad-encoded transgene in a gene therapy application.