bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutational Analysis of Influenza A Virus Nucleoprotein: Identification of Mutations That Affect RNA Replication
Authors: Mena Ignacio , Jambrina Enrique , Albo Carmen , Perales Beatriz , Ortín Juan , Arrese Marta , Vallejo Dolores , Portela Agustín 
Published: December 2019

ABSTRACT
The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis.
Mechanism of Suppression of Hepatitis B Virus Precore RNA Transcription by a Frequent Double Mutation
Authors: Li Jie , Buckwold Victor E. , Hon Man-wai , Ou Jing-hsiung 
Published: December 2019

ABSTRACT
A double mutation which converts nucleotide 1765 from A to T and nucleotide 1767 from G to A is frequently found in the hepatitis B virus (HBV) genome isolated from HBV patients with chronic hepatitis symptoms. This double mutation is located in the core promoter that controls the transcription of the precore RNA and the core RNA. In addition, this double mutation also resides in the X protein coding sequence, converting codon 130 from Lys to Met and codon 131 from Val to Ile. Previous studies indicate that this double mutation removes a nuclear receptor binding site in the core promoter, suppresses specifically precore RNA transcription, and enhances viral replication. In this study, we further investigated how this double mutation suppresses precore RNA transcription. We found that this double mutation not only removed the nuclear receptor binding site but also created an HNF1 transcription factor binding site. Further transfection studies using Huh7 hepatoma cells indicate that the removal of the nuclear receptor binding site has no effect on the transcription of HBV RNAs, the two-codon change in the X protein sequence suppresses the transcription of both precore and core RNAs, and the creation of the HNF1 binding site restores the core RNA level. Hence, the specific suppression of precore RNA transcription by this frequent double-nucleotide mutation is the combined result of multiple factors.
Human T-Cell Leukemia Virus Type 1 Tax Protein Abrogates Interleukin-2 Dependence in a Mouse T-Cell Line
Authors: Iwanaga Youichi , Tsukahara Tomonori , Ohashi Takashi , Tanaka Yuetsu , Arai Masaaki , Nakamura Masataka , Ohtani Kiyoshi , Koya Yoshihiro , Kannagi Mari , Yamamoto Naoki , Fujii Masahiro 
Published: December 2019

ABSTRACTHuman T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia. Tax, the viral protein, is thought to be crucial in the development of the disease, since it transforms healthy T cells in vitro and induces tumors in transgenic animals. We examined the effect of Tax activity on the growth of the interleukin-2 (IL-2)-dependent T-cell line CTLL-2. Stable expression of Tax in CTLL-2 transformed cell growth from being IL-2 dependent to IL-2 independent. Tax stimulated transcription through NF-κB and the cyclic AMP-responsive element-like sequence in the HTLV-1 promoter. The finding of Tax mutants segregating these two pathways suggested that the NF-κB pathway was essential for IL-2-independent growth of CTLL-2 cells while the CRE pathway was unnecessary. However, both pathways were necessary for another transformation-related activity (colony formation in soft agar) of CTLL-2/Tax. Our results show that Tax has at least two distinct activities on T cells, and suggest that Tax plays a crucial role in IL-2-independent T-cell transformation induced by HTLV-1, in addition to its well-known IL-2-dependent cell transformation.
Identification of Mutations Contributing to the Temperature-Sensitive, Cold-Adapted, and Attenuation Phenotypes of the Live-Attenuated Cold-Passage 45 (
cp
45) Human Parainfluenza Virus 3 Candidate Vaccine
Authors: Skiadopoulos Mario H. , Surman Sonja , Tatem Joanne M. , Paschalis Maribel , Wu Shin-Lu , Udem Stephen A. , Durbin Anna P. , Collins Peter L. , Murphy Brian R. 
Published: December 2019

ABSTRACT

The live-attenuated human parainfluenza virus 3 (PIV3) cold-passage 45 (
cp
45) candidate vaccine was shown previously to be safe, immunogenic, and phenotypically stable in seronegative human infants. Previous findings indicated that each of the three amino acid substitutions in the L polymerase protein of
cp
45 independently confers the temperature-sensitive (
ts
) and attenuation (
att
) phenotypes but not the cold-adaptation (
ca
) phenotype (29).
cp
45 contains 12 additional potentially important point mutations in other proteins (N, C, M, F, and hemagglutinin-neuraminidase [HN]) or in
cis
-acting sequences (the leader region and the transcription gene start [GS] signal of the N gene), and their contribution to these phenotypes was undefined. To further characterize the genetic basis for the
ts
,
ca
, and
att
phenotypes of this promising vaccine candidate, we constructed, using a reverse genetics system, a recombinant
cp
45 virus that contained all 15
cp
45-specific mutations mentioned above, and found that it was essentially indistinguishable from the biologically derived
cp
45 on the basis of plaque size, level of temperature sensitivity, cold adaptation, level of replication in the upper and lower respiratory tract of hamsters, and ability to protect hamsters from subsequent wild-type PIV3 challenge. We then constructed recombinant viruses containing the
cp
45 mutations in individual proteins as well as several combinations of mutations. Analysis of these recombinant viruses revealed that multiple
cp
45 mutations distributed throughout the genome contribute to the
ts
,
ca
, and
att
phenotypes. In addition to the mutations in the L gene, at least one other mutation in the 3′ N region (i.e., including the leader, N GS, and N coding changes) contributes to the
ts
phenotype. A recombinant virus containing all the
cp
45 mutations except those in L was more
ts
than
cp
45, illustrating the complex nature of this phenotype. The
ca
phenotype of
cp
45 also is a complex composite phenotype, reflecting contributions of at least three separate genetic elements, namely, mutations within the 3′ N region, the L protein, and the C-M-F-HN region. The
att
phenotype is a composite of both
ts
and non-
ts
mutations. Attenuating
ts
mutations are located in the L protein, and non-
ts
attenuating mutations are located in the C and F proteins. The presence of multiple
ts
and non-
ts
attenuating mutations in
cp
45 likely contributes to the high level of attenuation and phenotypic stability of this promising vaccine candidate.
Mutational Analysis of the Avian Adenovirus CELO, Which Provides a Basis for Gene Delivery Vectors
Authors: Michou Anne-Isabelle , Lehrmann Heike , Saltik Mediyha , Cotten Matt 
Published: December 2019

ABSTRACT

The avian adenovirus CELO is being developed as a gene transfer tool. Using homologous recombination in
Escherichia coli
, the CELO genome was screened for regions that could be deleted and would tolerate the insertion of a marker gene (luciferase or enhanced green fluorescent protein). For each mutant genome, the production of viable virus able to deliver the transgene to target cells was monitored. A series of mutants in the genome identified a set of open reading frames that could be deleted but which must be supplied in
trans
for virus replication. A region of the genome which is dispensable for viral replication and allows the insertion of an expression cassette was identified and a vector based on this mutation was evaluated as a gene delivery reagent. Transduction of avian cells occurs at 10- to 100-fold greater efficiency (per virus particle) than with an adenovirus type 5 (Ad5)-based vector carrying the same expression cassette. Most important for gene transfer applications, the CELO vector transduced mammalian cells as efficiently as an Ad5 vector. The CELO vector is exceptionally stable, can be grown inexpensively in chicken embryos, and provides a useful alternative to Ad5-based vectors.
Endoproteolytic Processing of the Ebola Virus Envelope Glycoprotein: Cleavage Is Not Required for Function
Authors: Wool-Lewis Rouven J. , Bates Paul 
Published: December 2019

ABSTRACT

Proteolytic processing is required for the activation of numerous viral glycoproteins. Here we show that the envelope glycoprotein from the Zaire strain of Ebola virus (Ebo-GP) is proteolytically processed into two subunits, GP
1
and GP
2
, that are likely covalently associated through a disulfide linkage. Murine leukemia virions pseudotyped with Ebo-GP contain almost exclusively processed glycoprotein, indicating that this is the mature form of Ebo-GP. Mutational analysis identified a dibasic motif, reminiscent of furin-like protease processing sites, as the Ebo-GP cleavage site. However, analysis of Ebo-GP processing in LoVo cells that lack the proprotein convertase furin demonstrated that furin is not required for processing of Ebo-GP. In sharp contrast to other viral systems, we found that an uncleaved mutant of Ebo-GP was able to mediate infection of various cell lines as efficiently as the wild-type, proteolytically cleaved glycoprotein, indicating that cleavage is not required for the activation of Ebo-GP despite the conservation of a dibasic cleavage site in all filoviral envelope glycoproteins.
Differential Effects of Human Immunodeficiency Virus Isolates on β-Chemokine and Gamma Interferon Production and on Cell Proliferation
Authors: Greco Giampaolo , Fujimura Sue H. , Mourich Dan V. , Levy Jay A. 
Published: December 2019

ABSTRACTAll human immunodeficiency virus (HIV) isolates can grow readily in primary CD4+T cells, but they can be distinguished by their ability to replicate in macrophages and established T-cell lines. The macrophage-tropic viruses are generally non-syncytium inducing (NSI), whereas the T-cell-line-tropic viruses are syncytium inducing (SI) in cultured cells. We now demonstrate that infection of CD4+T cells by NSI and SI viruses shows a differential effect on production of β-chemokines and gamma interferon. Infection by NSI viruses increased production of MIP-1α, MIP-1β, and gamma interferon, whereas infection by SI viruses had no effect or decreased production of these cytokines. Production of RANTES was slightly increased during infection by both virus phenotypes. This differential effect of NSI and SI viruses was observed at the level of β-chemokine mRNA as well as at the level of protein expression. Infection by NSI viruses also increased CD4+cell proliferation. These results may have relevance for a differential role of HIV strains in AIDS pathogenesis.
Induction of B-Cell Lymphoma in BALB/c Nude Mice with an Ecotropic, B-Tropic Helper Virus Present in the Murine AIDS Virus Stock
Authors: Tayar Lamin , Higo Kyoko , Kubo Yoshinao , Wang Yun , Lu Ling-Min , Zhang Fengmin , Iwatani Yasumasa , Wang Ling , Ono Takeshi , Maeda Michiyuki , Sakai Hiroyuki , Ishimoto Akinori 
Published: December 2019

ABSTRACTThe pathogenicities of the murine AIDS (MAIDS) virus complex (LP-BM5) and ecotropic helper virus (BM5eco) isolated from the complex to BALB/c nude mice were studied to elucidate the possible role of replication-competent helper virus in inducing the monoclonal outgrowth of lymphoid cells. Neither LP-BM5 nor BM5eco was pathogenic in adult BALB/c nude mice. However, B-cell lymphoma developed with a very high frequency when either virus was inoculated into newborn BALB/c nude (nu/nu) mice. The cells from the B-cell lymphoma were easily transplanted into nude mice. These results suggested that ecotropic helper virus in the MAIDS virus complex plays an important role in inducing the monoclonal outgrowth of lymphoid cells under immunodeficient conditions caused by defective virus.
Exponential Fitness Gains of RNA Virus Populations Are Limited by Bottleneck Effects
Authors: Novella Isabel S. , Quer Josep , Domingo Esteban , Holland John J. 
Published: December 2019

ABSTRACT
Fitness is a parameter that quantitatively measures adaptation of a virus to a given environment. We have previously reported exponential fitness gains of large populations of vesicular stomatitis virus replicating in a constant environment (I. S. Novella et al., Proc. Natl. Acad. Sci. USA 92:5841–5844, 1995). In this paper, we report that during long-term passage of such large viral populations, fitness values reached a high-fitness plateau during which stochastic fitness variations were observed. This effect appears likely to be due to bottleneck effects on very high fitness populations.
Nonstructural C Protein Is Required for Efficient Measles Virus Replication in Human Peripheral Blood Cells
Authors: Escoffier Carine , Manié Serge , Vincent Séverine , Muller Claude P. , Billeter Martin , Gerlier Denis 
Published: December 2019

ABSTRACTThe P gene of measles virus (MV) encodes the phosphoprotein, a component of the virus ribonucleoprotein complex, and two nonstructural proteins, C and V, with unknown functions. Growth of recombinant MV, defective in C or V expression, was explored in human peripheral blood mononuclear cells (PBMC). The production of infectious recombinant MV V−was comparable to that of parental MV tag in simian Vero fibroblasts and in PBMC. In contrast, MV C−progeny was strongly reduced in PBMC but not in Vero cells. Consistently, the expression of both hemagglutinin and fusion proteins, as well as that of nucleoprotein mRNA, was lower in MV C−-infected PBMC. Thus, efficient replication of MV in natural host cells requires the expression of the nonstructural C protein. The immunosuppression that accompanies MV infection is associated with a decrease in the in vitro lymphoproliferative response to mitogens. MV C−was as potent as MV tag or MV V−in inhibiting the phytohemagglutinin-induced proliferation of PBMC, indicating that neither the C protein nor the V protein is directly involved in this effect.
Polyomavirus Large T Antigen Binds the Transcriptional Coactivator Protein p300
Authors: Nemethova Maria , Wintersberger Erhard 
Published: December 2019

ABSTRACT

Using coimmunoprecipitation and glutathione
S
-transferase pulldown experiments, we found that polyomavirus large T antigen binds to p300 in vivo and in vitro. The N-terminal region of the viral protein, including the pRB binding motif, was dispensable for this interaction, which involved several regions within the C-terminal half of the large T antigen. Interestingly, anti-T antibody coimmunoprecipitated a subspecies of p300 which has high histone acetyltransferase activity.
Human Adeno-Associated Virus Type 5 Is Only Distantly Related to Other Known Primate Helper-Dependent Parvoviruses
Authors: Bantel-Schaal Ursula , Delius Hajo , Schmidt Rainer , zur Hausen Harald 
Published: December 2019

ABSTRACT
We have characterized 95% (4,404 nucleotides) of the genome of adeno-associated virus type 5 (AAV5), including part of the terminal repeats and the terminal resolution site. Our results show that AAV5 is different from all other described AAV serotypes at the nucleotide level and at the amino acid level. The sequence homology to AAV2, AAV3B, AAV4, and AAV6 at the nucleotide level is only between 54 and 56%. The positive strand contains two large open reading frames (ORFs). The left ORF encodes the nonstructural (Rep) proteins, and the right ORF encodes the structural (Cap) proteins. At the amino acid level the identities with the capsid proteins of other AAVs range between 51 and 59%, with a high degree of heterogeneity in regions which are considered to be on the exterior surface of the viral capsid. The overall identity for the nonstructural Rep proteins at the amino acid level is 54.4%. It is lowest at the C-terminal 128 amino acids (10%). There are only two instead of the common three putative Zn fingers in the Rep proteins. The Cap protein data suggest differences in capsid surfaces and raise the possibility of a host range distinct from those of other parvoviruses. This may have important implications for AAV vectors used in gene therapy.
Dual Stem Loops within the Poliovirus Internal Ribosomal Entry Site Control Neurovirulence
Authors: Gromeier Matthias , Bossert Birgit , Arita Mineo , Nomoto Akio , Wimmer Eckard 
Published: December 2019

ABSTRACT

In the human central nervous system, susceptibility to poliovirus (PV) infection is largely confined to a specific subpopulation of neuronal cells. PV tropism is likely to be determined by cell-external components such as the PV receptor CD155, as well as cell-internal constraints such as the availability of a suitable microenvironment for virus propagation. We reported previously that the exchange of the cognate internal ribosomal entry site (IRES) within the 5′ nontranslated region of PV with its counterpart from human rhinovirus type 2 (HRV2) can eliminate the neuropathogenic phenotype in a transgenic mouse model for poliomyelitis without diminishing the growth properties in HeLa cells. We now show that attenuation of neurovirulence of PV/HRV2 chimeras is not confined to CD155 transgenic mice but is evident also after intraspinal inoculation into
Cynomolgus
monkeys. We have dissected the PV and HRV2 IRES elements to determine those structures responsible for neurovirulence (or attenuation) of these chimeric viruses. We report that two adjacent stem loop structures within the IRES cooperatively determine neuropathogenicity.
Epstein-Barr Virus Nuclear Antigen 1 Sequences in Endemic and Sporadic Burkitt’s Lymphoma Reflect Virus Strains Prevalent in Different Geographic Areas
Authors: Habeshaw G. , Yao Q. Y. , Bell A. I. , Morton D. , Rickinson A. B. 
Published: December 2019

ABSTRACT
The Epstein-Barr virus (EBV) nuclear antigen EBNA1 is the only viral protein detectably expressed in virus genome-positive Burkitt’s lymphoma (BL); recent work has suggested that viral strains with particular EBNA1 sequence changes are preferentially associated with this tumor and that, within a patient, the tumor-associated variant may have arisen de novo as a rare mutant of the dominant preexisting EBV strain (K. Bhatia, A. Raj, M. J. Gutierrez, J. G. Judde, G. Spangler, H. Venkatesh, and I. T. Magrath, Oncogene 13:177–181, 1996). In the present work we first study 12 BL patients and show that the virus strain in the tumor is identical in EBNA1 sequence and that it is matched at several other polymorphic loci to the dominant strain rescued in vitro from the patient’s normal circulating B cells. We then analyze BL-associated virus strains from three different geographic areas (East Africa, Europe, and New Guinea) alongside virus isolates from geographically matched control donors by using sequence changes in two separate regions of the EBNA1 gene (N-terminal codons 1 to 60 and C-terminal codons 460 to 510) to identify the EBNA1 subtype of each virus. Different geographic areas displayed different spectra of EBNA1 subtypes, with only limited overlap between them; even type 2 virus strains, which tended to be more homogeneous than their type 1 counterparts, showed geographic differences at the EBNA1 locus. Most importantly, within any one area the EBNA1 subtypes associated with BL were also found to be prevalent in the general population. We therefore find no evidence that Burkitt lymphomagenesis involves a selection for EBV strains with particular EBNA1 sequence changes.
The Hypervariable Domain of the Murine Leukemia Virus Surface Protein Tolerates Large Insertions and Deletions, Enabling Development of a Retroviral Particle Display System
Authors: Kayman Samuel C. , Park Han , Saxon Maya , Pinter Abraham 
Published: December 2019

ABSTRACT
The surface proteins (SU) of murine type-C retroviruses have a central hypervariable domain devoid of cysteine and rich in proline. This 41-amino-acid region of Friend ecotropic murine leukemia virus SU was shown to be highly tolerant of insertions and deletions. Viruses in which either the N-terminal 30 amino acids or the C-terminal 22 amino acids of this region were replaced by the 7-amino-acid sequence ASAVAGA were fully infectious. Insertions of this 7-amino-acid sequence at the N terminus, center, and the C terminus of the hypervariable domain had little effect on envelope protein (Env) function, while this insertion at a position 10 amino acids following the N terminus partially destabilized the association between the SU and transmembrane subunits of Env. Large, complex domains (either a 252-amino-acid single-chain antibody binding domain [scFv] or a 96-amino-acid V1/V2 domain of HIV-1 SU containing eight N-linked glycosylation sites and two disulfides) did not interfere with Env function when inserted in the center or C-terminal portions of the hypervariable domain. The scFv domain inserted into the C-terminal region of the hypervariable domain was shown to mediate binding of antigen to viral particles, demonstrating that it folded into the active conformation and was displayed on the surface of the virion. Both positive and negative enrichment of virions expressing the V1/V2 sequence were achieved by using a monoclonal antibody specific for a conformational epitope presented by the inserted sequence. These results indicated that the hypervariable domain of Friend ecotropic SU does not contain any specific sequence or structure that is essential for Env function and demonstrated that insertions into this domain can be used to extend particle display methodologies to complex protein domains that require expression in eukaryotic cells for glycosylation and proper folding.