bluuebunny/crossref_metadata_2025_split huggingface dataset:
A novel negative cis-regulatory element on the hepatitis B virus S-(+)-strand
Authors: Wagner Markus , Alt Michael , Hofschneider Peter Hans , Renner Matthias 
Published: August 2015

Hepatitis B virus (HBV) has a double-stranded DNA genome. The minus-strand contains coding regions for all known HBV proteins and most of thecis-regulatory elements. Little is known about transcription from the S-(+)-strand and its regulation. Thus, the presence of regulatory elements located on the S-(+)-strand was investigated by inserting nt 1038–1783 of HBV in both orientations between the human cytomegalovirus (HCMV) promoter and a luciferase gene. Transfection experiments revealed that the plasmid containing this HBV DNA fragment in an orientation allowing expression from the S-(+)-strand (antisense) led to inhibition of luciferase gene expression compared to the plasmid containing this sequence in an orientation that allows gene expression from the L-(−)-strand (sense). Deletion analyses delimit the sequence essential for the inhibitory effect to a 150 bp region that also carries part of the enhancerII/core promoter complex. However, the possible influence of this regulatory element has been excluded in various experiments. The repressing HBV sequence acts in an orientation- and position-dependent manner; no inhibition was observed when this DNA element was inserted upstream of the HCMV promoter or downstream of the luciferase gene. Northern blot analyses revealed reduced luciferase mRNA steady-state levels in cells transfected with constructs containing the essential HBV sequence in antisense orientation compared to plasmids containing this sequence in sense orientation. Since nuclear run-on experiments showed similar transcription initiation rates with these plasmids, the diminished luciferase mRNA steady-state levels must be due to altered stabilities, suggesting that nt 1783–1638 of HBV encode an RNA-destabilizing element.
Temporal mapping of transcripts in human herpesvirus-7
Authors: Menegazzi Paola , Galvan Monica , Rotola Antonella , Ravaioli Tullia , Gonelli Arianna , Cassai Enzo , Di Luca Dario 
Published: August 2015

Transcription of human herpesvirus-7 (HHV-7) in cultures of productively infected T-cells was studied. Transcription of HHV-7 was regulated by the typical herpesvirus cascade in which α, β and γ genes are sequentially transcribed. Transcripts of U10, U14, U18, U31, U39, U41, U42, U53, U73 and U89/90 were detected 3 h after infection and were not inhibited by the absence of protein synthesis and therefore were α functions. U19 and U18/20 were β genes; their transcription was inhibited by cycloheximide but not by phosphonoacetate, an inhibitor of DNA synthesis. U60/66 and U98/100 were γ genes since their spliced transcripts were not detected in cells treated with phosphonoacetate. HHV-7 transcription was regulated by complex mechanisms, which involve the temporal coordinated activation of specific viral promoters and post-transcriptional processing. Splice mechanisms were also temporally regulated. Transcription of U89/90 pre-mRNA and splice took place simultaneously in the immediate-early phase. On the other hand, U16/17 pre-mRNA was synthesized with typical α kinetics, but the spliced product was regulated as a β function. Likewise, the primary transcripts of U60/66 and U98/100 were α and β, respectively, but both spliced products were synthesized in the late phase of virus replication. Finally, HHV-7 supported a bona fide latent infection in the adult population, since viral transcripts were not detected in peripheral blood mononuclear cells of healthy donors infected with HHV-7.
Identification and characterization of bovine herpesvirus type 5 glycoprotein H gene and gene products
Authors: Meyer G. , Bare O. , Thiry E. 
Published: August 2015

Bovine herpesvirus type 5 (BHV-5) is the causative agent of a fatal meningo-encephalitis in calves and is closely related to BHV-1 which causes infectious bovine rhinotracheitis. The gene encoding BHV-5 glycoprotein gH was sequenced. A high degree of conservation was found between BHV-1 and BHV-5 deduced gH amino acid sequences (86·4%), which is also observed for all alphaherpesvirus gH sequences. Transcriptional analysis revealed a 3·1 kb mRNA as the specific gH transcript which was detected 2 h post-infection (p.i.). Twelve out of twenty-one MAbs directed against BHV-1 gH immunoprecipitated a 108–110 kDa glycoprotein, which was then designated BHV-5 gH. Synthesis and intracellular processing of BHV- 5 gH was analysed in infected MDBK cells using gH cross-reacting MAbs. Glycoprotein gH was expressed as a beta-gamma protein, detected by radioimmunoprecipitation as early as 3 h p.i. Glycosylation studies indicated that BHV-5 gH contains N-linked carbohydrates which are essential for the recognition of the protein by the MAbs. This suggests that N-linked glycans are involved in protein folding or are targets for the gH cross-reacting MAbs. Plaque- reduction neutralization assays showed that at least one BHV-1 gH antigenic domain is lacking in BHV-5 which may possibly relate to in vivo differences in virus tropism.
Complete nucleotide sequence, genomic organization and phylogenetic analysis of a novel genital human papillomavirus type, HLT7474-S
Authors: Chow Vincent T. K. , Leong Peter W. F. 
Published: August 2015

A novel human papillomavirus (HPV) type, HLT7474-S, was isolated from a cervical scraping of a female sex worker with a wart virus infection. The complete DNA sequence of 7812 bp was derived from four overlapping PCR products and authenticated by RFLP analysis. The L1 gene exhibited 78% identity to those of its most closely related known HPV types in group A7, comprising HPV types 18, 39, 45, 59, 68 and 70. The genomic organization and phylogenetic analysis of HLT7474-S and group A7 HPVs reiterated their relatedness. Of significance were the strong sequence similarity, phylogenetic relationship and conservation of critical motifs between the transforming E6 and E7 of HLT7474-S and E6 of HPV-18 and E7 of HPV-59, respectively. These features clearly suggest that HLT7474-S is a high-risk genital HPV isolate, closely related to HPV-18 and other members of the A7 group of genital HPVs.
Transcriptional activation of human TR3/nur77 gene expression by human T-lymphotropic virus type I Tax protein through two AP-1-like elements
Authors: Liu Xiangdong , Chen Xiaolin , Zachar Vladimir , Chang Chawnshang , Ebbesen Peter 
Published: August 2015

The Tax transactivator of human T-lymphotropic virus type I (HTLV-I) is capable of inducing expression of the human immediate-earlyTR3/nur77gene. Deletion and mutation analyses of theTR3/nur77promoter demonstrated that multiple transcription elements in the 121 bp sequence proximal to the transcription start site are required for full Tax transactivation. Mutations of CArG-like, Ets and RCE motifs in this region severely decreased Tax transactivation. Mutation of either of the two identical AP-1-like elements (NAP 1 and 2) immediately upstream of the TATA box caused around 80% reduction of Tax transactivation. Mutation of both NAP elements blocked Tax-mediated activation totally. These two NAP elements could confer Tax-responsiveness on a heterologous basal promoter. Furthermore, the specific NAP-binding complex was only observed in HTLV-I-infected cells. Formation of this specific NAP-binding complex was correlated directly with Tax expression, as demonstrated in JPX-9 cells upon induction of Tax expression. The specific NAP binding could be competed for by consensus AP-1 and CREB elements, indicating that the NAP-binding proteins probably belong to the AP-1 and CREB/ATF transcription factor families. Supershift analysis with antibodies to both the AP-1 and CREB/ATF transcription factor families revealed that only anti-JunD antibody could partially shift this NAP-binding complex, indicating that JunD is a component of the NAP complex. This work suggests that JunD is involved in Tax-regulated TR3/nur77 expression.
Genetic stability of equine arteritis virus during horizontal and vertical transmission in an outbreak of equine viral arteritis
Authors: Balasuriya Udeni B. R. , Hedges Jodi F. , Nadler Steven A. , McCollum William H. , Timoney Peter J. , MacLachlan N. James 
Published: August 2015

An imported carrier stallion (A) from Europe was implicated in causing an extensive outbreak of equine viral arteritis (EVA) on a Warmblood breeding farm in Pennsylvania, USA. Strains of equine arteritis virus (EAV) present in the semen of two carrier stallions (A and G) on the farm were compared to those in tissues of foals born during the outbreak, as well as viruses present in the semen of two other stallions that became persistently infected carriers of EAV following infection during the outbreak. The 2822 bp segment encompassing ORFs 2–7 (nt 9807–12628; which encode the GS, GP3, GP4, GL, M and N proteins, respectively) was directly amplified by RT–PCR from semen samples and foal tissues. Nucleotide and phylogenetic analyses confirmed that virus present in the semen of stallion A initiated the outbreak. The genomes of viruses present in most foal tissues (10/11) and serum from an acutely infected mare collected during the outbreak were identical to that of virus present in the lung of the first foal that died of EVA. Virus in the placenta of one foal differed by one nucleotide (99·9% identity) from the predominant outbreak virus. The relative genetic stability of viruses that circulated during the outbreak contrasts markedly with the heterogeneous virus populations variously present in the semen of persistently infected stallions on the farm. These findings are consistent with the hypothesis that the carrier stallion can be a source of genetic diversity of EAV, and that outbreaks of EVA can be initiated by the horizontal aerosol transmission of specific viral variants that occur in the semen of particular carrier stallions.
Localization of a unique hepatitis B virus epitope sheds new light on the structure of hepatitis B virus surface antigen
Authors: Paulij W. P. , de Wit P. L. M. , Sünnen C. M. G. , van Roosmalen M. H. , Petersen-van Ettekoven A. , Cooreman M. P. , Heijtink R. A. 
Published: August 2015

In a search for monoclonal antibodies (MAbs) that can bind hepatitis B virus surface antigen (HBsAg) with amino acid substitutions in the immune dominant ‘a’ region (escape mutants) we investigated the epitope recognition site of the human MAb 4-7B. Pepscan analysis and experiments with alanine substitution as well as substitutions known from nature pointed to residues 178–186 in the small S protein with the amino acid sequence PFVQWFVGL (key amino acids in bold) as the minimal epitope. Single amino acid substitutions at positions 122(R/K)(d/y), 134(Y/F), 145(G/R), 148(T/A) and 160(K/R)(w/r), representing ‘a’ region variants in recombinant HBsAg COS-I cells, did not influence binding of MAb 4-7B. Synthetic peptides (residues 175–189) including the 4-7B epitope sequence were able to evoke an anti-HBs response in rabbits. According to established polypeptide models, the 4-7B epitope region is located in the lipid layer of 20 nm HBsAg particles. The present results, however, suggest that residues 178–186 are exposed on the surface of the 20 nm particle. This may change our view of the structure of HBsAg.
Kinetics of early molecular events in duck hepatitis B virus replication in primary duck hepatocytes
Authors: Qiao M. , Scougall C. A. , Duszynski A. , Burrell C. J. 
Published: August 2015

This paper describes the use of one-step growth conditions to study the kinetics of duck hepatitis B virus (DHBV) replication in primary duck hepatocytes. Synchronized infection was achieved using partially purified DHBV virions at an m.o.i. of 640 DHBV DNA-containing virions per cell, and these conditions were shown to produce a single cycle of infection. In this model, input purified DHBV DNA was rapidly internalized by cells at ⩾0·5 h, and localized to the nucleus by 4 h, but both covalently closed circular (CCC) DNA and single-stranded DNA were not detected until 48 h post-inoculation (p.i.), suggesting that there was a ⩾40 h delay between DHBV localization to the nucleus and formation of CCC DNA. In contrast, CCC DNA can be first detected in hepatocytes at 6 h p.i. in in vivo infection of ducks with the same DHBV strain. In an analysis of the nuclear transport of the DHBV genome, release of nuclear viral DNA from a particulate form to a soluble nucleoplasmic form was only 50% complete by 48 h p.i. However, this process occurred simultaneously with genome uncoating since all soluble nucleoplasmic DHBV DNA was free of nucleocapsid material; this suggests that nucleocapsid disassembly and genome uncoating may occur at the nuclear membrane and not within the nucleus. Quantitative analysis demonstrated inefficiency in a number of steps including virus uptake and internalization, translocation of nucleocapsid across the nuclear membrane and antigen expression from intranuclear viral DNA.
Identification and transcriptional analysis of the homologues of the herpes simplex virus type 1 UL30 to UL40 genes in the genome of nononcogenic Marek’s disease virus serotype 2
Authors: Izumiya Yoshihiro , Jang Hyung-Kwan , Sugawara Mie , Ikeda Yasuhiro , Miura Ryuichi , Nishimura Yorihiro , Nakamura Kazuya , Miyazawa Takayuki , Kai Chieko , Mikami Takeshi 
Published: August 2015

Studies on Marek’s disease virus serotype 2 (MDV2) are important for understanding the natural nononcogenic phenotype of MDV. This study reports a 27535 bp nucleotide sequence of part of the MDV2 genome located in the central unique long (UL) region. The analysis revealed 11 complete ORFs with high amino acid sequence identities to the products of other alphaherpesviruses. The MDV2 ORFs were arranged collinearly with the prototype sequence of herpes simplex virus type 1, ranging from the UL30 to UL40 genes. Sequences that were particularly well conserved among alphaherpesviruses were the putative functional domain of the DNA polymerase (UL30) and the ribonucleotide reductase large and small subunits (UL39 and UL40). On the other hand, in contrast to oncogenic MDV1, MDV2 did not contain the conserved proline-repeat region in the UL36 homologue. All the genes identified were confirmed to be transcribed as 3′-coterminal mRNAs and/or unique transcripts in virus-infected cells.
Identification and characterization of the UL14 gene product of herpes simplex virus type 2
Authors: Wada K. , Goshima F. , Takakuwa H. , Yamada H. , Daikoku T. , Nishiyama Y. 
Published: August 2015

The UL14 gene of herpes simplex virus type 2 (HSV-2) is predicted to encode a 219 amino acid protein with a molecular mass of 23 kDa. In this study, the HSV-2 UL14 gene product has been identified by using a rabbit polyclonal antiserum raised against a recombinant 6×His–UL14 fusion protein expressed in E. coli. The antiserum reacted specifically with 34, 33 and 28 kDa proteins in HSV-2-infected cell lysates and also with a 34 kDa protein produced by in vitro transcription and translation reactions, suggesting that the 34 kDa protein is the primary translation product of the UL14 gene. The protein was synthesized at late times post-infection (p.i.) and was not detectable in the presence of the viral DNA synthesis inhibitor acycloguanosine. Indirect immunofluorescence studies localized the UL14 protein both to the nucleus and to perinuclear regions of the cytoplasm, and the nuclear UL14 protein was found to co-localize with the scaffolding protein ICP35 at 9 h p.i. However, the protein accumulated in a perinuclear region of the cytoplasm at 12 h p.i., while most of the ICP35 protein localized within assemblons in the nucleus. Although no detectable UL14 protein was associated with intracellular capsids isolated in the presence of 0·5 M NaCl, it was detected in purified virions. Furthermore, the UL14 protein expressed alone was detected both in the nucleus and in the cytoplasm at 24 h after transfection, but was mainly localized to the cytoplasm at later times.
Highly regulated expression of adeno-associated virus large Rep proteins in stable 293 cell lines using the Cre/loxP switching system
Authors: Ogasawara Yoji , Mizukami Hiroaki , Urabe Masashi , Kume Akihiro , Kanegae Yumi , Saito Izumu , Monahan John , Ozawa Keiya 
Published: August 2015

Since the Rep proteins of adeno-associated virus (AAV) are harmful to cells, it is difficult to obtain stable cell lines that express them constitutively. In this study, stable 293 cell lines were obtained in which large Rep expression was inducible by using the Cre/loxP switching system. To determine the function of the induced Rep proteins, the packaging capacity was examined after supplementation with a plasmid expressing small Rep and Cap proteins. A significant amount of recombinant AAV (5·5×108 vector particles per 10 cm dish) was produced by transfection with a vector plasmid and infection with Cre-expressing recombinant adenovirus, indicating that the large Rep proteins retained the function required for packaging. These findings indicate that large Rep protein expression can be strictly regulated by the Cre/loxP system and will also serve as a basis for the development of an efficient AAV-packaging cell line.
The vaccinia virus A27L protein is needed for the microtubule-dependent transport of intracellular mature virus particles
Authors: Sanderson Christopher M. , Hollinshead Michael , Smith Geoffrey L. 
Published: August 2015

The vaccinia virus (VV) A27L gene encodes a 14 kDa protein that is required for the formation of intracellular enveloped virus (IEV) and, consequently, normal sized plaques. Data presented here show that A27L plays an additional role in VV assembly. When cells were infected with the VV WR32-7/Ind 14K, under conditions that repress A27L expression, transport of intracellular mature virus (IMV) from virus factories was inhibited and some IMV was found in aberrant association with virus crescents. In contrast, other VV mutants (vΔB5R and vΔF13L) that are defective in IEV formation produce IMV particles that are transported out of virus factories. This indicated a specific role for A27L in IMV transport. Induction of A27L expression at 10 h post-infection promoted the dispersal of clustered IMV particles, but only when microtubules were intact. Formation of IEV particles was also impaired when cells were infected with WR32-7/14K, a VV strain expressing a mutated form of the A27L protein; however, this mutation did not inhibit intracellular transport of IMV particles. Collectively, these data define two novel aspects of VV morphogenesis. Firstly, A27L is required for both IMV transport and the process of envelopment that leads to IEV formation. Secondly, movement of IMV particles between the virus factory and the site of IEV formation is microtubule-dependent.
Cloning and identification of regulatory gene UL76 of human cytomegalovirus
Authors: Wang Shang-Kwei , Duh Chang-Yih , Chang Tai-Tsung 
Published: August 2015

The major immediate-early promoter/enhancer (MIEP, −1139 to +52) of human cytomegalovirus (HCMV) is regulated by cell type-specific transcriptional factors, its own MIE proteins (IE2p40, IE1p55, IE1p72 and IE2p86) as well as viral proteins pUL69, pUL82 and pUL84. To investigate the hypothesis that the regulation of HCMV MIEP is modulated by additional viral genes, HCMV (AD169) genomic sublibraries were constructed and in vitro transient co-transfection assays were performed to assess the ability of these sublibraries to modulate MIEP expression. In this study, enhancement of MIEP expression was exhibited by a number of sublibraries, from one of which a genomic clone was selected for augmentation of expression. Subcloning the insert fragment led to the identification of the responsible locus, UL76. To generate a UL76-specific antibody for immunodetection, the UL76 ORF was constructed as a histidine-tagged fusion protein that was produced in prokaryotic cells. A polyclonal antibody raised against the UL76 fusion protein immunoreacts with a protein of 38 kDa (pUL76) in UL76 ORF-transfected cells. Additionally, pUL76 is present in HCMV-infected cells at the immediate-early to late stages of the reproductive cycle. Characterized by its highly basic composition (predicted pI 11·6), a free form of pUL76 tagged with green fluorescent protein was found to localize exclusively to the nucleus. In this report, pUL76 is defined as a novel regulatory protein that modulates both activation and repression of gene expression, depending on the promoter context and the ratio of transfected effector DNA.
Virus morphogenesis of Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea serum-free suspension culture
Authors: Lua Linda H. L. , Reid Steven 
Published: August 2015

Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) replication in Helicoverpa zea serum-free suspension culture was studied in detail and the sequence of virus morphogenesis was determined by transmission electron microscopy. By 16 h post-infection (p.i.), virus replication was observed in the virogenic stroma by the appearance of nucleocapsids. Polyhedron formation was detected by 24 h p.i. and the polyhedron envelope (PE) was completely formed by 72 h p.i. PE morphogenesis of HaSNPV is significantly different compared to the extensively studied Autograph californica (Ac)MNPV. In AcMNPV-infected cells, fibrillar structures are found in both cytoplasm and nuclei, and the fibrillar structures in nuclei are in close association with maturing polyhedra during PE formation. Fibrillar structures that resemble the AcMNPV fibrillar structures were detected only in the cytoplasm of HaSNPV-infected cells and appeared to interact with calyx precursors there, but their role in PE formation is unclear. However, prominent calyx precursor structures of various shapes and sizes were observed in the nuclei of HaSNPV-infected cells as well, and they appeared to interact with polyhedra during PE formation. Both the calyx precursor structure and the cytoplasmic fibrillar structure were detected only after HaSNPV virion occlusion had started, indicating that they might have a role in formation of PE. Similar calyx precursor structures and cytoplasmic fibrillar structures were observed in both serum-supplemented and serum-free suspension cultures, as well as in HaSNPV-infected larval tissues, indicating that the structures observed are not cell culture artefacts.
Characterization of VPg and the polyprotein processing of Cocksfoot mottle virus (genus Sobemovirus)
Authors: Mäkinen Kristiina , Mäkeläinen Katri , Arshava Natalya , Tamm Tiina , Merits Andres , Truve Erkki , Zavriev Sergei , Saarma Mart 
Published: August 2015

The polyprotein ofCocksfoot mottle virus(CfMV; genusSobemovirus) is translated from two overlapping open reading frames (ORFs) 2a and 2b by a −1 ribosomal frameshifting mechanism. In this study, a 12 kDa protein was purified from viral RNA-derived samples that appears to correspond to the CfMV genome-linked protein (VPg). According to the determined N-terminal amino acid sequence, the VPg domain is located between the serine proteinase and replicase motifs and the N terminus of VPg is cleaved from the polyprotein between glutamic acid and asparagine residues. Western blot analysis of infected plant material showed that the polyprotein is processed at several additional sites. An antiserum against the ORF 2a product recognized six distinct proteins, whereas, of these, the VPg antiserum clearly recognized only a 24 kDa protein. This indicates that the fully processed 12 kDa VPg detected in viral RNA-derived samples is a minor product in infected plants. An antiserum against the ORF 2b product recognized a 58 kDa protein, which indicates that the fully processed replicase is entirely or almost entirely encoded by ORF 2b. The origin of the detected cleavage products and a proposed polyprotein processing model are discussed.