bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of HLA DR13-restricted CD4+ T cell epitopes of hepatitis B core antigen associated with self-limited, acute hepatitis B
Authors: Cao Tinghua , Desombere Isabelle , Vanlandschoot Peter , Sällberg Matti , Leroux-Roels Geert
Published: August 2015

The HLA DR13 allele has been associated with a self-limited course of hepatitis B virus infection, possibly through the induction of a more vigorous hepatitis B core antigen (HBcAg) and/or hepatitis B e antigen-specific CD4+ T cell response. HBcAg-specific CD4+ T cell responses were investigated in three HLA DR13-positive subjects with self-limited, acute hepatitis B. HBcAg-specific, short-term T cell lines derived from these three subjects showed a dominant recognition of HBcAg peptides spanning aa 1–20 (P1), 11–30 (P2), 41–60 (P5), 111–131 (P12) and 141–160 (P15). In order to characterize these epitopes in more detail, CD4+ T cell clones and cell lines were generated using HBcAg. Surprisingly, 11 of 12 T cell clones examined recognized P15; one recognized P10 (aa 91–111). Of four T cell lines, two recognized P15 and two recognized P5. By peptide mapping, the minimal epitope of P15 was located to residues 147TVVRRRGRSP156.
Role of overlapping glycosylation sequons in antigenic properties, intracellular transport and biological activities of influenza A/H2N2 virus haemagglutinin
Authors: Tsuchiya Emi , Sugawara Kanetsu , Hongo Seiji , Matsuzaki Yoko , Muraki Yasushi , Nakamura Kiyoto
Published: August 2015

The haemagglutinin (HA) protein of influenza A/H2N2 virus possesses five oligosaccharide attachment sites, two of which have overlapping glycosylation sequons at positions 20–23 (NNST) and 169–172 (NNTS). Here, the role of these two oligosaccharide attachment sites is investigated with regard to antigenic property, intracellular transport and biological activity of the HA protein. Glycosylation-site HA mutants with mutation(s) in their overlapping glycosylated sequons, each of which had one or two oligosaccharide attachment sites removed, were constructed. Comparison of electrophoretic mobility between the wt and mutant HA proteins showed that both Asn residues 20 and 21 and Asn residues 169 and 170 could be used for glycosylation. Analysis of reactivity of the mutants with anti-HA monoclonal antibodies suggested that amino acid changes at these two positions result in a conformational change of the HA molecule. Even if oligosaccharide chains linked to Asn 20 or 21 and Asn 169 or 170 are eliminated, the antigenic properties, intracellular transport and biological activities are not influenced strongly. Thus it is reasonable to conclude that the two overlapping glycosylation sequons at positions 20–23 and 169–172 are conserved among all of the HAs of influenza A/H2N2 viruses because conservation of the amino acid sequence itself rather than that ofN-glycosylation is essential for the formation of the proper conformation, intracellular transport and biological activities of the H2 subtype HA.
Genetic recombination in wild-type poliovirus
Authors: Dahourou George , Guillot Sophie , Le Gall Olivier , Crainic Radu
Published: August 2015

Poliovirus isolates were screened for recombinants by combined analysis of two distant polymorphic segments of the poliovirus genome (one in the capsid and the other in the polymerase-coding region). Using a restriction fragment length polymorphism (RFLP) assay, a high number of recombinant genomes was found among vaccine-derived strains excreted by poliovirus vaccine vaccinees or vaccine-associated paralytic poliomyelitis cases. Some of these subjects carried a wild-type poliovirus (non-vaccine-specific) nucleotide sequence in the 3′ part of the genome. Using a similar approach, a collection of wild-type poliovirus strains isolated in South India between 1985 and 1993 was screened for recombinants. Genotypes were defined by the parallel application of RFLP assays and genomic sequencing of the capsid protein VP1 and the 3D polymerase polypeptide. Analyses revealed several instances where the position of an isolate on the phylogenic tree for the capsid protein-coding segment did not agree with its position on the tree for the polymerase-coding region. In this way, several wild-type/wild-type and wild-type/vaccine recombinants could be identified, indicating that recombination is encountered commonly in the natural evolution of poliovirus strains.
Development of infectious transcripts and genome manipulation of Black queen-cell virus of honey bees
Authors: Benjeddou Mongi , Leat Neil , Allsopp Mike , Davison Sean
Published: August 2015

The South African isolate of Black queen-cell virus (BQCV), a honey bee virus, was previously found to have an 8550 nucleotide genome excluding the poly(A) tail. Its genome contained two ORFs, a 5′-proximal ORF encoding a putative replicase protein and a 3′-proximal ORF encoding a capsid polyprotein. Long reverse transcription (RT)–PCR was used to produce infectious transcripts for BQCV and to manipulate its genome. Primers were designed for the amplification of the complete genome, the in vitro transcription of infectious RNA and PCR-directed mutagenesis. An 18-mer antisense primer was designed for RT to produce full-length single-stranded cDNA (ss cDNA). Unpurified ss cDNA from the RT reaction mixture was used directly as a template to amplify the full genome by long high-fidelity PCR. The SP6 promoter sequence was introduced into the sense primer to transcribe RNA directly from the amplicon. RNA was transcribed in vitro with and without the presence of a cap analogue and injected directly into bee pupae, which were then incubated for 8 days. In vitro transcripts were infectious but the presence of a cap analogue did not increase the amount of virus recovered. A single base mutation abolishing an EcoRI restriction site was introduced by fusion-PCR, to distinguish viral particles recovered from infectious transcripts from wild-type virus (wtBQCV). Mutant virus (mutBQCV) and wtBQCV were indistinguishable by electron microscopy and Western blot analysis. The EcoRI restriction site was present in wtBQCV and not in mutBQCV.
Capacity of simian immunodeficiency virus strain mac Nef for high-affinity Src homology 3 (SH3) binding revealed by ligand-tailored SH3 domains
Authors: Hiipakka Marita , Saksela Kalle
Published: August 2015

The simian immunodeficiency virus (SIV) Nef protein contains a consensus Src-homology 3 (SH3) binding motif. However, no SH3-domain proteins showing strong binding to SIV Nef have yet been found, and its potential capacity for high-affinity SH3 binding has therefore remained unproven. Here we have used phage-display-assisted protein engineering to develop artificial SH3 domains that bind tightly to SIV strain mac (SIVmac) Nef. Substitution of six amino acids in the RT loop region of Hck-SH3 with the sequence E/DGWWG resulted in SH3 domains that boundin vitroto SIVmac Nef much better than the natural Hck- or Fyn-SH3 domains. These novel SH3 domains also efficiently associated with SIVmac Nef when co-expressed in 293T cells and displayed a strikingly differential specificity when compared with SH3 domains similarly targeted for binding to human immunodeficiency virus type 1 (HIV-1) Nef. Thus, SIVmac Nef is competent for high-affinity SH3 binding, but its natural SH3 protein partners are likely to be different from those of HIV-1 Nef.
Thermostability of mouse-passaged BSE and scrapie is independent of host PrP genotype: implications for the nature of the causal agents
Authors: Taylor David M. , Fernie Karen , Steele Philip J. , McConnell Irene , Somerville Robert A.
Published: August 2015

Five experimentally maintained strains of scrapie and BSE agents have been passaged in two PrP genotypes of mice. Brain macerates were autoclaved at 126 °C and the levels of surviving infectivity were measured by titration. There was a large difference in the survival properties of transmissible spongiform encephalopathy (TSE) infectivity between TSE strains. PrP genotype had little effect. Phenotypic properties of the TSE strains were not affected with the exception that with one strain (ME7), incubation periods of the heated sample were longer than the controls given equivalent doses. It is concluded that PrP is probably not responsible for differences in thermostability between strains. More likely, a host-independent molecule which differs in covalent structure between strains accounts for these properties.
Replacing the SCR domains of vaccinia virus protein B5R with EGFP causes a reduction in plaque size and actin tail formation but enveloped virions are still transported to the cell surface
Authors: Rodger Gaener , Smith Geoffrey L.
Published: August 2015

A vaccinia virus (VV) recombinant is described in which the outer envelope of extracellular enveloped virus (EEV), cell-associated enveloped virus (CEV) and intracellular enveloped virus (IEV) is labelled with the enhanced green fluorescent protein (EGFP) derived fromAequorea victoria. To construct this virus, EGFP was fused to the VV B5R protein from which the four short consensus repeats (SCRs) of the extracellular domain had been deleted. Cells infected with the recombinant virus expressed a B5R–EGFP fusion protein of 40 kDa that was present on IEV, CEV and EEV, but was absent from IMV. The recombinant virus produced 2- and 3-fold reduced levels of IMV and EEV, respectively. Analysis of infected cells by confocal microscopy showed that actin tail formation by the mutant virus was reduced by 86% compared to wild-type (WT). The virus formed a small plaque compared to WT, consistent with a role for actin tails in promoting cell-to-cell spread of virus. However, the enveloped virions were still transported to the cell surface, confirming that this process is independent of actin tail formation. Lastly, we compared the mutant virus with a recombinant VV in which the B5R SCR domains were deleted and show that, contrary to a previous report, the plaque size of the latter virus was reduced compared to WT. This observation reconciles an inconsistency in the field and confirms that viruses deficient in formation of actin tails form small plaques.
Trans-complementation of a genetic defect in the coxsackie B3 virus 2B protein
Authors: van Kuppeveld Frank J. M. , van den Hurk Patrick J. J. C. , Schrama Ina W. J. , Galama Jochem M. D. , Melchers Willem J. G.
Published: August 2015

The enterovirus 2B protein contains a putative amphipathic α-helix that includes three positively charged and one negatively charged residue. Previously, we observed that replacement of the glutamic acid-40 residue with a lysine residue (mutation 2B-E[40]K) in the amphipathic α-helix of the coxsackie B3 virus 2B protein resulted in a quasi-infectious phenotype. On one occasion, however, transfection of 2B-E[40]K RNA transcripts gave rise to a virus stock in which the mutation was retained. This study was aimed at elucidating the molecular mechanism underlying this observation. Sequence analysis of the viral RNA provided no evidence for a second-site suppression mutation that rescued the defect of the 2B-E[40]K mutation in cis. Therefore, the possibility was considered that the defect caused by the 2B-E[40]K mutation was complemented in trans by viable revertants that had emerged in the virus population. The transfection-derived virus stock indeed contained a small fraction of (pseudo)revertant viruses, carrying the original glutamic acid-40, threonine-40 or asparagine-40, rather than the introduced lysine-40. Consistent with the idea that the 2B-E[40]K virus is unable to grow without the aid of trans-acting wild-type(-like) proteins, only the (pseudo)revertant viruses were able to produce individual plaques. Further support for the idea of trans-rescue was obtained using a genetic complementation assay, which revealed the occurrence of a low level of trans-complementation of the 2B-E[40]K mutation by wild-type virus. This is the first report that provides evidence that a genetic defect in the enterovirus 2B protein can be complemented in trans.
Efficient delivery and regulable expression of hepatitis C virus full-length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors
Authors: McCormick Christopher J. , Rowlands David J. , Harris Mark
Published: August 2015

Baculovirus vectors have been used as efficient delivery vehicles for constitutive gene expression in a variety of mammalian cells. We have further developed the system to allow for regulable expression by placing the gene of interest under the control of an inducible promoter, and complementing it with a second baculovirus vector providing the control elements necessary for promoter activity. We have used this system to express (a) the lacZ gene, (b) a ‘minigenome’ derived from hepatitis C virus (HCV) and carrying lacZ or (c) the full-length HCV viral genome, in human hepatocyte cell lines in an inducible fashion. Control systems that rely on either the absence of tetracycline or presence of ponasterone to induce gene expression were tested. Expression of lacZ was controlled by ponasterone, but β-galactosidase activity was limited to 10–20% of cells. In contrast, the tetracycline-controlled expression system gave a low basal activity and was highly inducible in almost 100% of cells. Inducible expression was also obtained in almost 100% of cells infected with baculoviruses in which an HCV minigenome was placed downstream of the tetracycline-inducible promoter and upstream of either a hammerhead or hepatitis δ virus ribozyme. Northern blot analysis was consistent with accurate cleavage of the minigenome transcript by the hepatitis δ virus ribozyme. Finally, regulable transcript production and viral polypeptide processing could be demonstrated in HepG2 cells infected with baculoviruses bearing the full-length HCV genome. This system thus provides a novel tool for the analysis of HCV replication and host–cell interactions.
The sialate-4-O-acetylesterases of coronaviruses related to mouse hepatitis virus: a proposal to reorganize group 2 Coronaviridae
Authors: Wurzer Walter Juergen , Obojes Karola , Vlasak Reinhard
Published: August 2015

Group 2 coronaviruses are characterized within the order Nidovirales by a unique genome organization. A characteristic feature of group 2 coronaviruses is the presence of a gene encoding the haemagglutinin–esterase (HE) protein, which is absent in coronaviruses of groups 1 and 3. At least three coronavirus strains within group 2 expressed a structural protein with sialate-4-O-acetylesterase activity, distinguishing them from other members of group 2, which encode an enzyme specific for 5-N-acetyl-9-O-acetylneuraminic acid. The esterases of mouse hepatitis virus (MHV) strains S and JHM and puffinosis virus (PV) specifically hydrolysed 5-N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2) as well as the synthetic substrates p-nitrophenyl acetate, 4-methylumbelliferyl acetate and fluorescein diacetate. The K

m
values of the MHV-like esterases for the latter substrates were two- to tenfold lower than those of the sialate-9-O-acetylesterases of influenza C viruses. Another unspecific esterase substrate, α-naphthyl acetate, was used for the in situ detection of the dimeric HE proteins in SDS–polyacrylamide gels. MHV-S, MHV-JHM and PV bound to horse serum glycoproteins containing Neu4,5Ac2. De-O-acetylation of the glycoproteins by alkaline treatment or incubation with the viral esterases resulted in a complete loss of recognition, indicating a specific interaction of MHV-like coronaviruses with Neu4,5Ac2. Combined with evidence for distinct phylogenetic lineages of group 2 coronaviruses, subdivision into subgroups 2a (MHV-like viruses) and 2b (bovine coronavirus-like viruses) is suggested.
Defective segment 1 RNAs that interfere with production of infectious influenza A virus require at least 150 nucleotides of 5′ sequence: evidence from a plasmid-driven system
Authors: Duhaut S. D. , Dimmock N. J.
Published: August 2015

The presence of at least 80–90 and more typically around 200 nucleotides (nt) at the 5′ end of the virion-sense RNA in all naturally occurring defective influenza A virus RNAs suggests that this is essential sequence, whereas the 3′-end sequence may be as short as 25 nt. The stability of defective RNA on serial passage with infectious helper virus also depends on the length of 5′-end sequence. Here, we have studied the influence of 5′-end sequences of a panel of six defective segment 1 RNAs from H3N8 and H7N7 viruses on their ability to interfere with the multiplication of plasmid-produced infectious A/WSN virus (H1N1). Four of the H3N8 defective RNAs are identical in overall length but vary in the length of 5′ sequence. Transfected defective RNAs interfered with infectious virus production in a concentration-dependent manner. The extent of interference also depended on the length of 5′-end sequence in the defective genome. This required at least 150 nt and was maximal with 220 nt of 5′ end sequence. The reduction in virus multiplication was highly significant and correlated with the presence of detectable intracellular defective RNA. Packaging of full-length segment 1 RNA by progeny virus was inversely proportional to the packaging of defective segment 1 RNA and may explain the reduction in infectivity. In summary, a critical length of 5′-end sequence is essential for the interfering properties of defective influenza virus RNAs, which indicates that this plays some vital role in the virus life cycle.
Virus-specific CTL responses induced by an H-2Kd-restricted, motif-negative 15-mer peptide from the fusion protein of respiratory syncytial virus
Authors: Jiang Shisong , Borthwick Nicola J. , Morrison Paul , Gao George F. , Steward Michael W.
Published: August 2015

We describe 15-mer peptide P8:F92–106 from the F protein of respiratory syncytial virus (RSV) that can act as an MHC class I-restricted (H-2Kd) epitope for RSV-specific CD8+CTL. This peptide is interesting because not only is it the first murine CTL epitope to be identified in the F protein but also because it does not contain a known allele-specific motif, as all 15 amino acids appear to be required for effective presentation to CTL. Inin vitroMHC class I refolding experiments, peptide P8:F92–106 induced complex formation with H-2Kdheavy chains and β2-microglobulin. Immunization of BALB/c mice with P8:F92–106 resulted in the induction of peptide and RSV-specific CTL responses as well as peptide-specific proliferative responses. Following intranasal challenge with RSV, P8:F92–106-immunized mice showed a significant reduction in viral load in the lungs compared to that seen in unimmunized mice. Furthermore, passive transfer of purified CD8+lymphocytes into BALB/cscidmice prior to challenge with RSV also resulted in a reduction in the virus load in lungs of challenged mice. These results indicate the potential of synthetic peptide epitopes for the induction of protective immune responses against RSV infection.
Fv1-like restriction of N-tropic replication-competent murine leukaemia viruses in mCAT-1-expressing human cells
Authors: Aagaard Lars , Mikkelsen Jacob Giehm , Warming Søren , Duch Mogens , Pedersen Finn Skou
Published: August 2015

To study the replication of murine leukaemia viruses in human cells we have used full-length as well as EGFP-tagged ecotropic viruses in combination with mCAT-1-expressing human cells. We present results showing that N-tropic murine leukaemia viruses are restricted in both infection and replication in such cells while B-tropic viruses, modified at capsid position 110, escape restriction. These results support a recently reported Fv1-like restriction in mammalian cells. We extend the analysis of Fv1-like restriction by demonstrating that NB-tropic viruses also escape restriction and human mCAT-1-expressing cells are thus similar to murine Fv1

b
cells with respect to infection though the ecotropic receptor pathway.
Assessment of foreign protein production by recombinant Heliothis (Helicoverpa) armigera entomopoxviruses in Spodoptera frugiperda cells
Authors: Olszewski Julie A. , Dall David J.
Published: August 2015

This report describes the first production of recombinant forms of Heliothis (Helicoverpa) armigera entomopoxvirus (HaEPV). These HaEPVs are engineered at either the spheroidin or fusolin locus, to produce the green fluorescent marker protein (GFP). The growth properties of these recombinant HaEPVs, in comparison to the parental HaEPV, were assessed in cultured Spodoptera frugiperda Sf9 cells. Additionally, GFP production by these recombinant HaEPVs was compared to that of a GFP-expressing recombinant of the baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) in the same in vitro system, at various multiplicities of infection. Expression of GFP from the HaEPV spheroidin locus produced up to 60% of that generated from the AcNPV polyhedrin locus, albeit over a longer period of infection. A considerably lower yield was recorded from the HaEPV fusolin locus, a result that contrasted markedly with the apparent activity of this promoter in caterpillar infections in vivo. The potential applications for further development of HaEPV expression systems are discussed.
Cytolytic viruses as potential anti-cancer agents
Authors: Ring Christopher J. A.
Published: August 2015

The resistance of cancers to conventional therapies has inspired the search for novel strategies. One such approach, namely gene therapy, is based upon the introduction of genes such as those encoding suicide proteins, tumour suppressor proteins or cytokines into tumour cells by means of a genetic vector. The efficiency with which viruses transfer their genes from one host cell to another has led to the widespread use of viruses as genetic vectors. For safety reasons, such virus vectors are generally replication-defective but, unfortunately, this has limited the efficacy of treatment by restricting the number of cells to which the therapeutic gene is delivered. For this reason, the use of replication-competent viruses has been proposed, since virus replication would be expected to lead to amplification and spread of the therapeutic genes in vivo. The replication of many viruses results in lysis of the host cells. This inherent cytotoxicity, together with the efficiency with which viruses can spread from one cell to another, has inspired the notion that replication-competent viruses could be exploited for cancer treatment. Some viruses have been shown to replicate more efficiently in transformed cells but it is unlikely that such examples will exhibit a high enough degree of tumour selectivity, and hence safety, for the treatment of patients. Our increasing knowledge of the pathogenesis of virus disease and the ability to manipulate specific regions of viral genomes have allowed the construction of viruses that are attenuated in normal cells but retain their ability to lyse tumour cells. Such manipulations have included modifying the ability of viruses to bind to, or replicate in, particular cell types, while others have involved the construction of replication-competent viruses encoding suicide proteins or cytokines. Naturally occurring or genetically engineered oncolytic viruses based upon adenovirus, herpes simplex virus, Newcastle disease virus, poliovirus, vesicular stomatitis virus, weasles virus and reovirus have been described. The results of animal studies are encouraging and a number of viruses are now being evaluated in clinical trials.
The hinge region of the human papillomavirus type 8 E2 protein activates the human p21WAF1/CIP1 promoter via interaction with Sp1
Authors: Steger Gertrud , Schnabel Carsta , Schmidt Hanns-Martin
Published: August 2015

The E2 proteins regulate papillomavirus (PV) gene expression by sequence-specific DNA binding. However, E2 is also able to activate in the absence of E2 binding sites. We show here that the E2 protein of human PV type 8 (HPV8) can activate the expression of p21WAF1/CIP1 via promoter-proximal 200 nucleotides, which contain several Sp1 binding sites and no E2 binding sites. HPV8 E2 lacking the activation domain, which is rather conserved among E2 proteins, cooperated with co-expressed Sp1 in stimulation of the p21WAF1/CIP1 promoter, in contrast to HPV18 E2 lacking the activation domain. We can demonstrate that the internal non-conserved hinge region of HPV8 E2 is sufficient for this functional cooperativity with Sp1. In correlation, the hinge of HPV8 E2 directly binds to Sp1. These results suggest that HPV8 E2 might be able to ‘super’-activate Sp1-mediated transcription by a direct interaction via the non-conserved hinge region.
Analysis of DNA binding by the adenovirus type 5 E1A oncoprotein
Authors: Avvakumov Nikita , Sahbegovic Majdina , Zhang Zhiying , Shuen Michael , Mymryk Joe S.
Published: August 2015

Adenovirus type 5 E1A proteins interact with cellular regulators of transcription to reprogram gene expression in the infected or transformed cell. Although E1A also interacts with DNA directlyin vitro, it is not clear how this relates to its functionin vivo. The N-terminal conserved regions 1, 2 and 3 and the C-terminal portions of E1A were prepared as purified recombinant proteins and analyses showed that only the C-terminal region bound DNAin vitro. Deletion of E1A amino acids 201–220 inhibited binding and a minimal fragment encompassing amino acids 201–218 of E1A was sufficient for binding single- and double-stranded DNA. This portion of E1A also bound the cation-exchange resins cellulose phosphate and carboxymethyl Sepharose. As this region contains six basic amino acids,in vitrobinding of E1A to DNA probably results from an ionic interaction with the phosphodiester backbone of DNA. Studies inSaccharomyces cerevisiaehave shown that expression of a strong transcriptional activation domain fused to a DNA-binding domain can inhibit growth. Although fusion of the C-terminal region of E1A to a strong transcriptional activation domain inhibited growth when expressed in yeast, this was not mediated by the DNA-binding domain identifiedin vitro. These data suggest that E1A does not bind DNAin vivo.
Conservation of substrate specificities among coronavirus main proteases
Authors: Hegyi Annette , Ziebuhr John
Published: August 2015

The key enzyme in coronavirus replicase polyprotein processing is the coronavirus main protease, 3CLpro. The substrate specificities of five coronavirus main proteases, including the prototypic enzymes from the coronavirus groups I, II and III, were characterized. Recombinant main proteases of human coronavirus (HCoV), transmissible gastroenteritis virus (TGEV), feline infectious peritonitis virus, avian infectious bronchitis virus and mouse hepatitis virus (MHV) were tested in peptide-basedtrans-cleavage assays. The determination of relative rate constants for a set of corresponding HCoV, TGEV and MHV 3CLprocleavage sites revealed a conserved ranking of these sites. Furthermore, a synthetic peptide representing the N-terminal HCoV 3CLprocleavage site was shown to be effectively hydrolysed by noncognate main proteases. The data show that the differential cleavage kinetics of sites within pp1a/pp1ab are a conserved feature of coronavirus main proteases and lead us to predict similar processing kinetics for the replicase polyproteins of all coronaviruses.
Factors determining vector competence and specificity for transmission of Tomato spotted wilt virus
Authors: Nagata Tatsuya , Inoue-Nagata Alice K. , van Lent Jan , Goldbach Rob , Peters Dick
Published: August 2015

The competence of a Frankliniella occidentalis and a Thrips tabaci population to transmit Tomato spotted wilt virus (TSWV) was analysed. Adults of the F. occidentalis population transmitted this virus efficiently, whereas those of the thelytokous T. tabaci population failed to transmit. TSWV replicated in the midgut of the larvae of both populations after ingestion of virus; however, lower amounts accumulated in T. tabaci larvae than in F. occidentalis larvae. The virus was almost undetectable in T. tabaci adults, whereas high titres were readily detected in the F. occidentalis adults. The first infections in F. occidentalis larvae were detected by immunocytochemical studies in midgut epithelial and subsequently in midgut muscle cells, the ligaments, and finally in the salivary glands. The infections were weaker in the midgut epithelial and muscle cells of T. tabaci larvae, followed by an almost complete absence of any infection in the ligaments, and a complete absence in the salivary glands. Studies by electron microscopy revealed the budding of some virus particles from the basal membrane of midgut epithelial cells of F. occidentalis larvae into the extracellular space of the basal labyrinth. Enveloped virus particles were also seen in midgut muscle cells of F. occidentalis larvae. They were not discerned in epithelial and muscle cells of T. tabaci larvae and adults. This study showed that the rate of virus replication in the midgut and the extent of virus migration from the midgut to the visceral muscle cells and the salivary glands are probably crucial factors in the determination of vector competence.
Expression of doppel in the CNS of mice does not modulate transmissible spongiform encephalopathy disease
Authors: Tuzi Nadia L. , Gall Elaine , Melton David , Manson Jean C.
Published: August 2015

Late onset ataxia reported in three independently derived PrP null lines of mice has been attributed to the overexpression of the doppel protein in the CNS of these mice rather than to the loss of PrP. The central role of PrP in the transmissible spongiform encephalopathies (TSEs), the proximity of the gene which encodes doppel (Prnd) to the PrP gene (Prnp) and the structural similarity shared by PrP and doppel have led to the proposition that ataxia which develops during TSE disease could, in part, be due to doppel. In order to address this hypothesis, we have crossed our two inbred lines of PrP null mice, which either express (RCM) or do not express (NPU) thePrndgene in the CNS, with mice expressing twoPrnpa[108F189V]alleles of the PrP gene. We have found that the TSE infection does not influence the level of expression ofPrndin the CNS at the terminal stages of disease. Moreover, we have demonstrated that the level of expression ofPrndin the CNS has no influence on the incubation period, vacuolar pathology nor amount or distribution of PrPScdeposition in the brains of the TSE-infected mice. Doppel has therefore no apparent influence on the outcome of TSE disease in transgenic mice, suggesting it is unlikely to be involved in the naturally occurring TSE diseases in other species.
Prion protein gene polymorphisms in natural goat scrapie
Authors: Billinis Charalambos , Panagiotidis Cynthia H. , Psychas Vassilios , Argyroudis Stamatis , Nicolaou Anna , Leontides Sotirios , Papadopoulos Orestis , Sklaviadis Theodoros
Published: August 2015

A total of 51 goats, including seven clinical cases, from the first herd in Greece reported to have scrapie was examined to discern an association between scrapie susceptibility and polymorphisms of the gene encoding the prion protein (PrP). Each animal was evaluated for clinical signs of the disease, histopathological lesions associated with scrapie, the presence of detectable protease-resistant PrP in the brain and PrP genotype. Eleven different PrP genotypes encoding at least five unique predicted mature PrP amino acid sequences were found. These genotypes included the amino acid polymorphisms at codons 143 (H→R) and 240 (S→P) and ‘silent’ nucleotide alterations at codons 42 (a→g) and 138 (c→t). Additionally, novel caprine amino acid polymorphisms were detected at codons 21 (V→A), 23 (L→P), 49 (G→S), 154 (R→H), 168 (P→Q) and 220 (Q→H) and new silent mutations were found at codons 107 (g→a) and 207 (g→a). The following variants were found in scrapie-affected goats: VV21, LL23, GG49, SS49, HH143, HR143, RR154, PP168, PP240, SP240 and SS240. All scrapie-affected animals carried the HH143RR154 genotype, with the exception of two goats (HR143), both of which had detectable protease-resistant PrP but showed no clinical signs or histopathological lesions characteristic of scrapie.
Hantavirus nucleocapsid protein interacts with the Fas-mediated apoptosis enhancer Daxx
Authors: Li Xiao-Dong , Mäkelä Tomi P. , Guo Deyin , Soliymani Rabah , Koistinen Vesa , Vapalahti Olli , Vaheri Antti , Lankinen Hilkka
Published: August 2015

Hantaviruses cause two severe diseases, haemorrhagic fever with renal syndrome in Eurasia and hantavirus pulmonary syndrome in the Americas. To understand more about the molecular mechanisms that lead to these diseases, the associations of Puumala virus nucleocapsid protein (PUUV-N) with cellular proteins were studied by yeast two-hybrid screening. Daxx, known as an apoptosis enhancer, was identified from a HeLa cDNA library and its interaction with PUUV-N was confirmed by GST pull-down assay, co-immunoprecipitation and co-localization studies. Furthermore, domains of interaction were mapped to the carboxyl-terminal region of 142 amino acids in Daxx and the carboxyl-terminal 57 residues in PUUV-N, respectively. In pepscan assays, the binding sites of Daxx to PUUV-N were mapped further to two lysine-rich regions, of which one overlaps the sequence of the predicted nuclear localization signal of Daxx. These data suggest a direct link between host cell machinery and a hantavirus structural component.
Sustained G→A hypermutation during reverse transcription of an entire human immunodeficiency virus type 1 strain Vau group O genome
Authors: Vartanian Jean-Pierre , Henry Michel , Wain-Hobson Simon
Published: August 2015

Two full-length human immunodeficiency virus type 1 O sequences are described, one of which was hypermutated in all regions of the genome. This indicates that the intracellular [dTTP]/[dCTP] bias conducive to G→A hypermutation may be sustained throughout the synthesis of minus-strand DNA. In turn, this suggests the possibility of mutation of host sequences.
Linkage on chromosome 10 of several murine retroviral integration loci associated with leukaemia
Authors: Haviernik Peter , Festin Stephen M. , Opavsky Rene , Koller Richard P. , Barr Nighean I. , Neil James C. , Wolff Linda
Published: August 2015

Mmlloci have been identified as provirus integration sites among a subset of monocytic tumours induced by murine leukaemia virus (MuLV) infection of BALB/c and DBA/2 mice. These myeloid leukaemias contain a retrovirus integrated on chromosome 10 in proximity to the c-myblocus; however, c-mybexpression was not altered. Detailed physical mapping enabled placement of the retroviral integration sites ∼25 kb (Mml1), ∼51 kb (Mml2), and ∼70 kb (Mml3) upstream of the c-myblocus. Furthermore, theFti1 (fit-1) locus, a common integration site in feline leukaemia virus-induced T cell lymphomas, was mapped upstream ofMml3. Sequence analysis ofMml1,Mml2 andMml3 loci (39·6, 16·4 and 5·9 kb, respectively) in conjunction with the BLAST (basic local alignment search tool) homology searches against the expressed sequence tag (EST) database and the use of gene/exon prediction programs revealed potential coding sequences that were not confirmed by Northern analysis or RT–PCR. The sequences between c-mybandFti1, which were shown to include two potential scaffold/matrix attachment regions (S/MARs), are most likely regulatory in nature. An extended search for transcribed sequences far upstream ofMml3 revealed five genes, four of which were expressed in multiple tissues in mice. These genes could not be linked to tumour formation by the virus but their homologous sequences were found on human chromosome 6, thus allowing extension of the syntenic region on mouse chromosome 10 to approximately 250 kb.
Human herpesvirus-6 rep/U94 gene product has single-stranded DNA-binding activity
Authors: Dhepakson Panadda , Mori Yasuko , Jiang Yun Bao , Huang Hong Lan , Akkapaiboon Pilailuk , Okuno Toshiomi , Yamanishi Koichi
Published: August 2015

The characterization is reported of the human herpesvirus-6B (HHV-6B) rep/U94 gene, which is a homologue of the adeno-associated virus type 2 rep. In this study, a monoclonal antibody was produced against HHV-6B REP (anti-REP mAb). Immunofluorescence staining using the anti-REP mAb showed that REP was localized to the nucleus in HHV-6-infected MT4 cells. It was first detected at 24 h post-infection (p.i.) and accumulated to higher levels by 72 h p.i. REP may be expressed only at very low levels in HHV-6-infected cells: even when the late protein glycoprotein H was detected in nearly 90% of HHV-6-infected cells, REP was detected in only a small percentage of them. Western blot analysis showed that the anti-REP mAb recognized a 56-kDa polypeptide in HHV-6B-infected MT4 cells. Furthermore, the REP protein was shown to bind single-stranded DNA.
The sequence of camelpox virus shows it is most closely related to variola virus, the cause of smallpox
Authors: Gubser Caroline , Smith Geoffrey L.
Published: August 2015

Camelpox virus (CMPV) and variola virus (VAR) are orthopoxviruses (OPVs) that share several biological features and cause high mortality and morbidity in their single host species. The sequence of a virulent CMPV strain was determined; it is 202182 bp long, with inverted terminal repeats (ITRs) of 6045 bp and has 206 predicted open reading frames (ORFs). As for other poxviruses, the genes are tightly packed with little non-coding sequence. Most genes within 25 kb of each terminus are transcribed outwards towards the terminus, whereas genes within the centre of the genome are transcribed from either DNA strand. The central region of the genome contains genes that are highly conserved in other OPVs and 87 of these are conserved in all sequenced chordopoxviruses. In contrast, genes towards either terminus are more variable and encode proteins involved in host range, virulence or immunomodulation. In some cases, these are broken versions of genes found in other OPVs. The relationship of CMPV to other OPVs was analysed by comparisons of DNA and predicted protein sequences, repeats within the ITRs and arrangement of ORFs within the terminal regions. Each comparison gave the same conclusion: CMPV is the closest known virus to variola virus, the cause of smallpox.
The vaccinia virus B9R protein is a 6 kDa intracellular protein that is non-essential for virus replication and virulence
Authors: Price Nicola , Tscharke David C. , Smith Geoffrey L.
Published: August 2015

Vaccinia virus (VV) strain Western Reserve gene B9R is shown to encode an intracellular 6 kDa protein that is expressed late during the infectious cycle.In vitrotranscription and translation produced two polypeptides in the presence of microsomal membranes, but only the larger protein in the absence of membranes. The smaller protein sedimented with microsomes during centrifugation, suggesting it was inserted into the lipid membrane or into the microsomal lumen via the N-terminal hydrophobic signal sequence that was subsequently cleaved proteolytically. A VV mutant lacking B9R was constructed and found to replicate normally in cell culture and twoin vivomodels.
Characterization of plant proteins that interact with cowpea mosaic virus ‘60K’ protein in the yeast two-hybrid system
Authors: Carette Jan E. , Verver Jan , Martens Joost , van Kampen Tony , Wellink Joan , van Kammen Ab
Published: August 2015

Cowpea mosaic virus (CPMV) replication occurs in close association with small membranous vesicles in the host cell. The CPMV RNA1-encoded 60 kDa nucleotide-binding protein (‘60K’) plays a role in the formation of these vesicles. In this study, five cellular proteins were identified that interacted with different domains of 60K using a yeast two-hybrid search of anArabidopsiscDNA library. Two of these host proteins (termed VAP27-1 and VAP27-2), with high homology to the VAP33 family of SNARE-like proteins from animals, interacted specifically with the C-terminal domain of 60K and upon transient expression colocalized with 60K in CPMV-infected cowpea protoplasts. eEF1-β, picked up using the central domain of 60K, was also found to colocalize with 60K. The possible role of these host proteins in the viral replicative cycle is discussed.
Adaptation from whitefly to leafhopper transmission of an autonomously replicating nanovirus-like DNA component associated with ageratum yellow vein disease
Authors: Saunders Keith , Bedford Ian D. , Stanley John
Published: August 2015

Ageratum yellow vein disease is caused by the whitefly-transmitted monopartite begomovirus Ageratum yellow vein virus and a DNA β satellite component. Naturally occurring symptomatic plants also contain an autonomously replicating nanovirus-like DNA 1 component that relies on the begomovirus and DNA β for systemic spread and whitefly transmission but is not required for maintenance of the disease. Here, we show that systemic movement of DNA 1 occurs in Nicotiana benthamiana when co-inoculated with the bipartite begomovirus Tomato golden mosaic virus and the curtovirus Beet curly top virus (BCTV), but not with the mastrevirus Bean yellow dwarf virus. BCTV also mediates the systemic movement of DNA 1 in sugar beet, and the nanovirus-like component is transmitted between plants by the BCTV leafhopper vector Circulifer tenellus. We also describe a second nanovirus-like component, referred to as DNA 2, that has only 47% nucleotide sequence identity with DNA 1. The diversity and adaptation of nanovirus components are discussed.
Shrimp Taura syndrome virus: genomic characterization and similarity with members of the genus Cricket paralysis-like viruses
Authors: Mari Jocelyne , Poulos Bonnie T. , Lightner Donald V. , Bonami Jean-Robert
Published: August 2015

The single-stranded genomic RNA of Taura syndrome virus (TSV) is 10205 nucleotides in length, excluding the 3′ poly(A) tail, and contains two large open reading frames (ORFs) that are separated by an intergenic region of 207 nucleotides. The ORFs are flanked by a 377 nucleotide 5′ untranslated region (UTR) and a 226 nucleotide 3′ UTR followed by a poly(A) tail. The predicted amino acid sequence of ORF1 revealed sequence motifs characteristic of a helicase, a protease and an RNA-dependent RNA polymerase, similar to the non-structural proteins of several plant and animal RNA viruses. In addition, a short amino acid sequence located in the N-terminal region of ORF1 presented a significant similarity with a baculovirus IAP repeat (BIR) domain of inhibitor of apoptosis proteins from double-stranded DNA viruses and from animals. The presence of this BIR-like sequence is the first reported in a single-stranded RNA virus, but its function is unknown. The N-terminal amino acid sequence of three TSV capsid proteins (55, 40 and 24 kDa) were mapped in ORF2, which is not in the same reading frame as ORF1 and possesses an AUG codon upstream of the structural genes. However, the intergenic region shows nucleotide sequence similarity with those of the genusCricket paralysis-like viruses, suggesting a similar non-AUG-mediated translation mechanism. The structure of the TSV genome [5′ UTR–non-structural proteins–intergenic UTR–structural proteins–3′ UTR–poly(A) tail] is similar to those of small insect-infecting RNA viruses, which were recently regrouped into a new virus genus,Cricket paralysis-like viruses.
Direct targeting of human cytomegalovirus protein kinase pUL97 by kinase inhibitors is a novel principle for antiviral therapy
Authors: Marschall Manfred , Stein-Gerlach Matthias , Freitag Martina , Kupfer Regina , van den Bogaard Miriam , Stamminger Thomas
Published: August 2015

The protein kinase pUL97, encoded by human cytomegalovirus (HCMV), is an important determinant of virus replication. Recently, indolocarbazoles were identified as a class of substances that inhibit the pUL97 kinase activityin vitro. In parallel, it was shown that indolocarbazoles interfere with HCMV replication; however, the causal relationship between inhibition of pUL97 kinase activity and virus replication has not been clarified. Here evidence is provided that indolocarbazole-mediated inhibition of virus replication is a direct result of diminished pUL97 protein kinase activity. In cell culture infections, a strong and selective antiviral activity was measured with respect to several strains of HCMV in contrast with other related or non-related viruses. For fine quantification, recombinant HCMVs expressing green fluorescent protein were used, demonstrating the high sensitivity towards compounds NGIC-I and Gö6976. Interestingly, a ganciclovir-resistant virus mutant (UL97-M460I) showed increased sensitivity to both compounds. Supporting this concept, transfection experiments with cloned pUL97 revealed that ganciclovir-resistant mutants were characterized by reduced levels of autophosphorylation compared with wild-type and possessed particularly high sensitivity to indolocarbazoles. Moreover, the Epstein–Barr virus-encoded homologous kinase, BGLF4, which showed a similar pattern of autophosphorylation and ganciclovir phosphorylation activities, was not inhibited. Importantly, a cytomegalovirus deletion mutant, lacking a functional UL97 gene and showing a severe impairment of replication, was completely insensitive to indolocarbazoles. Thus, our findings indicate that a specific block in the activity of pUL97 is the critical step in indolocarbazole-mediated inhibition of virus replication and that pUL97 might be targeted very efficiently by a novel antiviral therapy.
A CD46CD[55–46] chimeric receptor, eight short consensus repeats long, acts as an inhibitor of both CD46 (MCP)- and CD150 (SLAM)-mediated cell–cell fusion induced by CD46-using measles virus
Authors: Christiansen Dale , De Sousa Emmanuel R. , Loveland Bruce , Kyriakou Peter , Lanteri Marc , Wild Fabian T. , Gerlier Denis
Published: August 2015

According to their cellular receptor use, measles virus (MV) strains can be separated into two phenotypes, CD46-using and CD46-non-using. A long chimeric receptor, CD46CD[55–46], was generated from the CD46 backbone, encompassing the four short consensus repeat (SCR) domains of CD46 linked via a flexible glycine hinge to SCR1 and SCR2 of CD55, SCR3 and SCR4 of CD46 and the STP, transmembrane and cytoplasmic tail of CD46. This chimeric receptor was proficient for MV binding but deficient in mediating MV-induced cell-to-cell fusion and virus replication, possibly due to the extended distance between the MV haemagglutinin (H) binding site (CD46 SCR1–SCR2) and the cell membrane. When coexpressed with either wild-type CD46 or CD150, this fusion-incompetent receptor exerted a dominant negative effect and inhibited both cell-to-cell fusion and entry of MV with CD46-using, but not CD46-non-using, phenotype. A soluble octameric CD46–C4bpα exhibited similar CD46- and CD150-mediated fusion inhibition properties only against CD46-using MV. This suggests that the long CD46CD[55–46] receptor acts by sequestering incoming MV prior to its binding to the shorter functional CD46 or CD150 receptor.
High avidity cytotoxic T lymphocytes to a foreign antigen are efficiently activated following immunization with a recombinant paramyxovirus, simian virus 5
Authors: Parks Griffith D. , Alexander-Miller Martha A.
Published: August 2015

Our previous work has shown that high avidity cytotoxic T lymphocytes (CTL) are optimal for virus clearancein vivoand thus it is necessary that an effective vaccine is capable of eliciting high avidity CTL. To determine if vaccination with the paramyxovirus simian virus 5 (SV5) elicits a high avidity response to a model foreign antigen, a recombinant virus was engineered to express chicken ovalbumin (rSV5–Ova). To compare the CTL response elicited with rSV5–Ova and a recombinant vaccinia virus expressing ovalbumin (rVV–Ova), mice were vaccinated intranasally with various doses of each vector and the Ova-specific CTL response was determined by ELISPOT analysis. Here, it has been shown that rSV5 can be equally as effective as rVV in eliciting antigen-specific CTL, in terms of both the total number of CTL and the number of high avidity cells. This has implications for both the design of vaccine vectors and the route utilized for vaccine administration for the elicitation of high avidity CTL responses. The advantages and future potential use of rSV5 vaccine vectors are discussed.