bluuebunny/crossref_metadata_2025_split huggingface dataset:
The DNA form of a retroviroid-like element characterized in cultivated carnation species
Authors: Hegedűs Krisztina , Palkovics László , Tóth Endre Kristóf , Dallmann Géza , Balázs Ervin 
Published: August 2015

Carnation small viroid-like RNA (CarSV RNA) is a small (275 nt), circular molecule which is unique among plant viroid-like RNAs in having a tandemly repeated homologous DNA. This DNA form was found fused to DNA sequences of carnation etched ring caulimovirus (CERV) in certain Spanish carnation plants. The observation of a growth abnormality consisting of extensive shoot proliferation in cultivated carnations in Hungary prompted the molecular analysis of these plants, in which both CarSV RNA and DNA forms were detected. Several CarSV DNA sequences were characterized in various Dianthus caryophyllus cultivars which were symptomless or showed different symptoms. CarSV DNA forms showing minor sequence heterogeneities and deletions occurred in the same plant. Unit-length CarSV DNA sequences were proven to accumulate in the plant cell nucleus. The plants studied here were not infected by any of the viruses (including CERV) or other cellular pathogens described previously in carnation.
Persistent expression of a newly characterized Hyposoter didymator polydnavirus gene in long-term infected lepidopteran cell lines
Authors: Volkoff Anne-Nathalie , Rocher Janick , Cérutti Pierre , Ohresser Marc C. P. , d’Aubenton-Carafa Yves , Devauchelle Gérard , Duonor-Cérutti Martine 
Published: August 2015

An Hyposoter didymator ichnovirus (HdIV) gene was stably maintained and efficiently transcribed in lepidopteran cell lines more than 3 years after HdIV infection. This K-gene had two introns and the fully spliced cDNA, named K19, comprised a short open reading frame and a long 3′-untranslated region with 13 imperfectly repeated sequences (44 to 102 nt). Transcripts related to the K-gene were detected in several long-term infected cell lines (Sf9, Spodoptera littoralis haemocytes, Trichoplusia ni). Conversely, no transcripts related to seven other viral cDNAs were detected, suggesting that the K-related DNA is selectively retained in long-term infected Sf9 cells. The function of the K-gene product and its association with stably transformed insect cell lines remains to be investigated.
Phylogenetic analyses confirm the high prevalence of hepatitis C virus (HCV) type 4 in the Seine-Saint-Denis district (France) and indicate seven different HCV-4 subtypes linked to two different epidemiological patterns
Authors: Morice Yoann , Roulot Dominique , Grando Véronique , Stirnemann Jérome , Gault Elyanne , Jeantils Vincent , Bentata Michelle , Jarrousse Bernard , Lortholary Olivier , Pallier Coralie , Dény Paul 
Published: August 2015

Hepatitis C virus (HCV) has been classified into six clades as a result of high genetic variability. In the Seine-Saint-Denis district of north-east Paris, the prevalence of HCV-4, which usually infects populations from Africa or the Middle East, is twice as high as that recorded for the whole of continental France (10·2 versus 4·5%). Although the pathogenicity of HCV-4 remains unknown, resistance of HCV-4 to therapy appears to be similar to that observed for HCV-1. In order to characterize the epidemiology of HCV-4 in Paris, sequences of the non-structural 5B gene (332 bp) were obtained from 38 HCV-4-infected patients. Extensive phylogenetic analyses indicated seven different HCV-4 subtypes. Moreover, phylogenetic tree topologies clearly distinguished two epidemiological profiles. The first profile (52·6% of patients) reflects the intra-suburban emergence of two distinct HCV-4 subclades occurring mainly among intravenous drug users (65% of patients). The second profile shows six subclades [HCV-4a, -4f, -4h, -4k, -4a(B) and a new sequence] and accounts for patients from Africa (Egypt and sub-Saharan countries) who have unknown risk factors (77·8% of patients) and in whom no recent diffusion of HCV-4 is evident. This study indicates the high diversity of HCV-4 and the extension of HCV-4a and -4d subclades among drug users in France.
Retroviral recombination is temperature dependent
Authors: Li Ting , Zhang Jiayou 
Published: August 2015

Two conflicting in vitro observations suggest that retroviral recombinations are temperature dependent. Ouhammouch & Brody (Nucleic Acids Research 20, 5443–5450, 1992) suggested that retroviral recombination rates should increase as temperature increases. However, Shimomaye & Salvato (Gene Analysis Techniques 6, 25–28, 1989) and Brooks et al. (Biotechniques 19, 806–812, 814–815, 1985) found that at low temperature the tightly folded structure of RNAs may hinder reverse transcription proceeding along the RNA template, which increases its chance of dissociating from the template; therefore, raising the reaction temperature was the simplest way to overcome template secondary structure and prevent premature termination of cDNA synthesis. In this report, two vectors based on murine leukaemia virus (MLV) were constructed. The first contained two mutated gfp genes in tandem positions. The upstream gfp gene encoded a mutation at its 3′ end, while the downstream gfp gene encoded a mutation at its 5′ end. The recombination that occurred between the two mutated gfp genes restored a functional gfp gene. The cells that contained the functional gfp gene were green when observed under a fluorescence microscope. The second MLV vector contained a functional gfp gene with two identical sequences flanking either end. A recombination that occurred between the two identical sequences resulted in deletion of the gfp gene. Cells containing the vector with the gfp deletion were colourless or clear when observed under the microscope. Using these two vectors, we have demonstrated that retroviral recombination is temperature dependent and the rate of recombination decreases as temperature is raised from 31 to 43 °C.
Evaluation of the primary effect of brefeldin A treatment upon herpes simplex virus assembly
Authors: Dasgupta Anindya , Wilson Duncan W. 
Published: August 2015

Addition of the drug brefeldin A (BFA) to cells infected by herpes simplex virus (HSV) type 1 is known to result in a complex pattern of defects in particle assembly. BFA-treated, infected cells accumulate perinuclear enveloped virions and non-enveloped (‘naked’) cytoplasmic capsids, and it has been difficult to interpret these data in terms of the assembly pathway of HSV and the known effects of BFA on the secretory apparatus. Since BFA is a cytotoxic drug, and earlier studies commonly examined the effects of long-term BFA incubations on infected cells, it was hypothesized that the drug could have pleiotropic and indirect effects on HSV assembly. To test this, use was made of an HSV synchronized assembly assay, in which cells are infected with the virus mutant tsProt.A and maintained at 39 °C to induce reversible accumulation of a population of procapsids. By first adding BFA and then shifting these cells to 31 °C for 3 h to allow the accumulated procapsids to mature, it was possible to test the effect of short-term BFA treatment on only those HSV assembly events that are downstream of procapsid maturation. Under these conditions, it was found that procapsids matured and packaged the viral genome normally, but remained non-enveloped and failed to exit the nucleus. It is concluded that the primary effect of BFA on HSV replication is to inhibit budding at the inner nuclear membrane.
The UL41-encoded virion host shutoff (vhs) protein and vhs-independent mechanisms are responsible for down-regulation of MHC class I molecules by bovine herpesvirus 1
Authors: Koppers-Lalic Danijela , Rijsewijk Frans A. M. , Verschuren Sylvia B. E. , van Gaans-van den Brink Jacqueline A. M. , Neisig Anne , Ressing Maaike E. , Neefjes Jacques , Wiertz Emmanuel J. H. J. 
Published: August 2015

The virion host shutoff (vhs) protein of alphaherpesviruses causes a rapid shutoff of host cell protein synthesis. We constructed a bovine herpesvirus 1 (BHV1) deletion mutant in which the putative vhs gene, UL41, has been disrupted. Whereas protein synthesis is inhibited within 3 h after infection with wild-type BHV1, no inhibition was observed after infection with the BHV1vhs−deletion mutant. These results indicate that the BHV1 UL41 gene product is both necessary and sufficient for shutoff of host cell protein synthesis at early times post-infection. Using the vhs deletion mutant, we investigated the mechanism of BHV1-induced down-regulation of MHC class I cell surface expression. In contrast to BHV1 wild-type infection, the BHV1vhs−mutant allows detection of MHC class I molecules at much later time-points after infection. This illustrates the role the vhs protein plays in MHC class I down-regulation. However, even after infection with BHV1vhs−, MHC class I cell surface expression is impaired. In BHV1vhs−-infected cells, MHC class I molecules are retained within the endoplasmic reticulum (ER). Moreover, the transporter associated with antigen presentation (TAP) is still blocked. Temporal control of viral protein expression using chemical inhibitors shows that viral protein(s) expressed within the early phase of BHV1 infection are responsible for ER retention of MHC class I molecules. These results indicate that multiple mechanisms are responsible for down-regulation of MHC class I molecules in BHV1-infected cells.
Leucine at position 278 of the AIK-C measles virus vaccine strain fusion protein is responsible for reduced syncytium formation
Authors: Nakayama Tetsuo , Komase Katsuhiro , Uzuka Rina , Hoshi Akiyoshi , Okafuji Takao 
Published: August 2015

The live measles virus (MV) vaccine strain AIK-C was attenuated from the wild-type strain Edmonston by plaque purification at 33 °C. Strain AIK-C grew well at 33 °C with a mixture of small-and medium-sized plaques in Vero cells, but did not grow well at 40 °C. To investigate fusion inducibility, expression plasmids for the fusion (F) and haemagglutinin (H) protein regions of MV strains AIK-C (pAIK-F01 and pAIK-H) and Edmonston (pEdm-F and pEdm-H) were constructed. pEdm-F induced extensive cell fusion in B95a and Vero cells under the control of T7 RNA polymerase, whereas a sharp reduction in syncytium formation was observed when pAIK-F01 was used. Six amino acid differences were determined between pAIK-F01 and pEdm-F. Direct sequencing showed that the seed strain AIK-C contained either Leu or Phe at position 278 of the F protein. Experiments using recombinant F protein plasmids demonstrated that those with Leu at position 278 induced poor syncytium formation, while those with Phe at position 278 (Edmonston type) induced extensive cell fusion. Replacement of Phe with Leu at position 278 of pEdm-F reduced fusion-inducing capability. A full-length infectious clone of AIK-C with Leu at position 278 of the F protein was constructed. The rescued virus produced small plaques in Vero cells. However, the same rescued virus with Phe at position 278 produced large plaques. It was concluded that Leu at position 278 of the F protein of the MV vaccine strain AIK-C is responsible for the formation of small plaques.
Complete nucleotide sequence of avian paramyxovirus type 6 isolated from ducks
Authors: Chang P.-C. , Hsieh M.-L. , Shien J.-H. , Graham D. A. , Lee M.-S. , Shieh H. K. 
Published: August 2015

There are nine serotypes of avian paramyxovirus (APMV). Only the genome of APMV type 1 (APMV-1), also called Newcastle disease virus (NDV), has been completely sequenced. In this study, the complete nucleotide sequence of an APMV-6 serotype isolated from ducks is reported. The 16236 nt genome encodes eight proteins, nucleocapsid protein (NP), phosphoprotein (P), V protein, matrix protein (M), fusion protein (F), small hydrophobic (SH) protein, haemagglutinin–neuraminidase (HN) protein and large (L) protein, which are flanked by a 55 nt leader sequence and a 54 nt trailer sequence. Sequence comparison reveals that the protein sequences of APMV-6 are most closely related to those of APMV-1 (NDV) and -2, with sequence identities ranging from 22 to 44%. However, APMV-6 contains a gene that might encode the SH protein, which is absent in APMV-1, but present in the rubulaviruses simian virus type 5 and mumps virus. The presence of an SH gene in APMV-6 might provide a link between the evolution of APMV and rubulaviruses. Phylogenetic analysis demonstrates that APMV-6, -1, -2 (only the F and HN sequences were available for analysis) and -4 (only the HN sequences were available for analysis) all cluster into a single lineage that is distinct from other paramyxoviruses. This result suggests that APMV should constitute a new genus within the subfamily Paramyxovirinae.
The human herpesvirus-8 (Kaposi’s sarcoma-associated herpesvirus) ORF 40/41 region encodes two distinct transcripts
Authors: AuCoin David P. , Pari Gregory S. 
Published: August 2015

The human herpesvirus-8 (HHV-8) locus encoding ORFs 40/41 is a candidate homologue for the Epstein–Barr virus BBLF 2/3 gene, which encodes the putative primase-associated factor. Northern blot data revealed that two transcripts originated from the HHV-8 ORF 40/41 region. The sizes of these transcripts (2.2 and 0.7 kb) suggested that one transcript was the result of a spliced form of ORFs 40 and 41 and the second transcript originated from a region within ORF 41. cDNA sequence and 5′ RACE analysis revealed the removal of an intron between ORFs 40 and 41 and a transcriptional start site 82 nt upstream of ORF 40. The start of transcription for the smaller transcript was mapped to within ORF 41. Regions upstream of the transcriptional start sites were subcloned into a luciferase reporter vector, and transient luciferase assays indicated that distinct promoters drive the expression of each transcript.
The vaccinia virus F12L protein is associated with intracellular enveloped virus particles and is required for their egress to the cell surface
Authors: van Eijl Henriette , Hollinshead Michael , Rodger Gaener , Zhang Wei-Hong , Smith Geoffrey L. 
Published: August 2015

The vaccinia virus (VV) F12L gene encodes a 65 kDa protein that is expressed late during infection and is important for plaque formation, EEV production and virulence. Here we have used a recombinant virus (vF12LHA) in which the F12L protein is tagged at the C terminus with an epitope recognized by a monoclonal antibody to determine the location of F12L in infected cells and whether it associates with virions. Using confocal and electron microscopy we show that the F12L protein is located on intracellular enveloped virus (IEV) particles, but is absent from immature virions (IV), intracellular mature virus (IMV) and cell-associated enveloped virus (CEV). In addition, F12L shows co-localization with endosomal compartments and microtubules. F12L did not co-localize with virions attached to actin tails, providing further evidence that actin tails are associated with CEV but not IEV particles. In vΔF12L-infected cells, virus morphogenesis was arrested after the formation of IEV particles, so that the movement of these virions to the cell surface was inhibited and CEV particles were not found. Previously, virus mutants lacking IEV- or EEV-specific proteins were either unable to make IEV particles (vΔF13L and vΔB5R), or were unable to form actin tails after formation of CEV particles (vΔA36R, vΔA33R, vΔA34R). The F12L deletion mutant therefore defines a new stage in the morphogenic pathway and the F12L protein is implicated as necessary for microtubule-mediated egress of IEV particles to the cell surface.
Solution structures of potato virus X and narcissus mosaic virus from Raman optical activity
Authors: Blanch Ewan W. , Robinson David J. , Hecht Lutz , Syme Christopher D. , Nielsen Kurt , Barron Laurence D. 
Published: August 2015

Potato virus X (PVX) and narcissus mosaic virus (NMV) were studied using vibrational Raman optical activity (ROA) in order to obtain new information on the structures of their coat protein subunits. The ROA spectra of the two intact virions are very similar to each other and similar to that of tobacco mosaic virus (TMV) studied previously, being dominated by signals characteristic of proteins with helix bundle folds. In particular, PVX and NMV show strong positive ROA bands at ∼1340 cm−1 assigned to hydrated α-helix and perhaps originating in surface exposed helical residues, together with less strong positive ROA intensity in the range ∼1297–1312 cm−1 assigned to α-helix in a more hydrophobic environment and perhaps originating in residues at helix–helix interfaces. The positive ∼1340 cm−1 ROA band of TMV is less intense than those of PVX and NMV, suggesting that TMV contains less hydrated α-helix. Small differences in other spectral regions reflect differences in some loop, turn and side-chain compositions and conformations among the three viruses. A pattern recognition program based on principal component analysis of ROA spectra indicates that the coat protein subunit folds of PVX and NMV may be very similar to each other and similar to that of TMV. These results suggest that PVX and NMV may have coat protein subunit structures based on folds similar to the TMV helix bundle and hence that the helical architecture of the PVX and NMV particles may be similar to that of TMV but with different structural parameters.
Characterization of Chelonus inanitus polydnavirus segments: sequences and analysis, excision site and demonstration of clustering
Authors: Wyder Stefan , Tschannen Adrian , Hochuli Anita , Gruber Andreas , Saladin Verena , Zumbach Sonja , Lanzrein Beatrice 
Published: August 2015

Polydnaviruses (genera Ichnovirus and Bracovirus) have a segmented genome of circular double-stranded DNA molecules, replicate in the ovary of parasitic wasps and are essential for successful parasitism of the host. Here we show the first detailed analysis of various segments of a bracovirus, the Chelonus inanitus virus (CiV). Four segments were sequenced and two of them, CiV12 and CiV14, were found to be closely related while CiV14.5 and CiV16.8 were unrelated. CiV12, CiV14.5 and CiV16.8 are unique while CiV14 occurs also nested in another larger segment. All four segments are predicted to contain genes and predictions could be substantiated in most cases. Comparison with databases revealed no significant similarities at either the nucleotide or amino acid level. Inverted repeats with identities between 77% and 92% and lengths between 26 bp and 100 bp were found on all segments outside of predicted genes. Hybridization experiments indicate that CiV12 and CiV14 are both flanked by other virus segments, suggesting that proviral CiV segments are clustered in the genome of the wasp. The integration/excision site of CiV14 was analysed and compared to that of CiV12. On both termini of proviral CiV12 and CiV14 as well as in the excised circular molecule and the rejoined DNA a very similar repeat of 14 bp was found. A model to illustrate where the terminal repeats might recombine to yield the circular molecule is presented. Excision of CiV12 and CiV14 is restricted to the female and sets in at a very specific time-point in pupal–adult development.
Multiple glycosylated forms of the respiratory syncytial virus fusion protein are expressed in virus-infected cells
Authors: Rixon Helen W. McL. , Brown Craig , Brown Gaie , Sugrue Richard J. 
Published: August 2015

Analysis of the respiratory syncytial virus (RSV) fusion (F) protein in RSV-infected Vero cells showed the presence of a single F1 subunit and at least two different forms of the F2 subunit, designated F2a (21 kDa) and F2b (16 kDa), which were collectively referred to as [F2]a/b. Enzymatic deglycosylation of [F2]a/b produced a single 10 kDa product suggesting that [F2]a/b arises from differences in the glycosylation pattern of F2a and F2b. The detection of [F2]a/b was dependent upon the post-translational cleavage of the F protein by furin, since its appearance was prevented in RSV-infected Vero cells treated with the furin inhibitor dec-RVKR-cmk. Analysis by protein cross-linking revealed that the F1 subunit interacted with [F2]a/b, via disulphide bonding, to produce equivalent F protein trimers, which were expressed on the surface of infected cells. Collectively, these data show that multiple F protein species are expressed in RSV-infected cells.
Characterization of a human immunodeficiency virus type 1 pre-integration complex in which the majority of the cDNA is resistant to DNase I digestion
Authors: Khiytani Dheeraj K. , Dimmock Nigel J. 
Published: August 2015

The human immunodeficiency virus type 1 (HIV-1) pre-integration complex (PIC) is a cytoplasmic nucleoprotein structure derived from the core of the virion and is responsible for reverse transcription of viral RNA to cDNA, transport to the nucleus and integration of the cDNA into the genome of the infected target cell. Others have shown by Mu phage-mediated PCR footprinting that only the LTRs of the cDNA of PICs isolated early in infection are protected by bound protein, while the rest of the genome is susceptible to nuclease attack. Here, using DNase I footprinting, we confirmed that the majority of the cDNA of PICs isolated at 8·5 h after infection with cell-free virus was sensitive to digestion with DNase I and that only part of the LTRs (approximately 6% of the total cDNA) was protected. However, PICs isolated 90 min later (at 10 h post-infection) were very different in that the majority (approximately 90%) of cDNA was protected from nuclease degradation. These late PICs were integration active in vitro. We conclude that HIV-1 has at least two types of PIC, an early PIC characterized by protein bound only at the LTRs, and a late, and possibly more mature form, in which protein is bound along the length of the cDNA.
A release-competent influenza A virus mutant lacking the coding capacity for the neuraminidase active site
Authors: Gubareva Larisa V. , Nedyalkova Marina S. , Novikov Dmitri V. , Murti K. Gopal , Hoffmann Erich , Hayden Frederick G. 
Published: August 2015

Both influenza A virus surface glycoproteins, the haemagglutinin (HA) and neuraminidase (NA), interact with neuraminic acid-containing receptors. The influenza virus A/Charlottesville/31/95 (H1N1) has shown a substantially reduced sensitivity to NA inhibitor compared with the A/WSN/33 (H1N1) isolate by plaque-reduction assays in Madin–Darby canine kidney (MDCK) cells. However, there was no difference in drug sensitivity in an NA inhibition assay. The replacement of the HA gene of A/WSN/33 with the HA gene of A/Charlottesville/31/95 led to a drastic reduction in sensitivity of A/WSN/33 to NA inhibitor in MDCK cells. Passage of A/Charlottesville/31/95 in cell culture in the presence of an NA inhibitor resulted in the emergence of mutant viruses (delNA) whose genomes lacked the coding capacity for the NA active site. The delNA mutants were plaque-to-plaque purified and further characterized. The delNA-31 mutant produced appreciable yields (∼106 p.f.u./ml) in MDCK cell culture supernatants in the absence of viral or bacterial NA activity. Sequence analysis of the delNA mutant genome revealed no compensatory substitutions in the HA or other genes compared with the wild-type. Our data indicate that sialylation of the oligosaccharide chains in the vicinity of the HA receptor-binding site of A/Charlottesville/31/95 virus reduces the HA binding efficiency and thus serves as a compensatory mechanism for the loss of NA activity. Hyperglycosylation of HA is common in influenza A viruses circulating in humans and has the potential to reduce virus sensitivity to NA inhibitors.