bluuebunny/crossref_metadata_2025_split huggingface dataset:
Replicative Functions of Minute Virus of Mice NS1 Protein Are Regulated In Vitro by Phosphorylation through Protein Kinase C
Authors: Nüesch Jürg P. F. , Dettwiler Sabine , Corbau Romuald , Rommelaere Jean 
Published: December 2019

ABSTRACT

NS1, the major nonstructural protein of the parvovirus minute virus of mice, is a multifunctional phosphoprotein which is involved in cytotoxicity, transcriptional regulation, and initiation of viral DNA replication. For coordination of these various functions during virus propagation, NS1 has been proposed to be regulated by posttranslational modifications, in particular phosphorylation. Recent in vitro studies (J. P. F. Nüesch, R. Corbau, P. Tattersall, and J. Rommelaere, J. Virol. 72:8002–8012, 1998) provided evidence that distinct NS1 activities, notably the intrinsic helicase function, are modulated by the phosphorylation state of the protein. In order to study the dependence of the initiation of viral DNA replication on NS1 phosphorylation and to identify the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on plasmid substrates containing the minimal left-end origins of replication. Cellular components necessary to drive NS1-dependent rolling-circle replication (RCR) were freed from endogenous serine/threonine protein kinases by affinity chromatography, and the eukaryotic DNA polymerases were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1
P
) supported RCR under these conditions, dephosphorylated NS1 (NS1
O
) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1
O
, are enriched in protein kinase C (PKC), and, when present together, reactivate NS1
O
for replication. One of these components, containing atypical PKC, was sufficient to restore NS1
O
helicase activity. The requirement of NS1
O
reactivation for characteristic PKC cofactors such as Ca
2+
/phosphatidylserine or phorbol esters strongly suggests the involvement of this protein kinase family in regulation of NS1 replicative functions in vitro.
A New System for Stringent, High-Titer Vesicular Stomatitis Virus G Protein-Pseudotyped Retrovirus Vector Induction by Introduction of Cre Recombinase into Stable Prepackaging Cell Lines
Authors: Arai Tohru , Matsumoto Kazuyuki , Saitoh Kanako , Ui Motoyasu , Ito Taiji , Murakami Masao , Kanegae Yumi , Saito Izumu , Cosset François-Loïc , Takeuchi Yasuhiro , Iba Hideo 
Published: December 2019

ABSTRACT

We report here on stable prepackaging cell lines which can be converted into packaging cell lines for high-titer vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors by the introduction of Cre recombinase-expressing adenovirus. The generated prepackaging cell lines constitutively express the
gag-pol
genes and contain an inducible transcriptional unit for the VSV-G gene. From this unit, the introduced Cre recombinase excised both a neomycin resistance (Neo
r
) gene and a poly(A) signal flanked by a tandem pair of
loxP
sequences and induced transcription of the VSV-G gene from the same promoter as had been used for Neo
r
expression. By inserting an mRNA-destabilizing signal into the 3′ untranslated region of the Neo
r
gene to reduce the amount of Neo
r
transcript, we were able efficiently to select the clones capable of inducing VSV-G at high levels. Without the introduction of Cre recombinase, these cell lines produce neither VSV-G nor any detectable infectious virus at all, even after the transduction of a murine leukemia virus-based retrovirus vector encoding β-galactosidase. They reproducibly produced high-titer virus stocks of VSV-G-pseudotyped retrovirus (1.0 × 10
6
infectious units/ml) from 3 days after the introduction of Cre recombinase. We also present evidence that VSV-G-producing cells are still fully susceptible to transduction by VSV-G pseudotypes. However, in this vector-producing system, which regulates VSV-G pseudotype production in an all-or-none manner, the integration of vector DNA into packaging cell lines would be minimized. We further show that heparin significantly inhibits retransduction of VSV-G pseudotypes in the culture fluids of packaging cell lines, leading to a two- to fourfold increase in the yield of the pseudotypes after induction. This vector-producing system was very stable and should be advantageous in human gene therapy.
Overexpression of Nonconvertible PrP

c

Δ114–121 in Scrapie-Infected Mouse Neuroblastoma Cells Leads to
trans
-Dominant Inhibition of Wild-Type PrP

Sc

Accumulation
Authors: Hölscher Christina , Delius Hajo , Bürkle Alexander 
Published: December 2019

ABSTRACT

One hallmark of prion diseases is the accumulation of the abnormal isoform PrP

Sc

of a normal cellular glycoprotein, PrP

c

, which is characterized by a high content of β-sheet structures and by its partial resistance to proteinase K. It was hypothesized that the PrP region comprising amino acid residues 109 to 122 [PrP(109–122)], which spontaneously forms amyloid when it is synthesized as a peptide but which does not display significant secondary structure in the context of the full-length PrP

c

molecule, should play a role in promoting the conversion into PrP

Sc

. By using persistently scrapie-infected mouse neuroblastoma (Sc
+
-MNB) cells as a model system for prion replication, we set out to design dominant-negative mutants of PrP

c

that are capable of blocking the conversion of endogenous, wild-type PrP

c

into PrP

Sc

. We constructed a deletion mutant (PrP

c

Δ114–121) lacking eight codons that span most of the highly amyloidogenic part, AGAAAAGA, of PrP(109–122). Transient transfections of mammalian expression vectors encoding either wild-type PrP

c

or PrP

c

Δ114–121 into uninfected mouse neuroblastoma cells (Neuro2a) led to overexpression of the respective PrP

c

versions, which proved to be correctly localized on the extracellular face of the plasma membrane. Transfection of Sc
+
-MNB cells revealed that PrP

c

Δ114–121 was not a substrate for conversion into a proteinase K-resistant isoform. Furthermore, its presence led to a significant reduction in the steady-state levels of PrP

Sc

derived from endogenous PrP

c

. Thus, we showed that the presence of amino acids 114 to 121 of mouse PrP

c

plays an important role in the conversion process of PrP

c

into PrP

Sc

and that a deletion mutant lacking these codons indeed behaves as a dominant-negative mutant with respect to PrP

Sc

accumulation. This mechanism could form a basis for a new gene therapy and/or a prevention concept for prion diseases.
The Application of a Homologous Recombination Assay Revealed Amino Acid Residues in an LTR-Retrotransposon That Were Critical for Integration
Authors: Atwood Angela , Choi Jeannie , Levin Henry L. 
Published: December 2019

ABSTRACT

Retroviruses and their relatives, the LTR-retrotransposons, possess an integrase protein (IN) that is required for the insertion of reverse transcripts into the genome of host cells.
Schizosaccharomyces pombe
is the host of Tf1, an LTR-retrotransposon with integration activity that can be studied by using techniques of yeast genetics. In this study, we sought to identify amino acid substitutions in Tf1 that specifically affected the integration step of transposition. In addition to seeking amino acid substitutions in IN, we also explored the possibility that other Tf1 proteins contributed to integration. By comparing the results of genetic assays that monitored both transposition and reverse transcription, we were able to seek point mutations throughout Tf1 that blocked transposition but not the synthesis of reverse transcripts. These mutant versions of Tf1 were candidates of elements that possessed defects in the integration step of transposition. Five mutations in Tf1 that resulted in low levels of integration were found to be located in the IN protein: two substitutions in the N-terminal Zn domain, two in the catalytic core, and one in the C-terminal domain. These results suggested that each of the three IN domains was required for Tf1 transposition. The potential role of these five amino acid residues in the function of IN is discussed. Two of the mutations that reduced integration mapped to the RNase H (RH) domain of Tf1 reverse transcriptase. The Tf1 elements with the RH mutations produced high levels of reverse transcripts, as determined by recombination and DNA blot analysis. These results indicated that the RH of Tf1 possesses a function critical for transposition that is independent of the accumulation of reverse transcripts.
Development and Characterization of an In Vivo Pathogenic Molecular Clone of Equine Infectious Anemia Virus
Authors: Cook R. Frank , Leroux Caroline , Cook Sheila J. , Berger Sandra L. , Lichtenstein Drew L. , Ghabrial Nadia N. , Montelaro Ronald C. , Issel Charles J. 
Published: December 2019

ABSTRACT

An infectious nonpathogenic molecular clone (19-2-6A) of equine infectious anemia virus (EIAV) was modified by substitution of a 3.3-kbp fragment amplified by PCR techniques from a pathogenic variant (EIAV
PV
) of the cell culture-adapted strain of EIAV (EIAV
PR
). This substitution consisted of coding sequences for 77 amino acids at the carboxyl terminus of the integrase, the S1 (encoding the second exon of
tat
), S2, and S3 (encoding the second exon of
rev
) open reading frames, the complete
env
gene (including the first exon of
rev
), and the 3′ long terminal repeat (LTR). Modified 19-2-6A molecular clones were designated EIAV
PV3.3
, and infection of a single pony (678) with viruses derived from a mixture of five of these molecular clones induced clinical signs of acute equine infectious anemia (EIA) at 23 days postinfection (dpi). As a consequence of this initial study, a single molecular clone, EIAV
PV3.3#3
(redesignated EIAV
UK
), was selected for further study and inoculated into two ponies (613 and 614) and two horses (700 and 764). Pony 614 and the two horses developed febrile responses by 12 dpi, which was accompanied by a 48 to 64% reduction in platelet number, whereas pony 613 did not develop fever (40.6°C) until 76 dpi. EIAV could be isolated from the plasma of these animals by 5 to 7 dpi, and all became seropositive for antibodies to this virus by 21 dpi. Analysis of the complete nucleotide sequence demonstrated that the 3.3-kbp 3′ fragment of EIAV
UK
differed from the consensus sequence of EIAV
PV
by just a single amino acid residue in the second exon of the rev gene. Complete homology with the EIAV
PV
consensus sequence was observed in the hypervariable region of the LTR. However, EIAV
UK
was found to contain an unusual 68-bp nucleotide insertion/duplication in a normally conserved region of the LTR sequence. These results demonstrate that substitution of a 3.3-kbp fragment from the EIAV
PV
strain into the infectious nonpathogenic molecular clone 19-2-6A leads to the production of progeny virus particles with the ability to induce clinical signs of EIA. Therefore, EIAV
UK
, which is the first pathogenic, cell culture-adapted molecular clone of EIAV to be described, should be of value in identifying viral determinants of pathogenicity.
A Vaccinia Virus Late Transcription Factor Copurifies with a Factor That Binds to a Viral Late Promoter and Is Complemented by Extracts from Uninfected HeLa Cells
Authors: Wright Cynthia F. , Hubbs Alan E. , Gunasinghe Sajeevani K. , Oswald Betty W. 
Published: December 2019

ABSTRACT
We have previously described a vaccinia virus late transcription factor, VLTF-X, which we found to be present in cells at early and late times in infection. In this study, transcription complementation assays were used to demonstrate that VLTF-X activity is also present in virion extracts and in the cytoplasm of uninfected HeLa cells. Mobility shift assays performed on various VLTF-X preparations revealed that a late promoter DNA-binding activity cochromatographed and cosedimented with VLTF-X activity. Competition experiments demonstrated that this binding was specific for the late promoter region of the probe and that late transcription was dramatically reduced by an oligonucleotide that blocked factor-DNA complex formation but was only minimally affected by an oligonucleotide that did not inhibit complex formation. These results suggest that a cellular factor may participate in vaccinia virus late transcription. These findings also confirm the requirement for VLTF-X and distinguish it from any of the previously described vaccinia virus late transcription factors, which have all been mapped to the viral genome. Finally, these studies also suggest that the biochemical role for VLTF-X may be in late promoter recognition.
Moesin Is Not a Receptor for Measles Virus Entry into Mouse Embryonic Stem Cells
Authors: Doi Yoshinori , Kurita Mitsue , Matsumoto Misako , Kondo Takahisa , Noda Tetsuo , Tsukita Sachiko , Tsukita Shoichiro , Seya Tsukasa 
Published: December 2019

ABSTRACTThe involvement of moesin in measles virus (MV) entry was investigated with moesin-positive and -negative mouse embryonic stem (ES) cells. MV infection of these cells was very ineffective and was independent of moesin expression. Furthermore, when these cells were transfected to express human CD46, a 100-fold increase in syncytium formation was observed with these cells and was independent of the expression of moesin. The only obvious difference between moesin-positive and -negative ES cells was the shape of the syncytia formed. Moesin-negative ES cells expressing or not expressing human CD46 formed separate pieces of fragmented syncytia which were torn apart during spreading, whereas ES cells expressing moesin exhibited typical syncytia. In addition, moesin was not detected on the surface of any murine cells or cell lines that we have tested by a flow cytometric assay with moesin-specific antibodies. These findings indicate that murine moesin is neither a receptor nor a CD46 coreceptor for MV entry into mouse ES cells. Moesin is involved in actin filament-plasma membrane interactions as a cross-linker, and it affects only the spreading and shape of MV-mediated syncytia.
Functional Analysis of Ground Squirrel Hepatitis Virus Enhancer II
Authors: Fourel Geneviève , Ringeisen François , Flajolet Marc , Tiollais Pierre , Buendia Marie Annick 
Published: December 2019

ABSTRACT
We have characterized a major regulatory element of ground squirrel hepatitis virus (GSHV) located within a 90-nucleotide fragment of the core promoter upstream sequences and have compared its organization to that of woodchuck hepatitis virus (WHV) enhancer II (We2). The GSHV element (Ge2) stimulates transcription from the viral core promoter and heterologous promoters in an orientation-independent manner but displays a lower level of activity than We2 in transient transfection assays in human hepatoma cells. The general organization of Ge2 into binding sites for the liver-enriched HNF-1 and HNF-4 proteins and for ubiquitous factors of the NF1 and Oct families was found to be mostly conserved with respect to the homologous We2 region. Accordingly, transactivation by HNF-1 and HNF-4 plays an essential role in the liver-specific transcriptional activity of both the GSHV and WHV core promoters. Distinctive features of the GSHV enhancer consist of its ability to bind C/EBP family factors in a central motif that overlaps with one of the two HNF-4 sites and its differential binding affinities for HNF-4.
Visna Virus dUTPase Is Dispensable for Neuropathogenicity
Authors: Pétursson Gudmundur , Turelli Priscilla , Matthíasdóttir Sigrídur , Georgsson Gudmundur , Andrésson Ólafur S. , Torsteinsdóttir Sigurbjörg , Vigne Robert , Andrésdóttir Valgerdur , Gunnarsson Eggert , Agnarsdóttir Gudrún , Quérat Gilles 
Published: December 2019

ABSTRACT
The major part of the dUTPase-encoding region of the visna virus genome was deleted. Intracerebral injection of the mutant virus resulted in a somewhat reduced viral load compared to that resulting from injection of the wild type, especially in the lungs, but the neuropathogenic effects were comparable. The dUTPase gene is dispensable for induction of lesions in the brain.
Enzymatic Characterization of Refolded Human Rhinovirus Type 14 2A Protease Expressed in
Escherichia coli
Authors: Wang Q. May , Johnson Robert B. , Cox Gregory A. , Villarreal Elcira C. , Churgay Lisa M. , Hale John E. 
Published: December 2019

ABSTRACT

Reported here is the production of recombinant human rhinovirus 14 (HRV14) 2A protease from bacterial cells transformed with a heat-inducible plasmid containing the HRV14 2A cDNA sequence. Overexpressed 2A protein partitioned into the inclusion bodies was solubilized in urea and then refolded in the presence of Zn
2+
. Transition metals were required for the restoration of 2A protease activity as a structural component, but appeared to be inhibitory if added exogenously once the enzyme was refolded. Based on the cleavage specificity studies, a colorimetric assay was developed for the highly purified HRV14 2A protease. A peptide with the sequence RKGDIKSY–
p
-nitroanilide was found to be cleaved by the 2A protease with a
k
cat
/

K
m

ratio of ∼335 M
−1
s
−1
, which allows its activity to be measured continuously with a spectrophotometer or a microplate reader.
Cloning and Characterization of a Novel Hepatitis B Virus x Binding Protein That Inhibits Viral Replication
Authors: Melegari Margherita , Scaglioni Pier Paolo , Wands Jack R. 
Published: December 2019

ABSTRACT
The hepatitis B virus and the mammalian hepadnavirus genomes encode for a short open reading frame called x. Expression of the protein product (HBx) appears necessary for establishment of natural infection. However, in vitro studies have suggested a multifunctional role for HBx as an indirect transcriptional transactivator of a variety of different viral and cellular promoters. Indeed, HBx has no known direct DNA binding properties but may interact with transcription factors as well as activate intracellular signaling pathways associated with cell growth. To further address the possible functional role of HBx in the life cycle of hepatitis B virus, we performed an analysis using the yeast two-hybrid system to screen a cDNA library derived from a hepatocellular carcinoma cell line with a HBx fusion bait in an attempt to identify cellular partners that may bind to and alter the biologic properties of HBx. A HBx-interacting protein that specifically complexes with the carboxy terminus of wild-type HBx was identified and designated XIP. This 9.6-kDa protein is capable of binding to HBx in vitro, and transient and stable expression in hepatocellular carcinoma cells abolishes the transactivation properties of HBx on luciferase constructs driven by AP-1 and endogenous hepatitis B virus enhancer/promoter elements. Investigation of the role of XIP in hepatitis B virus replication in differentiated hepatocellular carcinoma cells revealed that XIP expression reduces wild-type hepatitis B virus replication to levels observed following transfection with an HBx-minus virus. In contrast, the replication levels of the duck hepatitis B virus, a hepadnavirus that lacks the x open reading frame, were unchanged in the context of XIP expression. We propose that one of the physiologic functions of the cellular protein XIP is to negatively regulate HBx activity and thus to alter the replication life cycle of the virus.
Effect of Passive Immunization or Maternally Transferred Immunity on the Antibody Response to a Genetic Vaccine to Rabies Virus
Authors: Wang Yijie , Xiang Zhiquan , Pasquini Susanna , Ertl Hildegund C. J. 
Published: December 2019

ABSTRACTA plasmid vector, termed pSG5rab.gp, expressing the glycoprotein of rabies virus was tested in young adult or neonatal mice in the presence of maternally transferred immunity or passively administered antibodies to rabies virus for induction of an antibody response. Mice born to rabies virus-immune dams developed an impaired antibody response to genetic immunization at 6 weeks of age, as had been previously observed upon vaccination with an inactivated viral vaccine. Similarly, mice passively immunized with hyperimmune serum showed an inhibited B-cell response upon vaccination with the pSG5rab.gp vector, resulting in both cases in vaccine failures upon challenge with a virulent strain of rabies virus. In contrast, the immune responses of mice vaccinated as neonates in the presence of maternal immunity or upon passive immunization to rabies virus with the pSG5rab.gp construct were only marginally affected.
Polyadenylation of Vesicular Stomatitis Virus mRNA Dictates Efficient Transcription Termination at the Intercistronic Gene Junctions
Authors: Hwang Leroy N. , Englund Nathan , Pattnaik Asit K. 
Published: December 2019

ABSTRACT

The intercistronic gene junctions of vesicular stomatitis virus (VSV) contain conserved sequence elements that are important for polyadenylation and transcription termination of upstream transcript as well as reinitiation of transcription of downstream transcript. To examine the role of the putative polyadenylation signal 3′AUACU
7
5′ at the gene junctions in polyadenylation and transcription termination, we constructed plasmids encoding antigenomic minireplicons containing one or two transcription units. In plasmid-transfected cells, analyses of the bicistronic minireplicon containing the wild-type or mutant intercistronic gene junctions for the ability to direct synthesis of polyadenylated upstream, downstream, and readthrough mRNAs showed that the AUACU
7
sequence element is required for polyadenylation of VSV mRNA. Deletion of AUAC or U
7
resulted in templates that did not support polyadenylation of upstream mRNA. Interestingly, we found that the loss of polyadenylation function led to antitermination of the upstream transcript and resulted in a readthrough transcript that contained the upstream and downstream mRNA sequences. Mutations that blocked polyadenylation also blocked transcription termination and generated mostly readthrough transcript. Reverse transcription-PCR of readthrough transcripts and subsequent nucleotide sequencing of the amplified product revealed no extra adenosine residues at the junction of the readthrough transcript. These results indicate that polyadenylation is required for transcription termination of VSV mRNA. The intergenic dinucleotide GA did not appear to be necessary for transcription termination. Furthermore, we found that insertion of the polyadenylation signal sequence AUACU
7
alone was sufficient to direct polyadenylation and efficient transcription termination at the inserted site. Taken together, the data presented here support the conclusion that polyadenylation is the major determinant of transcription termination at the intercistronic gene junctions of VSV.
Extended Minus-Strand DNA as Template for R-U5-Mediated Second-Strand Transfer in Recombinational Rescue of Primer Binding Site-Modified Retroviral Vectors
Authors: Mikkelsen Jacob Giehm , Lund Anders H. , Dybkær Karen , Duch Mogens , Pedersen Finn Skou 
Published: December 2019

ABSTRACT
We have previously demonstrated recombinational rescue of primer binding site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen et al., J. Virol. 70:1439–1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of different structures, suggesting that PBS knockout vectors may be rescued through initial priming on endogenous virus RNA, read-through of the mutated PBS during minus-strand synthesis, and subsequent second-strand transfer mediated by the R-U5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms for R-U5-mediated second-strand transfer and its possible role in retrovirus replication and evolution are discussed.