Botchway, Stan

Graves, Leo

Irons, Sarah

King, Linda

Possee, Robert

Raza, Farheen

English

OA OpenAgrar repository

    131  acid substitution to attempt to retain a functional enzyme.  Bioassays showed that the deletion or alteration of just the zinc binding site of the metalloprotease, but not the entire enhancin gene, did not change viral potency. For example, a construct with E1 deleted/E2zinc modified had the same potency as E1deleted/E2 intact. These results suggest that the enhancins of LdNPV do not improve viral potency through the activity of metalloproteases, but appear to have a different mechanism, which has yet to be identified..   Poster / Viruses. Wednesday, 16:30. VI-17   A Cypovirus VP5 Displays the RNA Chaperone-like Activity that Destabilizes RNA Helices and Accelerates Strand Annealing Jie Yang, Jiamin Zhang, Yuehua Kuang and Yuanyang Hu State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.  Address for Correspondence: yyhu@whu.edu.cn  For double-stranded RNA (dsRNA) viruses in the family Reoviridae, their inner capsids function as the machinery for viral RNA (vRNA) replication. Unlike other multishelled reoviruses, cypovirus has a single-layered capsid, thereby representing a simplified model for studying vRNA replication of reoviruses. VP5 is one of the three major cypovirus capsid proteins and functions as a clamp protein to stabilize cypovirus capsid. Here, we expressed VP5 from Helicoverpa armigera cypovirus-5 (HaCPV-5) in a eukaryotic system and determined that this VP5 possesses RNA chaperone-like activity, which destabilizes RNA helices and accelerates strand annealing independent of ATP. Our further characterization of VP5 revealed that its helix-destabilizing activity is RNA specific, lacks directionality, and could be inhibited by divalent ions, such as Mg2+, Mn2+, Ca2+ or Zn2+, to varying degrees. Furthermore, we found that HaCPV-5 VP5 facilitates the replication initiation of an alternative polymerase (i.e. reverse transcriptase) through a panhandle-structured RNA template, which mimics the 5’-3’ cyclization of cypoviral positive-stranded RNA. Given that the replication of negative-stranded vRNA on the positive-stranded vRNA template necessitates the dissociation of the 5’-3’ panhandle, the RNA chaperone activity of VP5 may play a direct role in the initiation of reoviral dsRNA synthesis..    Poster / Viruses. Wednesday, 16:30. VI-18   A recombinant Autographa californica nucleopolyhedrosis virus expressing a Cyt1A/GFP chimera in Trichoplusia ni larvae Miguel A. Salas-Marina 1, Cristina Del Rincón-Castro 2 and Jorge E. Ibarra 1 1CINVESTAV-Irapuato, Irapuato, GTO, Mexico; 2División de Ciencias de la Vida, Universidad de Guanajuato, Irapuato, GTO., Mexico Address for Correspondence: jibarra@ira.cinvestav.mx  A novel approach was followed in order to achieve the expression of the Cyt1A toxin of Bacillus thuringiensis in a recombinant strain of AcNPV, trying to increase its virulence. First, the reporter GFP protein was used as a means to identify recombinant viruses; and second, biolistisc was used to achieve co-transfection. The recombinant construction pAccyta-GFP, containing the B. thuringiensis gene cyt1A fused with the GFP gene under the control of the p10 promoter from the pacuW31 vector was generated. Successful co-transfection by biobalistics was achieved with the AcNPV genome, when neonate Trichoplusia ni larvae were bombarded with DNA-coated gold micro-projectiles. Treated larvae showed the typical NPV infection symptoms, although only a thorough inspection detected fluorescent points in the fat body. Microscopic corroboration indicated that a  recombinant AcNPV (AcNPV-cyt1a-GFP) was generated, showing glowing polyhedra in the infected cells, under fluorescence microscopy. This observation may indicate that the putative chimeric protein is incorporated into the polyhedron structure during its integration. Interestingly, the nuclei holding the recombinant polyhedra appeared less compact than those holding the wild-type polyhedra. A series of purification cycles of AcNPV-cyt1a-GFP rendered larvae showing fluorescence throughout the whole body. Preliminary observations indicate that AcNPV-cyt1a-GFP kills larvae faster than the wild-type strain; however, accurate LT50s are still to be estimated.   Poster / Viruses. Wednesday, 16:30. VI-19   iLOV baculovirus: Using a novel small fluorescent protein for imaging virus proteins during infection Farheen Raza1, Sarah Irons1, Leo Graves1, Stan Botchway2, Robert Possee1,3, Linda King1  1 Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, UK; 2 Central Laser Facility, STFC, Harwell, UK; 3 Oxford Expression Technologies, Oxford, UK Address for Correspondence: sarahirons@brookes.ac.uk  Imaging of living cells is now a common place approach in cell biology and virus research, however addition of conventional fluorescent proteins such as green fluorescent protein (GFP) and its derivatives, can lead to alterations in the location and behaviour of target proteins. Such problems in mis-targeting have previously been observed on fusion of GFP to Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) P10, thus hindering imaging of P10 dynamics during virus infection of insect cells. Here we report use of iLOV, a small (~11kDa) genetically encoded fluorescent protein based on the LOV domain of plant phototropin 2, fused to P10 in AcMNPV. Expression of the P10-iLOV fusion during infection showed presence of filaments and nuclear structures, comparable to those seen in previous immunofluorescence images. We have also looked at the fluorescence lifetime of iLOV in P10 structures, where we established that the P10-iLOV fusion shows a very long fluorescence decay of ~4ns, compared to ~2.5ns for GFP. This work shows the successful use of iLOV in the baculovirus system, and provides an opportunity to tag proteins where GFP has previously failed. In addition the long fluorescence lifetime makes iLOV a promising candidate for use in protein interaction studies using Förster resonance energy transfer-fluorescence lifetime imaging (FRET-FLIM).     Poster / Viruses. Wednesday, 16:30. VI-20   Expression analysis of the nsd-2 gene encoding the putative densovirus receptor in the midgut Katsuhiko Ito 1, Hiroko Tabunoki 1, Takeshi Yokoyama 1, Keiko Kadono-Okuda 2 1Tokyo University of Agriculture and Technology, Tokyo, Japan; 2 National Institute of Agrobiological Sciences, Ibaraki, Japan Address for Correspondence: katsuito@cc.tuat.ac.jp  Bombyx mori densovirus type 2 (BmDNV-2) is a pathogen that replicates only in the midgut columnar cells and causes fatal disease in the silkworm. The resistance to BmDNV-2 is determined by a single gene, nsd-2, which is characterized as non-susceptibility irrespective of the viral dose. Previously we have identified nsd-2 by positional cloning and found that this gene encodes a putative amino acid transporter which might work as a receptor for BmDNV-2. In this study, we investigated the relationship between the part of the midgut expressing nsd-2 and the BmDNV-2 infection. To investigate the expression pattern of nsd-2 in the midgut, we divided the midgut into three parts, anterior, middle, and posterior part, and performed the RT-PCR analysis with total RNA isolated from each part. nsd-2 transcript  

iLOV baculovirus: Using a novel small fluorescent protein for imaging virus proteins during infection

Irons, Sarah L.

Possee, Robert D.

King, Linda A.

    130  LC50 was 1.3x103 and 3.1x103 OBs/ml for the tested 2nd and 4th instars, respectively. The study reports a new baculovirus that might be used as a promising agent for biological control of the jasmine moth.   Poster / Viruses. Wednesday, 16:30. VI-13   A single baculovirus for the production of recombinant Adeno-Associated Virus 8 vectors  Lionel Galibert; Aurélien Jacob; Bérangère Bertin; Marjorie Boutin Fontaine; Delphine Bonnin; Christophe Lecomte; Christel Rivière; Otto-Wilhelm Merten Genethon, 1bis, rue de l’Internationale, 91002 Evry, France Address for correspondence: galibert@genethon.fr  We have developed a single baculovirus, named “Monobac”, for the production of recombinant Adeno-Associated Virus vectors of serotype 8 (rAAV8) using the Sf9 cell/baculovirus system. In an AcMNPV bacmid devoid of the chitinase and cathepsin a genes, the AAV rep2 and cap8 genes have been inserted at the egt locus, while the recombinant AAV was cloned in the Tn7 site. This system was used for the production rAAV8 encoding the human γ-sarcoglycan gene, of clinical interest for the treatment of LGMD2C (γ-sarcoglycanopathy) myopathy disease. Enhanced rAAV8 productivity was observed in the cell culture and was maintained after purification, compared to production system based on the use of 2 baculoviruses. The produced rAAV8 capsids displayed a reduced degradation profile of the capsid proteins VP1/VP2 due to the elimination of the baculovirus cathepsin protease gene. This optimized system allows the production of an improved quantity of rAAV vectors with improved vector quality, resulting in enhanced infectivity of the rAAV. .    Poster / Viruses. Wednesday, 16:30. VI-14 Determining the role of P10 during baculovirus infection through the development of novel mutants in Autographa californica multicapsid nucleopolyhedrovirus Leo Graves1, Farheen Raza1, Sarah L. Irons1; Robert D Possee1,2 & Linda A King1 1Department of Biological and Medical Sciences, Oxford Brookes University, Oxford UK, 2Oxford Expression Technologies Ltd, Oxford, UK. Address for Correspondence: 12083983@brookes.ac.uk  P10 is a fibrous protein that forms complex networks of filaments and a distinct perinuclear tubular structure around the nucleus during the later stages of infection of cells with baculovirus.  Previous research has suggested possible roles of P10 in nuclear stability, polyhedron formation and cell lysis, but distinct functional roles for the protein have yet to be determined. In order to investigate the role of P10 during infection, a variety of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) mutants have been constructed, which include a p10 deletion and associated rescue virus, phosphorylation mutants and a virus in which the AcMNPV p10 coding region has been replaced with that from Spodoptera frugiperda (Sf)NPV. Mass spectrometry was used to confirm the phosphorylation of P10 serine 93.  Mutation of serine 93 to alanine affected the structure of P10 tubules as evidenced by confocal microscopy.  The distinctive tubular structure surrounding the nucleus that is observed in wild-type virus infected cells failed to form correctly.  Circular dichroism analysis confirmed a distinct change in the protein secondary structure.  These data suggest that phosphorylation plays a key role in P10 function. Replacement of the AcMNPV p10 coding region with that from SfNPV resulted in a virus with low budded virus titre and aberrant rearrangement of microtubules in comparison to AcMNPV-infected cells, suggesting that the SfNPV P10 may be affecting microtubules and translocation of nucleocapsids to the plasma membrane for budding.     Poster / Viruses. Wednesday, 16:30. VI-15   Evaluation of the transcriptional transactivation of betabaculovirus regulatory elements in transformed cell lines by alphabaculovirus transcription factors Santiago Haase1, M. Leticia Ferrelli1; Matías L. Pidre1; Alicia Sciocco-Cap2, Víctor Romanowski1 1IBBM-UNLP-CONICET, La Plata, AR; 2IMyZA-INTA, Castelar, AR; Address for Correspondence: santiagohaase@biol.unlp.edu.ar  The narrow host range of baculoviruses is one of their advantages for sustainable pest control of a single insect species with minimal or no effect on non-target organisms. However, the limited host range may be less desirable from the economical point of view since more than one pest can be present at the same time on most crops. Engineering baculovirus genomes with an expanded host range by design would be an answer to this type of scenarios. Nevertheless, genetic determinants of host range have not been widely characterized and the mechanisms of host recognition are still not well understood. In this context, the generation of hybrid or chimaeric baculoviruses may be an empirical approach to generate viruses with expanded host range. The expression of viral genes in this context will require the transcriptional transactivation of their promoters by heterologous transcription factors (TFs). However, the recognition of baculovirus promoters in different species has not been systematically studied so far. The aim of our work is to evaluate the transcriptional transactivation of betabaculovirus promoters by alphabaculovirus TFs. It has been noted before that late promoters require the replication of the DNA to be activated. Therefore, we generated stably transformed cell lines expressing the red fluorescent protein (DsRed) as a reporter gene under the control of immediate-early, early and late gene promoters of the Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) and Epinotia aporema granulovirus (EpapGV), respectively. These cell lines were infected with AgMNPV to evaluate the transcriptional transactivation of these promoters. Our results showed that the AgMNPV transcription factors activate early and late EpapGV promoters.     Poster / Viruses. Wednesday, 16:30. VI-16   Enhancin Genes of Lymantria dispar NPV Do Not Increase Potency Via Metalloprotease Activity Kelli Hoover1, James Slavicek2, Algimantas P. Valaitis2,3, Nancy Hayes-Plazolles2, and Elizabeth McCarthy1 1 Department of Entomology, Penn State University, University Park, PA USA, 2 USDA Forest Service, Delaware, OH USA, 3Retired Address for Correspondence: kxh25@psu.edu  The baculovirus encoded enhancins characterized so far are metalloproteases that digest proteins in the peritrophic matrix (PM) of their host midgut, increasing viral potency in many systems. Lymantria dispar NPV has two enhancin genes (E1 and E2) that are distributed in the ODV envelopes, placing them in a position to interact with the PM and the and possibly midgut cells. Deletion of either enhancin or both reduces viral potency 2-fold and 12-fold, respectively, compared to wildtype. Removal of the PM with optical brightener treatment did not alter these differences in potencies, suggesting that the enhancins do not affect the PM. The results of an in vitro PM digestion assay found that although the PM was degraded, it was not affected by inhibitors of metalloproteases, whereas treatment with serine protease inhibitors showed little or no PM degradation. Mutant LdNPV viruses were generated by altering the region that encodes the zinc binding site of the metalloprotease; this region of E1 and E2 was either deleted or altered by homologous amino  

Determining the role of P10 during baculovirus infection through the development of novel mutants in Autographa californica multicapsid nucleopolyhedrovirus

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Determining the role of P10 during baculovirus infection through the development of novel mutants in Autographa californica multicapsid nucleopolyhedrovirus

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