bluuebunny/crossref_metadata_2025_split huggingface dataset:
Zinc-binding properties of Junín virus nucleocapsid protein
Authors: Tortorici M. Alejandra , Ghiringhelli P. Daniel , Lozano Mario E. , Albariño César G. , Romanowski Víctor 
Published: August 2015

The arenavirus nucleocapsid protein (N) is a highly basic 63 kDa protein with a dual function during the virus life-cycle. First, it is involved in essential steps of genome replication, promoting the synthesis of the full-length antigenomic copy of S RNA, and second it associates with the genomic RNA to form the nucleocapsid. We have expressed the N protein of Junín virus inE. coliand shown that it binds zincin vitro. This property is in agreement with the presence in the carboxy-terminal region of the N protein of the CX2HX23CX4C sequence, which resembles a classical zinc-finger motif. The specificity for zinc binding was demonstrated by competition with other divalent metal ions. The ability of the predicted motif to bind zinc was established by analysis of a series of N mutants, including truncated variants and amino acid substitutions. In addition, alternative zinc-binding sites were found.
RNA-binding properties of the 63 kDa protein encoded by the triple gene block of poa semilatent hordeivirus
Authors: Kalinina N. O. , Rakitina D. A. , Yelina N. E. , Zamyatnin A. A. , Stroganova T. A. , Klinov D. V. , Prokhorov V. V. , Ustinova S. V. , Chernov B. K. , Schiemann J. , Solovyev A. G. , Morozov S. Yu. 
Published: August 2015

The 63 kDa ‘63K’ movement protein encoded by the triple gene block of poa semilatent virus (PSLV) comprises the C-terminal NTPase/helicase domain and the N-terminal extension domain, which contains two positively charged sequence motifs, A and B. In this study, the in vitro RNA-binding properties of PSLV 63K and its mutants were analysed. Membrane-immobilized 63K and N-63K (isolated N-terminal extension domain) bound RNA at high NaCl concentrations. In contrast, C-63K (isolated NTPase/helicase domain) was able to bind RNA only at NaCl concentrations of up to 50 mM. In gel-shift assays, C-63K bound RNA to form complexes that were unable to enter an agarose gel, whereas complexes formed by N-63K could enter the gel. Full-length 63K formed both types of complexes. Visualization of the RNA–protein complexes formed by 63K, N-63K and C-63K by atomic force microscopy demonstrated that each complex had a different shape. Collectively, these data indicate that 63K has two distinct RNA-binding activities associated with the NTPase/helicase domain and the N-terminal extension domain. Mutations in either of the positively charged sequence motifs A and B had little effect on the RNA binding of the N-terminal extension domain, whereas mutations in both motifs together inhibited RNA binding. Hybrid viruses with mutations in motifs A and B were able to infect inoculated leaves of Nicotiana benthamiana plants, but were unable to move systemically to uninoculated leaves, suggesting that the RNA-binding activity of the N-terminal extension domain of PSLV 63K is associated with virus long-distance movement.
Establishment of an infectious RNA transcription system for Striped jack nervous necrosis virus, the type species of the betanodaviruses
Authors: Iwamoto Tokinori , Mise Kazuyuki , Mori Koh-ichiro , Arimoto Misao , Nakai Toshihiro , Okuno Tetsuro 
Published: August 2015

A system has been established to produce infectious RNA transcripts for Striped jack nervous necrosis virus (SJNNV), the type species of the betanodaviruses, which infect fish. An enzymological analysis suggested that both RNA1 and RNA2 of SJNNV have a 5′ cap. Both RNAs were largely resistant to 3′ polyadenylation and ligation, suggesting the presence of an interfering 3′ structure, while a small quantity of viral RNAs were polyadenylated in vitro. The complete 5′ and 3′ non-coding sequences of both segments were determined using the rapid amplification of cDNA ends method. Based on the terminal sequences obtained, RT–PCR was carried out and plasmid clones containing full-length cDNA copies of both RNAs, positioned downstream of a T7 promoter, were constructed. These plasmids were cleaved at a unique restriction site just downstream of the 3′ terminus of each SJNNV sequence and were transcribed in vitro into RNA with a cap structure analogue. A mixture of the transcripts was transfected into the fish cell line E-11. Using indirect immunofluorescence staining with anti-SJNNV serum, fluorescence was observed specifically in these transfected cells; this culture supernatant exhibited pathogenicity to striped jack larvae. Northern blot analysis of E-11 cells infected with the recombinant virus or SJNNV showed small RNA (ca. 0·4 kb) that was newly synthesized and corresponded to the 3′-terminal region of RNA1. Finally, the complete nucleotide sequences of these functional cDNAs (RNA1, 3107 nt; RNA2, 1421 nt) were determined. This is the first report of betanodavirus cDNA clones from which infectious genomic RNAs can be transcribed.
Movements of vaccinia virus intracellular enveloped virions with GFP tagged to the F13L envelope protein
Authors: Geada María M. , Galindo Inmaculada , Lorenzo María M. , Perdiguero Beatriz , Blasco Rafael 
Published: August 2015

Vaccinia virus produces several forms of infectious virions. Intracellular mature virions (IMV) assemble in areas close to the cell nucleus. Some IMV acquire an envelope from intracellular membranes derived from the trans-Golgi network, producing enveloped forms found in the cytosol (intracellular enveloped virus; IEV), on the cell surface (cell-associated enveloped virus) or free in the medium (extracellular enveloped virus; EEV). Blockage of IMV envelopment inhibits transport of virions to the cell surface, indicating that enveloped virus forms are required for virion movement from the Golgi area. To date, the induction of actin tails that propel IEV is the only well-characterized mechanism for enveloped virus transport. However, enveloped virus transport and release occur under conditions where actin tails are not formed. In order to study these events, recombinant vaccinia viruses were constructed with GFP fused to the most abundant protein in the EEV envelope, P37 (F13L). The P37–GFP fusion, like normal P37, accumulated in the Golgi area and was incorporated efficiently into enveloped virions. These recombinants allowed the monitoring of enveloped virus movementsin vivo. In addition to a variety of relatively slow movements (<0·4 μm/s), faster, saltatory movements both towards and away from the Golgi area were observed. These movements were different from those dependent on actin tails and were inhibited by the microtubule-disrupting drug nocodazole, but not by the myosin inhibitor 2,3-butanedione monoxime. Video microscopy (5 frames per s) revealed that saltatory movements had speeds of up to, and occasionally more than, 3 μm/s. These results suggest that a second, microtubule-dependent mechanism exists for intracellular transport of enveloped vaccinia virions.
Gene transfer using human polyomavirus BK virus-like particles expressed in insect cells
Authors: Touzé Antoine , Bousarghin Latifa , Ster Céline , Combita Alba-Lucia , Roingeard Philippe , Coursaget Pierre 
Published: August 2015

The major structural protein (VP1) of the BK polyomavirus (BKV) was expressed in the recombinant baculovirus expression system. Recombinant BKV VP1 was shown to self-assemble into virus-like particles (VLPs) with a diameter of 45–50 nm. As for other polyomaviruses, BKV VP1 has the capacity to bind to exogenous DNA. Furthermore, the potential of BKV VP1 VLPs was investigated for gene transfer into COS-7 cells using three methods for the formation of pseudo-virions: disassembly/reassembly, osmotic shock and direct interaction between VLPs and reporter plasmid DNA. The latter method was shown to be the most efficient when using linearized plasmid. Gene transfer efficiency with BKV pseudo-virions was of the same order as that observed with human papillomavirus type 16 L1 protein VLPs. In addition, it is demonstrated that cellular entry of BKV pseudo-virions is dependent on cell surface sialic acid.
Genome organization of Tobacco leaf curl Zimbabwe virus, a new, distinct monopartite begomovirus associated with subgenomic defective DNA molecules
Authors: Paximadis M. , Rey M. E. C. 
Published: August 2015

The complete DNA A of the begomovirus Tobacco leaf curl Zimbabwe virus (TbLCZWV) was sequenced: it comprises 2767 nucleotides with six major open reading frames encoding proteins with molecular masses greater than 9 kDa. Full-length TbLCZWV DNA A tandem dimers, cloned in binary vectors (pBin19 and pBI121) and transformed into Agrobacterium tumefaciens, were systemically infectious upon agroinoculation of tobacco and tomato. Efforts to identify a DNA B component were unsuccessful. These findings suggest that TbLCZWV is a new member of the monopartite group of begomoviruses. Phylogenetic analysis identified TbLCZWV as a distinct begomovirus with its closest relative being Chayote mosaic virus. Abutting primer PCR amplified ca. 1300 bp molecules, and cloning and sequencing of two of these molecules revealed them to be subgenomic defective DNA molecules originating from TbLCZWV DNA A. Variable symptom severity associated with tobacco leaf curl disease and TbLCZWV is discussed.
Morphological and molecular evidence that Culex nigripalpus baculovirus is an unusual member of the family Baculoviridae
Authors: Moser Bettina A. , Becnel James J. , White Susan E. , Afonso Claudio , Kutish Gerald , Shanker Savita , Almira Ernesto 
Published: August 2015

We present evidence that a newly discovered mosquito virus fromCulex nigripalpusis an unusual member of the familyBaculoviridae. Development of this virus was restricted to nuclei of midgut epithelial cells in the gastric caeca and posterior stomach. The globular occlusion bodies were not enveloped, measured around 400 nm in diameter, occurred exclusively in nuclei of infected cells and typically contained four, sometimes up to eight, virions. The developmental sequence involved two virion phenotypes: an occluded form (ODV) that initiated infection in the midgut epithelial cells, and a budded form that spread the infection in the midgut. Each ODV contained one rod-shaped enveloped nucleocapsid (40×200 nm). The double-stranded DNA genome was approximately 105–110 kbp with an estimated GC content of 52%. We have sequenced approximately one-third of the genome and detected 96 putative ORFs of 50 amino acids or more including several genes considered to be unique to baculoviruses. Phylogenetic analysis of the amino acid sequences ofDNApoland p74 placed this virus in a separate clade from the generaNucleopolyhedrovirusandGranulovirus. We provisionally assign this virus in the genusNucleopolyhedrovirus, henceforth abbreviated as CuniNPV (forCulex nigripalpusnucleopolyhedrovirus), and suggest that, awaiting additional data to clarify its taxonomic status, it may be a member of a new genus within the familyBaculoviridae.
Sequence comparison of an Australian duck hepatitis B virus strain with other avian hepadnaviruses
Authors: Triyatni Miriam , Ey Peter L. , Tran Thien , Le Mire Marc , Qiao Ming , Burrell Christopher J. , Jilbert Allison R. 
Published: August 2015

The genome of an Australian strain of duck hepatitis B virus (AusDHBV) was cloned from a pool of congenitally DHBV-infected-duck serum, fully sequenced and found by phylogenetic analyses to belong to the ‘Chinese’ DHBV branch of the avian hepadnaviruses. Sequencing of the Pre-S/S gene of four additional AusDHBV clones demonstrated that the original clone (pBL4.8) was representative of the virus present in the pool, and a head-to-tail dimer of the clone was infectious when inoculated into newly hatched ducks. When the published sequences of 20 avian hepadnaviruses were compared, substitutions or deletions in the polymerase (POL) gene were most frequent in the 500 nt segment encoding the ‘spacer’ domain that overlaps with the Pre-S domain of the Pre-S/S gene in a different reading frame. In contrast, substitutions and deletions were rare within the adjacent segment that encodes the reverse transcriptase domain of the POL protein and the S domain of the envelope protein, presumably because they are more often deleterious.
The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus
Authors: Sawicki Dorothea L. , Wang Tao , Sawicki Stanley G. 
Published: August 2015

In addition to the RI (replicative intermediate RNA) and native RF (replicative form RNA), mouse hepatitis virus-infected cells contained six species of RNA intermediates active in transcribing subgenomic mRNA. We have named these transcriptive intermediates (TIs) and native transcriptive forms (TFs) because they are not replicating genome-sized RNA. Based on solubility in high salt solutions, approximately 70% of the replicating and transcribing structures that accumulated in infected cells by 5–6 h post-infection were multi-stranded intermediates, the RI/TIs. The other 30% were in double-stranded structures, the native RF/TFs. These replicating and transcribing structures were separated by velocity sedimentation on sucrose gradients or by gel filtration chromatography on Sepharose 2B and Sephacryl S-1000, and migrated on agarose gels during electrophoresis, according to their size. Digestion with RNase T1 at 1–10 units/μg RNA resolved RI/TIs into RF/TF cores and left native RF/TFs intact, whereas RNase A at concentrations of 0·02 μg/μg RNA or higher degraded both native RF/TFs and RI/TIs. Viral RI/TIs and native RF/TFs bound to magnetic beads containing oligo(dT)25, suggesting that the poly(A) sequence on the 3′ end of the positive strands was longer than any poly(U) on the negative strands. Kinetics of incorporation of [3H]uridine showed that both the RI and TIs were transcriptionally active and the labelling of RI/TIs was not the dead-end product of aberrant negative-strand synthesis. Failure originally to find TIs and TF cores was probably due to overdigestion with RNase A.
Heat stability of prion rods and recombinant prion protein in water, lipid and lipid–water mixtures
Authors: Appel Thomas Raul , Wolff Michael , von Rheinbaben Friedrich , Heinzel Michael , Riesner Detlev 
Published: August 2015

Prion rods, i.e. insoluble infectious aggregates of the N-terminally truncated form of the prion protein, PrP 27–30, and the corresponding recombinant protein, rPrP(90–231), were autoclaved in water, bovine lipid or lipid–water mixtures for 20 min at temperatures from 100 to 170 °C. A protocol was developed for the quantitative precipitation of small amounts of protein from large excesses of lipid. PrP remaining undegraded after autoclaving was quantified by Western blot and degradation factors were calculated. The Arrhenius plot of the rate of degradation vs temperature yielded linear relationships for prion rods in water or lipid–water as well as for rPrP(90–231) in lipid–water. The presence of lipids increased the heat stability of prion rods, especially at lower temperatures. Prion rods had a much higher thermal stability compared to rPrP. Autoclaving of prion rods in pure lipid gave different results – not simple degradation but bands indicative of covalently linked dimers, tetramers and higher aggregates. The heat stability of prion rods in pure lipid exceeded that in lipid–water mixtures. Degradation factors larger than 104 were reached at 170 °C in the presence of lipids and at 150 °C in the absence of lipids. The linear correlation of the data allows cautious extrapolation to conditions not tested, i.e. temperatures higher than 170 °C. A factual basis for assessing the biological safety of industrial processes utilizing potentially BSE-or scrapie-contaminated animal fat is provided.
The size of encapsidated single-stranded DNA determines the multiplicity of African cassava mosaic virus particles
Authors: Frischmuth Thomas , Ringel Margit , Kocher Cornelia 
Published: August 2015

Transgenic Nicotiana benthamiana plants harbouring a defective interfering (DI) DNA of African cassava mosaic virus (ACMV) and control plants were inoculated with ACMV. Virus particles were purified from infected plants, separated in sucrose gradients and fractions were analysed by Southern blotting. Transgenic plant-derived virus particles taken from the top fractions of sucrose gradients contained DI DNA, middle fractions contained a mixture of genomic and DI DNA and bottom fractions contained a mixture of multimeric, genomic and DI DNA. Virus particles from selected top, middle and bottom fractions were analysed by electron microscopy. In fractions containing only DI DNA, isometric particles of 18–20 nm were detected. In fractions containing DI DNA as well as genomic size DNA, isometric and geminate particles were found. Fractions containing multimeric size DNA were found to comprise particles consisting of three subunits adjacent to geminate particles. From these data, it is concluded that the size of encapsidated DNA determines the multiplicity of ACMV particles.
A novel variant genotype C of hepatitis B virus identified in isolates from Australian Aborigines: complete genome sequence and phylogenetic relatedness
Authors: Sugauchi Fuminaka , Mizokami Masashi , Orito Etsuro , Ohno Tomoyoshi , Kato Hideaki , Suzuki Seiji , Kimura Yoshihide , Ueda Ryuzo , Butterworth L. A. , Cooksley W. G. E. 
Published: August 2015

There have been no reports of DNA sequences of hepatitis B virus (HBV) strains from Australian Aborigines, although the hepatitis B surface antigen (HBsAg) was discovered among them. To investigate the characteristics of DNA sequences of HBV strains from Australian Aborigines, the complete nucleotide sequences of HBV strains were determined and subjected to molecular evolutionary analysis. Serum samples positive for HBsAg were collected from five Australian Aborigines. Phylogenetic analysis of the five complete nucleotide sequences compared with DNA sequences of 54 global HBV isolates from international databases revealed that three of the five were classified into genotype D and were most closely related in terms of evolutionary distance to a strain isolated from a healthy blood donor in Papua New Guinea. Two of the five were classified into a novel variant genotype C, which has not been reported previously, and were closely related to a strain isolated from Polynesians, particularly in the X and Core genes. These two strains of variant genotype C differed from known genotype C strains by 5·9–7·4% over the complete nucleotide sequence and 4·0–5·6% in the small-S gene, and had residues Arg122, Thr127 and Lys160, characteristic of serotype ayw3, which have not been reported previously in genotype C. In conclusion, this is the first report of the characteristics of complete nucleotide sequences of HBV from Australian Aborigines. These results contribute to the investigation of the worldwide spread of HBV, the relationship between serotype and genotype and the ancient common origin of Australian Aborigines.
Characterization and functional analysis of Serp3: a novel myxoma virus-encoded serpin involved in virulence
Authors: Guerin Jean-Luc , Gelfi Jacqueline , Camus Christelle , Delverdier Maxence , Whisstock James C. , Amardeihl Marie-France , Py Robert , Bertagnoli Stéphane , Messud-Petit Frédérique 
Published: August 2015

Myxoma virus (MV), a member of the familyPoxviridae, is the causative agent of myxomatosis, a fatal disease of the European rabbit. The MV genome is a linear, double-stranded DNA molecule that encodes several factors important for evasion of the host immune system. Sequencing the right-end region of the MV genome identified an 801 bp open reading frame (ORF) encoding a polypeptide that belongs to the serpin superfamily. To date, two MV-encoded serpins have been characterized: SERP-1 binds to several targets and is an anti-inflammatory molecule, whereas Serp2 is essential for virus virulence and has both anti-inflammatory and anti-apoptotic effects. Thus, Serp3 is the third MV-encoded serpin. DNA sequence analysis of Serp3 indicated a similarity to poxvirus late promoters, which was confirmed by mRNA expression analysis. Serp3 has an atypical serpin motif and has significant sequence deletions as compared to most cellular and viral serpins. However, molecular modelling studies suggested that Serp3 can retain the overall serpin fold. Insertional inactivation of theserp3ORF led to a significant attenuation of virulencein vivo(as measured by the increase in survival of infected rabbits) and limited dissemination of the virus to secondary sites of infection. In rabbits infected with a Serp3 deletion mutant (MV-Serp3−), the main histopathological feature is the absence of secondary myxomas. Both wild-type MV and MV-Serp3−replicate at comparable levelsin vivo. Serp3 may represent a significant virulence factor of MV and probably acts in synergy with other viral proteins.
Characterization of a novel genital human papillomavirus by overlapping PCR: candHPV86 identified in cervicovaginal cells of a woman with cervical neoplasia
Authors: Terai Masanori , Burk Robert D. 
Published: August 2015

A novel human papillomavirus (HPV), candHPV86, was cloned and characterized from cervicovaginal cells obtained from a 37-year-old Hispanic woman with cervical intraepithelial neoplasia grade 1 (CIN1) using an overlapping PCR technique. Primers were designed by phylogenetic alignment of closely related HPV genomes using the L1 fragment sequence amplified by GP5+/6+. The 7983 bp complete nucleotide sequence of the HPV genome was determined by sequence walking. A basic local alignment sequence tool (BLAST) homology search using the L1 open reading frame demonstrated that this HPV was most closely related to HPVHAN2294 (GenBank, AJ400628; 86% homology) and HPV84 (84% homology). candHPV86 was placed in the HPV genome homology group A3 by phylogenetic analyses. The overlapping PCR technique is applicable for characterizing the complete spectrum and variation of HPVs in a population.
Mutagenesis of hepatitis C virus E1 protein affects its membrane-permeabilizing activity
Authors: Ciccaglione A. R. , Costantino A. , Marcantonio C. , Equestre M. , Geraci A. , Rapicetta M. 
Published: August 2015

The E1 glycoprotein of hepatitis C virus is a transmembrane glycoprotein with a C-terminal anchor domain. When expressed inEscherichia coli, E1 induces a change in membrane permeability that is toxic to the bacterial cell. The C-terminal hydrophobic region (aa 331–383) of E1 is mainly responsible for membrane association and for inducing changes in membrane permeability. These observed changes are similar to those produced inE. coliby influenza virus M2, human immunodeficiency virus gp41 and poliovirus 3AB proteins, whose hydrophobic domains are thought to cause pore formation in biological membranes. To further characterize the activity of E1 at a molecular level, the membrane-permeabilizing ability of a second internal hydrophobic region (aa 262–291) was examined by expressing different deletion mutants of E1 in anE. colisystem that is widely used for analysing membrane-active proteins from other animal viruses. Moreover, highly conserved amino acids in the C-terminal hydrophobic region were mutated to identify residues that are critical for inducing changes in membrane permeability. Analysis of cell growth curves of recombinant cultures and membrane-permeability assays revealed that synthesis of this fragment increased the flux of small compounds through the membrane and caused progressive cell lysis, suggesting that this domain has membrane-active properties. Furthermore, analysis of C-terminal mutants indicated that the conserved amino acids Arg339, Trp368and Lys370play a critical role in protein function, as both cell lysis and changes in membrane permeability induced by the wild-type clone could be blocked by substitutions in these positions.