bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression and localization of LEF-11 in Autographa californica nucleopolyhedrovirus-infected Sf9 cells
Authors: Lin Guangyun , Slack Jeffrey M. , Blissard Gary W. 
Published: August 2015

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-11 gene was found previously to be necessary to support optimal levels of transient expression from an AcMNPV late promoter. The lef-11 gene is unusual in that it overlaps both upstream (orf38) and downstream (pp31) genes. In this study, the expression and cellular localization of LEF-11 were examined. The lef-11 transcripts were detected from 4 to 36 h post-infection (p.i.). The 1·5 kb lef-11 mRNA initiates 196 nt upstream of the lef-11 translation initiation codon, within the upstream orf38 gene. This relatively long 5′ upstream region encodes a potential small upstream open reading frame (ORF) of 58 amino acids that overlaps the lef-11 ORF. The 3′ end of the lef-11 mRNA was mapped as co-terminal with mRNAs from the downstream pp31 gene. Using affinity purified anti-LEF-11 antibodies, levels of LEF-11 expression were found to be maximal between approximately 8 and 24 h p.i., although LEF-11 could be detected as late as 72 h p.i. Using immunofluorescence microscopy, it was determined that LEF-11 localized to dense regions of infected cell nuclei, consistent with its role as a possible late transcription factor.
Phylogenetic analysis of conserved genes within the ecdysteroid UDP-glucosyltransferase gene region of the slow-killing Adoxophyes orana granulovirus
Authors: Wormleaton S. L. , Winstanley D. 
Published: August 2015

A physical map of the genome of Adoxophyes orana granulovirus (AoGV) was constructed for the restriction enzymes BamHI, BglII, EcoRI, PstI and SacI using restriction endonuclease analysis and DNA hybridization techniques. This enabled the size of the AoGV genome to be estimated at 100·9 kbp. A plasmid library covering 99·9% of the AoGV genome was constructed using five restriction enzymes. The ecdysteroid UDP-glucosyltransferase gene (egt) was located by hybridization with the egt gene of Cydia pomonella granulovirus. The sequence of 6000 bp of the egt region is presented and compared to the equivalent area in other GVs. Database searches showed that this region contained eight open reading frames (ORFs) similar to the baculovirus genes egt, granulin, pk-1, me53 and four ORFs of Xestia c-nigrum granulovirus (ORF 178, ORF 2, ORF 7 and ORF 8). The egt gene was shown to encode an active EGT using an EGT assay. Phylogenetic trees of the granulovirus genes egt, granulin, pk-1 and me53 were constructed using maximum parsimony and distance analyses. These analyses indicated that AoGV genes may be more closely related to other tortricid-infecting GVs than to GVs that infect other lepidopteran families.
Genomic and phylogenetic analyses of an adenovirus isolated from a corn snake (Elaphe guttata) imply a common origin with members of the proposed new genus Atadenovirus
Authors: Farkas Szilvia L. , Benkő Mária , Élő Péter , Ursu Krisztina , Dán Ádám , Ahne Winfried , Harrach Balázs 
Published: August 2015

Approximately 60% of the genome of an adenovirus isolated from a corn snake (Elaphe guttata) was cloned and sequenced. The results of homology searches showed that the genes of the corn snake adenovirus (SnAdV-1) were closest to their counterparts in members of the recently proposed new genus Atadenovirus. In phylogenetic analyses of the complete hexon and protease genes, SnAdV-1 indeed clustered together with the atadenoviruses. The characteristic features in the genome organization of SnAdV-1 included the presence of a gene homologous to that for protein p32K, the lack of structural proteins V and IX and the absence of homologues of the E1A and E3 regions. These characteristics are in accordance with the genus-defining markers of atadenoviruses. Comparison of the cleavage sites of the viral protease in core protein pVII also confirmed SnAdV-1 as a candidate member of the genus Atadenovirus. Thus, the hypothesis on the possible reptilian origin of atadenoviruses (Harrach, Acta Veterinaria Hungarica 48, 484–490, 2000) seems to be supported. However, the base composition of DNA sequence (>18 kb) determined from the SnAdV-1 genome showed an equilibrated GC content of 51%, which is unusual for an atadenovirus.
Molecular epidemiology of Rabbit haemorrhagic disease virus
Authors: Moss S. R. , Turner S. L. , Trout R. C. , White P. J. , Hudson P. J. , Desai A. , Armesto M. , Forrester N. L. , Gould E. A. 
Published: August 2015

Millions of domestic and wild European rabbits (Oryctolagus cuniculus) have died in Europe, Asia, Australia and New Zealand during the past 17 years following infection by Rabbit haemorrhagic disease virus (RHDV). This highly contagious and deadly disease was first identified in China in 1984. Epidemics of RHDV then radiated across Europe until the virus apparently appeared in Britain in 1992. However, this concept of radiation of a new and virulent virus from China is not entirely consistent with serological and molecular evidence. This study shows, using RT–PCR and nucleotide sequencing of RNA obtained from the serum of healthy rabbits stored at 4 °C for nearly 50 years, that, contrary to previous opinions, RHDV circulated as an apparently avirulent virus throughout Britain more than 50 years ago and more than 30 years before the disease itself was identified. Based on molecular phylogenetic analysis of British and European RHDV sequences, it is concluded that RHDV has almost certainly circulated harmlessly in Britain and Europe for centuries rather than decades. Moreover, analysis of partial capsid sequences did not reveal significant differences between RHDV isolates that came from either healthy rabbits or animals that had died with typical haemorrhagic disease. The high stability of RHDV RNA is also demonstrated by showing that it can be amplified and sequenced from rabbit bone marrow samples collected at least 7 weeks after the animal has died.
Initiation of hepatitis delta virus (HDV) replication: HDV RNA encoding the large delta antigen cannot replicate
Authors: Sheu Gwo-Tarng 
Published: August 2015

The hepatitis delta virus (HDV) nucleocapsid consists of a genomic-length RNA of 1·7 kb and approximately equimolar amounts of the small and large forms of the hepatitis delta antigen (S-HDAg and L-HDAg, respectively). Since HDV RNA particles contain not only a genomic RNA species encoding S-HDAg but also an RNA species encoding L-HDAg, which is produced by an RNA-editing process, the question arises as to whether RNAs encoding either L-HDAg or S-HDAg can initiate replication. To study this, two cDNA-free transfection methods were employed: HDV RNA cotransfected with either the S-HDAg-encoding mRNA species or the ribonucleocapsid protein complex, comprising HDV RNA and recombinant S-HDAg. Results showed that the genomic-sense RNA encoding S-HDAg could promote HDV replication, whereas the L-HDAg-encoding RNA species was unable to replicate under the same conditions. The antigenomic RNA species encoding either S-HDAg or L-HDAg could not replicate by either of these procedures. In addition, L-HDAg alone could not promote replication of the genomic RNA but, by supplementing an equal amount of S-HDAg, replication occurred. These data indicate that L-HDAg-encoding RNA species are probably not involved in the initiation of HDV RNA synthesis; instead, their main function may be to serve as template for producing L-HDAg, which regulates HDV RNA synthesis and virion assembly. These results suggest that the genomic RNA species encoding S-HDAg is the only functional genome for HDV infection and explain why the presence of the edited HDV RNA encoding L-HDAg does not interfere with HDV infection.
Modification of primary and recurrent genital herpes in guinea pigs by passive immunization
Authors: Bourne Nigel , Pyles Richard B. , Bernstein David I. , Stanberry Lawrence R. 
Published: August 2015

Guinea pigs were administered antiserum 24 h (As+24) or 72 h (As+72) after intravaginal herpes simplex virus type 2 (HSV-2) challenge. Treatment at either time reduced acute virus replication in the dorsal root ganglia and the overall magnitude of replication in the genital tract. In two studies, As+24 treatment significantly reduced the severity of primary genital skin disease and the frequency of subsequent spontaneous recurrent disease. In contrast, As+72 treatment produced a modest reduction in primary disease severity but did not impact on recurrent disease. Quantitative PCR analysis of dorsal root ganglia DNA from latently infected animals showed that As+24 treatment produced a significantly reduced viral DNA burden, which appeared to correlate with the reduction in recurrent disease. The amount of DNA in the ganglia of As+72-treated animals was not significantly lower than that of controls. These observations have implications for both the dynamics of latency establishment and desirable vaccine characteristics.
The vaccinia virus soluble interferon-γ receptor is a homodimer
Authors: Alcamí Antonio , Smith Geoffrey L. 
Published: August 2015

The vaccinia virus (VV) interferon (IFN)-γ receptor (IFN-γR) is a 43 kDa soluble glycoprotein that is secreted from infected cells early during infection. Here we demonstrate that the IFN-γR from VV, cowpox virus and camelpox virus exists naturally as a homodimer, whereas the cellular IFN-γR dimerizes only upon binding the homodimeric IFN-γ. The existence of the virus protein as a dimer in the absence of ligand may provide an advantage to the virus in efficient binding and inhibition of IFN-γ in solution.
N-Glycans attached to the stem domain of haemagglutinin efficiently regulate influenza A virus replication
Authors: Wagner Ralf , Heuer Dagmar , Wolff Thorsten , Herwig Astrid , Klenk Hans-Dieter 
Published: August 2015

The haemagglutinin (HA) protein of fowl plague virus A/FPV/Rostock/34 (H7N1) contains three N-linked oligosaccharide side chains in its stem domain. These stem glycans, which are attached to the Asn residues at positions 12, 28 and 478, are highly conserved throughout all HA protein sequences analysed to date. In a previous study, in which mutant HA proteins lacking individual stem glycosylation sites had been expressed from an SV-40 vector, it was shown that these glycans maintain the HA protein in the metastable form required for fusion activity. In the present study, the functional role of the stem N-glycans for virus replication was investigated using recombinant influenza viruses generated by an RNA polymerase I-based system. Studies in Madin–Darby canine kidney cells and embryonated chickens’ eggs revealed that the N-glycan at Asn12 is crucial for virus replication. In both culture systems, growth of virus lacking this glycan (mutant cg1) was completely blocked at 37 °C and inhibited at 33 °C. Loss of the glycan from Asn478 (mutant cg3) caused less striking, but still measurable, effects. Interestingly, it was not possible to generate mutant viruses containing the HA protein lacking the N-glycan at Asn28. It is concluded from this that the N-glycan at Asn28 is indispensable for the formation of replication-competent influenza viruses. When compared to viruses containing wild-type HA protein, mutants cg1 and cg3 showed a significantly decreased pH stability. Taken together, these data show that the HA stem glycans are potent regulators of influenza virus replication.
Six-helix bundle assembly and characterization of heptad repeat regions from the F protein of Newcastle disease virus
Authors: Yu Ming , Wang Enxiu , Liu Youfang , Cao Dianjun , Jin Ningyi , Zhang Catherine W.-H. , Bartlam Mark , Rao Zihe , Tien Po , Gao George F. 
Published: August 2015

Paramyxoviruses may adopt a similar fusion mechanism to other enveloped viruses, in which an anti-parallel six-helix bundle structure is formed post-fusion in the heptad repeat (HR) regions of the envelope fusion protein. In order to understand the fusion mechanism and identify fusion inhibitors of Newcastle disease virus (NDV), a member of the Paramyxoviridae family, we have developed an E. coli system that separately expresses the F protein HR1 and HR2 regions as GST fusion proteins. The purified cleaved HR1 and HR2 have subsequently been assembled into a stable six-helix bundle heterotrimer complex. Furthermore, both the GST fusion protein and the cleaved HR2 show virus–cell fusion inhibition activity (IC50 of 1·07–2·93 μM). The solubility of the GST–HR2 fusion protein is much higher than that of the corresponding peptide. Hence this provides a plausible method for large-scale production of HR peptides as virus fusion inhibitors.
Dual-colour imaging of membrane protein targeting directed by poa semilatent virus movement protein TGBp3 in plant and mammalian cells
Authors: Zamyatnin A. A. , Solovyev A. G. , Sablina A. A. , Agranovsky A. A. , Katul L. , Vetten H. J. , Schiemann J. , Hinkkanen A. E. , Lehto K. , Morozov S. Yu. 
Published: August 2015

The movement function of poa semilatent hordeivirus (PSLV) is mediated by the triple gene block (TGB) proteins, of which two, TGBp2 and TGBp3, are membrane proteins. TGBp3 is localized to peripheral bodies in the vicinity of the plasma membrane and is able to re-direct TGBp2 from the endoplasmic reticulum (ER) to the peripheral bodies. For imaging of TGBp3-mediated protein targeting, PSLV TGBp3 tagged with a red fluorescent protein (DsRed) was used. Coexpression of DsRed-TGBp3 with GFP targeted to the ER lumen (ER-GFP) demonstrated that ER-GFP was contained in typical ER structures and peripheral bodies formed by TGBp3 protein, suggesting an ER origin for these bodies. In transient coexpression with viral membrane proteins tagged with GFP, DsRed-TGBp3 directed to the peripheral bodies the homologous TGBp2 protein and two unrelated membrane proteins, the 6 kDa movement protein of beet yellows closterovirus and the putative movement protein encoded by the genome component 4 of faba bean necrotic yellows nanovirus. However, coexpression of TGBp3 with GFP derivatives targeted to the ER membranes by artificial hydrophobic tail sequences suggested that targeting to the ER membranes per se was not sufficient for TGBp3-directed protein trafficking to peripheral bodies. TGBp3-induced targeting of TGBp2 also occurred in mammalian cells, indicating the universal nature of the protein trafficking signals and the cotargeting mechanism.
Partial redistribution of the Autographa californica nucleopolyhedrovirus chitinase in virus-infected cells accompanies mutation of the carboxy-terminal KDEL ER-retention motif
Authors: Saville Giles P. , Thomas Carole J. , Possee Robert D. , King Linda A. 
Published: August 2015

During virus infection of insect cells, the Autographa californica nucleopolyhedrovirus chitinase is localized primarily within the endoplasmic reticulum (ER), which is consistent with the presence of a carboxy-terminal ER retention motif (KDEL). Release of chitinase into the extracellular medium appears to be concomitant with terminal cell lysis, rather than by active secretion. In this study, we have shown that mutation of the KDEL motif induces a partial redistribution of the chitinase at both early and late times post-infection. Deletion of the KDEL motif or substitution with glycine residues allowed chitinase to move through the secretory pathway, accumulating to detectable levels in the extracellular medium by 24 h post-infection; more than 48 h prior to cell lysis. Deletion of the KDEL motif did not compromise enzyme activity, with the modified enzyme exhibiting characteristic endo- and exo-chitinolytic activity. Trichoplusia ni larvae infected with the modified virus were found to liquefy approximately 24 h earlier than larvae infected with a control virus in which the chitinase KDEL motif had not been deleted.
Transcriptional mapping of two genes encoding baculovirus envelope-associated proteins
Authors: Pearson Margot N. , Russell Rebecca L. Q. , Rohrmann George F. 
Published: August 2015

Genes encoding two representatives of the LD130 family of baculovirus envelope-associated proteins were transcriptionally mapped. These included ld130, which encodes a low pH-induced envelope fusion protein of the Lymantria dispar multinucleocapsid nucleopolyhedrovirus, and op21, which is related to ld130 but is encoded by Orgyia pseudotsugata MNPV and appears to lack an envelope fusion activity. The size and temporal expression of mRNA of both genes were examined by Northern blot analysis of RNA extracted from infected cells at selected timepoints. In addition, 5′ rapid amplification of cDNA ends (RACE) in combination with DNA sequence analysis was used to map the start sites of mRNA. Ld130 predominately utilized its early promoter at 24 h post-infection but by 72 h post-infection ld130 expression was almost exclusively from its late promoter. In contrast, op21 was expressed predominantly from its early promoter throughout the timecourse, even though a consensus late promoter sequence was present within 100 bp of the translation start codon. A significant fraction of late transcripts that mapped to op21 were spliced transcripts originating in the op18 gene region. The 3′ termini of the transcripts were also mapped using 3′ RACE.
Epstein–Barr virus latent membrane protein 2B (LMP2B) co-localizes with LMP2A in perinuclear regions in transiently transfected cells
Authors: Lynch David T. , Zimmerman Jeffrey S. , Rowe David T. 
Published: August 2015

Epstein–Barr virus is a human gammaherpesvirus that infects and establishes latency in B lymphocytes in vivo. The latent membrane protein 2 (LMP2) gene is expressed in latently infected B cells and encodes two protein isoforms, LMP2A and LMP2B, that are identical except for an additional N-terminal 119 aa cytoplasmic domain which is present in the LMP2A isoform. A panel of fusion proteins was constructed in which the fluorescent enhanced green fluorescent protein and DsRed protein domains were fused to the N- and C-termini of LMP2A and LMP2B. By fluorescence microscopy, LMP2B localized to perinuclear regions of both live and fixed transiently transfected cells. Co-localization was detected with markers for the endoplasmic reticulum and the trans-Golgi network. No evidence of co-localization of LMP2B with endosomes or surface expression was obtained. Transiently expressed LMP2B co-localized with transiently or constitutively expressed LMP2A. Confocal microscopy confirmed that LMP2A proteins localized to intracellular perinuclear compartments with markers for the trans-Golgi network. Only LMP2A proteins with C-terminal truncations were detected in the plasma membrane with extracellular loop1 epitope tags. These results indicate that the transmembrane domain of LMP2 proteins possess intracellular retention signals and suggest that LMP2A-mediated signalling effects are likely to be ectopic, originating from sites inside the cell close to the nucleus.
Identification and localization of a prawn white spot syndrome virus gene that encodes an envelope protein
Authors: Zhang Xiaobo , Huang Canhua , Xu Xun , Hew Choy L. 
Published: August 2015

Among the important challenges to shrimp aquaculture worldwide are the diseases caused by viruses, in particular by white spot syndrome virus (WSSV), which has a genome estimated to contain 305 kb. By analysis and comparison of the WSSV genomic DNA and cDNA libraries, an ORF (vp28 gene) was identified. The gene, encoding a novel 204-amino-acid protein, was expressed in Escherichia coli and purified. A specific antibody was raised using the purified VP28 protein. After inoculation of healthy adult Penaeus monodon shrimp with WSSV, the gene transcript and VP28 protein were first detected at low levels at 6 and 18 h post-infection, respectively. These experiments suggest that it might be a late gene. Immuno-electron microscopy with gold-labelled antibody revealed that the gold particles were distributed in the outer envelope of WSSV virions and showed that vp28 encodes a virus envelope protein.
Long-range RNA–RNA interactions between distant regions of the hepatitis C virus internal ribosome entry site element
Authors: Lafuente Esther , Ramos Ricardo , Martínez-Salas Encarnación 
Published: August 2015

Efficient internal initiation of translation from the hepatitis C virus (HCV) internal ribosome entry site (IRES) requires sequences of domain II, but the precise role of these sequences is still unknown. In this study, the formation of RNA–RNA complexes in the HCV IRES was evaluated. Using transcripts that contain the sequences of the structural HCV IRES domains II, IIIabcd, IIIabc, IV and IIIef-IV, specific long-range interactions between domains II and IV, as well as domains II and IIIabcd, have been found. These interactions were readily detected in a gel mobility-shift assay and required the presence of magnesium ions. A high concentration of nonspecific competitors, an 80 nt fragment of 18S rRNA or poly(I:C), did not interfere with the formation of RNA complexes. Interestingly, an RNA oligonucleotide bearing the sequence of stem–loop IIId interacted with domain II but not with domain IV or IIIef-IV, strongly suggesting that the interaction between domains II and IIIabcd was mediated by the IIId hairpin. Interaction between domains IIIabcd and IV was barely detected, consistent with the result that the apical part of domain III folds independently of the rest of the IRES. Moreover, the addition of stem–loop IIIef sequences to domain IV significantly reduced its ability to interact, which is in agreement with the formation of a compact RNA structure of domain IV with IIIef. The interactions observed in the absence of proteins between domains II and IV as well as stem–loop IIId and domain II may be transient, having a regulatory role in the translation efficiency of the HCV IRES.
The products of human cytomegalovirus genes UL23, UL24, UL43 and US22 are tegument components
Authors: Adair Richard , Douglas Elaine R. , Maclean Jean B. , Graham Susan Y. , Aitken James D. , Jamieson Fiona E. , Dargan Derrick J. 
Published: August 2015

We have investigated the human cytomegalovirus (HCMV) US22 gene family members UL23, UL24, UL43 and US22. Specific antibodies were generated to identify pUL23 (33 kDa), pUL24 (40 kDa) and pUL43 (48 kDa), while pUS22 was identified by monoclonal antibody HWLF1. A C-terminally truncated UL43 product (pUL43t; 21 kDa) produced by a deletion mutant was also investigated. The UL24 and UL43 genes were expressed with early-late (γ1) and true-late (γ2) kinetics, respectively. Immunoblot and immuno-EM studies demonstrated that pUL23, pUL24, pUL43 and pUS22 were virion tegument components. Immunofluorescence and immuno-EM studies showed that pUL23, pUL24, pUL43 and pUL43t were located in cytoplasmic protein aggregates, manifesting two forms: complex juxtanuclear structures and smaller, membrane-bound aggregates resembling dense bodies. The complex-type aggregate is a putative site of particle maturation. Because pUL43t was present in protein aggregates, but under-represented in virus particles compared to pUL43, it was concluded that N-terminal sequences target pUL43 to protein aggregates and that C-terminal sequences are important for incorporation into particles. Since three other US22 family products (pUL36, pTRS1 and pIRS1) are documented tegument components, at least seven of the twelve US22 family genes encode tegument proteins, suggesting that the products of the remaining five genes might be similarly located. These findings demonstrate a common biological feature among most, if not all, US22 family proteins and implicate the family in events occurring immediately after virus penetration.