Characterization of the replication of a baculovirus mutant lacking the DNA polymerase gene

AbstractIn a previous study, the DNA polymerase gene (dnapol) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was identified as one of six genes required for plasmid replication in a transient replication assay (M. Kool, C. Ahrens, R.W. Goldbach, G.F. Rohrmann, J.M. Vlak, Identification of genes involved in DNA replication of the Autographa californica, Proc. Natl. Acad. Sci. U.S.A. 91, (1994) 11212–11216); however, another study based on a similar approach reported that the virally encoded polymerase was only stimulatory (A. Lu, L.K. Miller, The roles of 18 baculovirus late expression factor genes in transcription and DNA replication, J. Virol. 69, (1995) 975–982). To reconcile the conflicting data and determine if the AcMNPV DNA polymerase is required for viral DNA replication during the course of an infection, a dnapol-null virus was generated using bacmid technology. To detect viral DNA replication, a highly sensitive assay was designed based on real-time PCR and SYBR green chemistry. Our results indicate that a bacmid in which the dnapol ORF was deleted is unable to replicate its DNA when transfected into Spodoptera frugiperda (Sf-9) cells, although when the dnapol ORF was introduced into the polyhedrin (polh) locus, this repaired virus could propagate at levels similar to the control virus. These results confirm that the AcMNPV-encoded DNA polymerase is required for viral DNA replication and the host DNA polymerases cannot substitute for the viral enzyme in this process

Characterization of baculovirus constructs lacking either the Ac 101, Ac 142, or the Ac 144 open reading frame

AbstractTo investigate the role of the gene products encoded from the open reading frames 101, 142, and 144 of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a set of bacmid knockout and repair constructs were generated. The repair genes were engineered to contain an HA epitope tag at their C-termini. The results of transfection–infection assays and growth curve analyses showed that the Ac 101, 142, and 144 genes were required for infectious virus production. To better characterize the role of these genes in the baculovirus replication cycle, quantitative DNA replication assays were performed and demonstrated that in cells transfected with the Ac 101, 142, or 144 knockouts, DNA replicated with similar kinetics as a control virus. Western blot analyses of budded virus from cells infected with the repair viruses showed that these proteins are associated with the viral nucleocapsid. Furthermore, immunoelectron microscopy of cells transfected with the knockout bacmids revealed defects in nucleocapsid production for all three constructs. From these results we concluded that the gene products encoded from these open reading frames are essential for virus production and may be involved in DNA processing, packaging, or nucleocapsid morphogenesis

PDGF Receptor-α Does Not Promote HCMV Entry into Epithelial and Endothelial Cells but Increased Quantities Stimulate Entry by an Abnormal Pathway

Epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFRα) were reported to mediate entry of HCMV, including HCMV lab strain AD169. AD169 cannot assemble gH/gL/UL128–131, a glycoprotein complex that is essential for HCMV entry into biologically important epithelial cells, endothelial cells, and monocyte-macrophages. Given this, it appeared incongruous that EGFR and PDGFRα play widespread roles in HCMV entry. Thus, we investigated whether PDGFRα and EGFR could promote entry of wild type HCMV strain TR. EGFR did not promote HCMV entry into any cell type. PDGFRα–transduction of epithelial and endothelial cells and several non-permissive cells markedly enhanced HCMV TR entry and surprisingly, promoted entry of HCMV mutants lacking gH/gL/UL128–131 into epithelial and endothelial cells. Entry of HCMV was not blocked by a panel of PDGFRα antibodies or the PDGFR ligand in fibroblasts, epithelial, or endothelial cells or by shRNA silencing of PDGFRα in epithelial cells. Moreover, HCMV glycoprotein induced cell-cell fusion was not increased when PDGFRα was expressed in cells. Together these results suggested that HCMV does not interact directly with PDGFRα. Instead, the enhanced entry produced by PDGFRα resulted from a novel entry pathway involving clathrin-independent, dynamin-dependent endocytosis of HCMV followed by low pH-independent fusion. When PDGFRα was expressed in cells, an HCMV lab strain escaped endosomes and tegument proteins reached the nucleus, but without PDGFRα virions were degraded. By contrast, wild type HCMV uses another pathway to enter epithelial cells involving macropinocytosis and low pH-dependent fusion, a pathway that lab strains (lacking gH/gL/UL128–131) cannot follow. Thus, PDGFRα does not act as a receptor for HCMV but increased PDGFRα alters cells, facilitating virus entry by an abnormal pathway. Given that PDGFRα increased infection of some cells to 90%, PDGFRα may be very useful in overcoming inefficient HCMV entry (even of lab strains) into the many difficult-to-infect cell types

Characterization of Baculovirus genes involved in genome replication and processing

The Baculoviridae comprise a diverse group of occluded DNA viruses that contain large double-stranded DNA genomes of 80 - 180 kb and may encode up to 180 gene products. To understand how baculoviruses replicate and process their genomes and the gene products that are involved in these events, a series of mutant virus constructs were generated using the Autographa californica multinucleopolyhedrovirus (AcMNPV) genome propagated as a bacteria artificial chromosome (bacmid). The gene products investigated were very late expression factor-1 (VLF-1), DNA polymerase, alkaline nuclease, the single-stranded DNA binding protein (DBP), and those encoded from the open reading frames, Ac 101, Ac 142, and Ac 144. Using the Spodoptera frugiperda cell line as a tissue culture model system, bacmid constructs lacking any of the open reading frames encoding these seven gene products were shown to be non-infectious. In addition, based on a series of assays to determine the cause for the non-infectious phenotype, it was revealed that VLF-1, a putative recombinase and transcriptional activator was also an essential structural component of the capsid and appeared to possess an enzymatic function related to its role as a recombinase during the final stages of DNA encapsidation. A bacmid lacking the alkaline nuclease gene was able to replicate DNA to normal levels, however, this construct produced aberrant genomes and defective nucleocapsids, suggesting that alkaline nuclease was involved in processing viral genomes during replication. Similarly, analysis of a bacmid lacking the single-stranded DNA binding protein DBP revealed that, although not absolutely required for DNA synthesis, DNA synthesis was reduced in the absence of DBP and no unit-length genomes could be detected from transfected cell extracts. Moreover, although not a structural component of budded virus, the DBP knockout was deficient in nucleocapsid production which is predicted to result from a defect in genome maturation. Finally, the gene products encoded by open reading frames Ac 101, Ac 142, and Ac144, while expendable for DNA synthesis, were shown to be components of the nucleocapsid from budded virus, and bacmids lacking these gene products were deficient in nucleocapsid production

Characterization of the Role of Very Late Expression Factor 1 in Baculovirus Capsid Structure and DNA Processing

Very late expression factor 1 (VLF-1) of Autographa californica multiple nucleopolyhedrovirus is a putative tyrosine recombinase and is required for both very late gene expression and budded virus production. In this report, we show that a vlf-1 knockout bacmid was able to synthesize viral DNA at levels similar to that detected for a gp64 knockout bacmid that served as a noninfectious control virus. Additionally, analysis of replicated bacmid DNA by field-inversion gel electrophoresis indicated that VLF-1 is not required for synthesizing high-molecular-weight intermediates that could be resolved into unit-length genomes when cut at a unique restriction site. However, immunoelectron microscopic analysis revealed that in cells transfected with a vlf-1 knockout bacmid, aberrant tubular structures containing the capsid protein vp39 were observed, suggesting that this virus construct was defective in producing mature capsids. In contrast, rescuing the vlf-1 knockout bacmid construct with a copy of VLF-1 that carries a mutation of a highly conserved tyrosine (Y355F) was sufficient to restore the production of nucleocapsids with a normal appearance, but not infectious virus production. Furthermore, the results of a DNase I protection assay indicated that the DNA packaging efficiency of the VLF-1(Y355F) virus construct was similar to that of the gp64 knockout control. Finally, a recombinant virus containing a functional hemagglutinin epitope-tagged version of VLF-1 was constructed to investigate the association of VLF-1 with the nucleocapsid. Analysis by immunoelectron microscopy of Sf-9 cells infected with this virus showed that VLF-1 localized to an end region of the nucleocapsid. Collectively, these results indicate that VLF-1 is required for normal capsid assembly and serves an essential function during the final stages of the DNA packaging process

Characterization of a Baculovirus Lacking the Alkaline Nuclease Gene

The Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) alkaline nuclease (AN) associates with the baculovirus single-stranded DNA binding protein LEF-3 and possesses both a 5′→3′ exonuclease and an endonuclease activity. These activities are thought to be involved in DNA recombination and replication. To investigate the role of AN in AcMNPV replication, the λ Red system was used to replace the an open reading frame with a chloramphenicol acetyltransferase gene (cat) and a bacmid containing the AcMNPV genome in Escherichia coli. The AcMNPV an knockout bacmid (vAcAN-KO/GUS) was unable to propagate in Sf9 cells, although an an-rescued bacmid (vAcAN-KO/GUS-Res) propagated normally. In addition, the mutant did not appear to produce budded virions. These data indicated that an is an essential baculovirus gene. Slot blot and DpnI assays of DNA replication in Sf9 cells transfected with vAcAN-KO/GUS, vAcAN-KO/GUS-Res, and a wild-type bacmid showed that the vAcAN-KO/GUS bacmid was able to replicate to levels similar to those seen with the vAcAN-KO/GUS-Res and wild-type bacmids at early stages posttransfection. However, at later time points DNA did not accumulate to the levels seen with the repaired or wild-type bacmids. Northern analysis of Sf9 cells transfected with bacmid vAcAN-KO/GUS showed that transcription of late and very late genes was lower at later times posttransfection relative to the results seen with wild-type and vAcAN-KO/GUS-Res bacmids. These data suggest that the an gene might be involved in the maturation of viral DNA or packaging of the DNA into virions

Cellular FLIP can substitute for the herpes simplex virus type 1 latency-associated transcript gene to support a wild-type virus reactivation phenotype in mice

Zadatak rada je projektirati i konstruirati uređaj za ispitivanje BMI penetracija na dnu reaktorske posude. Za potrebe raščišćavanja zadatka napravljen je tehnički upitnik iz kojega je proizašla lista zahtjeva. U fazi koncipiranja napravljena je funkcijska dekompozicija i morfološka matrica iz koje su generirana dva koncepta od kojih je nakon provedene evaluacije odabran jedan. Za sve kritične dijelove provedena je provjera s obzirom na mehaničku čvrstoću. Za kraj je izrađen 3D model cijelog uređaja u programu Autodesk Inventor 2014, kao i pripadajući sklopni crtež.Main task of this thesis is to design a device for inspection of BMI penetrations at the bottom of reactor vessel. For clearing the task specifications, technical questionnaire was used to get request list. In conceptual phase functional decomposition was done along with morphological matrix from which two concepts were generated. After concept evaluation only one was chosen. Strength check was done for all critical parts. Finally, 3D model of whole device was made in Autodesk Inventor 2014, along with main assembly drawing. Key words: Manipulator, inspection, BMI penetration

HCMV gB-gH/gL complexes detected by IP-western blots.

<p>ARPE-19 or MRC-5 cells were transduced with Ad vectors expressing HCMV gB, gH/gL, both gB and gH/gL, or with Ad-tet-trans (tet, as a negative control) as indicated at the top part of each panel. After 20 hrs the cells were lysed in IP buffer containing 1% NP-40. (A) Proteins were IP’d from ARPE-19 or MRC-5 cell extracts with anti-gH MAb 14-4b, separated by SDS-PAGE under reducing conditions, transferred to membranes, and then analyzed by western blot using rabbit polyclonal sera specific to gB or anti-gH MAb AP86. (B) Proteins from ARPE-19 cell lysates were IP’d with anti-gB MAbs 9C1, 13H10, or 15H7 and the IPs analyzed by western blot as described above using anti-gH AP86. Input represents 5% of the extract loaded directly onto gels then blotted. (C) ARPE-19 cells were transfected with an Ad vector expressing gB with a C-terminal FLAG epitope tag or wild type gB and co-transduced with Ad vectors to express gH/gL as indicated along the top of the panel. Proteins were IP’d with an anti-FLAG antibody and analyzed by western blot as described above with rabbit polyclonal gB-specific serum or anti-gH MAb AP-86 to detect gB and gH, respectively. The percent of gB and gH/gL that was co-IP in these experiments was quantified using NIH ImageJ software by comparing the relative band intensities from the IP’d proteins to the 5% input and are indicated under the lanes. Molecular mass (MW) markers are indicated on the left.</p