Infected Sf9 Cells Research Articles

DSP-PP Precursor Protein Cleavage by Tolloid-Related-1 Protein and by Bone Morphogenetic Protein-1

Dentin sialoprotein (DSP) and phosphophoryn (PP), acidic proteins critical to dentin mineralization, are translated from a single transcript as a DSP-PP precursor that undergoes specific proteolytic processing to generate DSP and PP. The cleavage mechanism continues to be controversial, in part because of the difficulty of obtaining DSP-PP from mammalian cells and dentin matrix. We have infected Sf9 cells with a recombinant baculovirus to produce large amounts of secreted DSP-PP240, a variant form of rat DSP-PP. Mass spectrometric analysis shows that DSP-PP240 secreted by Sf9 cells undergoes specific cleavage at the site predicted from the N-terminal sequence of PP extracted from dentin matrix: SMQG447↓D448DPN. DSP-PP240 is cleaved after secretion by a zinc-dependent activity secreted by Sf9 cells, generating DSP430 and PP240 products that are stable in the medium. DSP-PP processing activity is constitutively secreted by Sf9 cells, but secretion is diminished 3 days after infection. Using primers corresponding to the highly conserved catalytic domain of Drosophila melanogaster tolloid (a mammalian BMP1 homolog), we isolated a partial cDNA for a Spodopotera frugiperda tolloid-related-1 protein (TLR1) that is 78% identical to Drosophila TLR1 but only 65% identical to Drosophila tolloid. Tlr1 mRNA decreased rapidly in Sf9 cells after baculovirus infection and was undetectable 4d after infection, paralleling the observed decrease in secretion of the DSP-PP240 processing activity after infection. Human BMP1 is more similar to Sf9 and Drosophila TLR1 than to tolloid, and Sf9 TLR1 is more similar to BMP1 than to other mammalian homologs. Recombinant human BMP1 correctly processed baculovirus-expressed DSP-PP240 in a dose-dependent manner. Together, these data suggest that the physiologically accurate cleavage of mammalian DSP-PP240 in the Sf9 cell system represents the action of a conserved processing enzyme and support the proposed role of BMP1 in processing DSP-PP in dentin matrix.

An ac34 Deletion Mutant of Autographa californica Nucleopolyhedrovirus Exhibits Delayed Late Gene Expression and a Lack of Virulence In Vivo

Ac34 and its homologs are highly conserved in all sequenced alphabaculoviruses. In this paper, we show that ac34 transcripts were detected from 6 to 48 h postinfection (p.i.) in Autographa californica nucleopolyhedrovirus (AcMNPV)-infected Sf9 cells. Ac34 localized to both the cytoplasm and the nuclei of infected cells but was not a viral structural protein. To determine the function of ac34 in the viral life cycle, an ac34 knockout AcMNPV (vAc34KO) was constructed. Compared with wild-type and repair viruses, vAc34KO exhibited an approximately 100-fold reduction in infectious virus production. Further investigations showed that the ac34 deletion did not affect the replication of viral DNA, polyhedron formation, or nucleocapsid assembly but delayed the expression of late genes, such as vp39, 38k, and p6.9. Bioassays revealed that vAc34KO was unable to establish a fatal infection in Trichoplusia ni larvae via per os inoculation. Few infectious progeny viruses were detected in the hemolymph of the infected larvae, indicating that the replication of vAc34KO was attenuated. These results suggest that Ac34 is an activator protein that promotes late gene expression and is essential for the pathogenicity of AcMNPV.

Autographa californica multiple nucleopolyhedrovirus orf114 is not essential for virus replication in vitro, but its knockout reduces per os infectivity in vivo

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf114 (ac114) is one of the highly conserved unique genes in the lepidopteran group I nucleopolyhedrovirus. So far, the biological function of ac114 is unknown. To study the function of ac114 in the virus life cycle, an ac114 knockout baculovirus shuttle vector (bacmid) was generated. Fluorescence and light microscopy showed that the ac114 knockout mutant was able to produce infectious budded viruses (BVs) and occlusion bodies (OBs). Titration assays demonstrated that the ac114 knockout virus had similar growth kinetics to the control virus during the infection phase. Electron microscopy indicated that ac114 did not affect the morphogenesis of BVs and occlusion-derived viruses (ODVs); however, the numbers of ODVs per OB of the ac114 knockout virus were significantly lower than those of the control virus. RT-PCR demonstrated that ac114 was a late stage expression gene and that its transcription initiated at an A residue, 16 nucleotides upstream of the ATG start codon. Intracellular localization analysis revealed that the Ac114-GFP fusion protein localized predominantly as punctate patches in the cytoplasm of infected Sf9 cells. Bioassays showed that the ac114 knockout did not change the killing speed of AcMNPV in Spodoptera exigua larvae, but reduced its viral infectivity significantly. Taken together, these data indicate that ac114 is an auxiliary gene that facilitates embedding of ODVs into OBs, thus affecting the per os infectivity of the virus.

Preparation of immunoaffinity column for rapid purification of human DNA polymerase delta and its subassemblies

Mammalian deoxyribonucleic acid (DNA) polymerase delta (pol δ) is well characterized as a tightly associated heterotetrameric complex. It is thought to play a central role in chromosomal DNA replication and various DNA repair processes. However, the availability of highly purified active pol δ becomes one of the major barriers for its in-depth functional and structural analysis. In this work, a powerful immunoaffinity column was prepared. The human DNA pol δ and its subassemblies were reconstituted with a novel MultiBac system, over-expressed in insect cells, and followed by purification using immunoaffinity chromatography in combination with ion-exchange chromatography. Starting from 500 ml of infected Sf-9 cells, as much as 5 mg of recombinant pol δ with different subunit combinations was isolated near homogeneity with active forms that were conformed by the assays both on sparsely primed poly (dA)/oligo (dT) template-primer and on singly primed M13 DNA template. Thus, our home-made immunoaffinity column provides a significant advance in the isolation of pol δ, allowing its facile isolation from over-expressed insect cells or natural source in good yield and high purity.

Autographa californica multiple nucleopolyhedrovirus odv-e66 is an essential gene required for oral infectivity

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e66 is a core gene and encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E66. The N-terminal 23 amino acid of the envelope protein ODV-E66 are sufficient to direct native and fusion proteins to induced membrane microvesicles and the viral envelope during infection with AcMNPV. In this study, an odv-e66-knockout bacmid can not express N-terminal hydrophobic domains was constructed via homologous recombination in Escherichia coli. The odv-e66 deletion had no effect on budded virus (BV) production and viral DNA replication in infected Sf9 cells. Larval bioassays demonstrated that injection of odv-e66 deletion BV into the hemocoel could kill P. xylostella larvae as efficiently as repaired and control viruses; however, odv-e66 deletion mutant resulted in a 50% lethal dose that was 10 3 higher than that of the repaired and control viruses when inoculated per os. These results indicated that ODV-E66 envelope protein most likely played an important role in the oral infectivity of AcMNPV, but is not essential for virus replication.

TubeSpin bioreactor 50 for the high-density cultivation of Sf-9 insect cells in suspension

Here we present the TubeSpin bioreactor 50 (TubeSpins) as a simple and disposable culture system for Sf-9 insect cells in suspension. Sf-9 cells had substantially better growth in TubeSpins than in spinner flasks. After inoculation with 10(6) cells/ml, maximal cell densities of 16×10(6) and 6×10(6) cells/ml were reached in TubeSpins and spinner flasks, respectively. In addition the cell viability in these batch cultures remained above 90% for 10 days in TubeSpins but only for 4 days in spinner flasks. Inoculation at even higher cell densities reduced the duration of the lag phase. After inoculation at 2.5×10(6) cells/ml, the culture reached the maximum cell density within 3 days instead of 7 days as observed for inoculation with 10(6) cells/ml. Infection of Sf-9 cells in TubeSpins or spinner flasks with a recombinant baculovirus coding for green fluorescent protein (GFP) resulted in similar GFP-specific fluorescence levels. TubeSpins are thus an attractive option for the small-scale cultivation of Sf-9 cells in suspension and for baculovirus-mediated recombinant protein production.

Enterovirus 71 virus-like particle vaccine: Improved production conditions for enhanced yield

To develop the enterovirus 71 (EV71) vaccine, we previously constructed a recombinant baculovirus (Bac-P1-3CD) co-expressing EV71 P1 (under polyhedrin promoter) and 3CD (under p10 promoter) proteins, which caused P1 cleavage by 3CD protease and self-assembly of virus-like particles (VLPs) in Sf-9 cells. Assuming that reducing the 3CD expression can alleviate the competition with P1 expression and elevate the VLPs yield, hereby we constructed Bac-P1-C3CD and Bac-P1-I3CD expressing 3CD under weaker CMV and IE-1 promoters, respectively. Western blot and ELISA analyses revealed that Bac-P1-C3CD and Bac-P1-I3CD led to the VLPs release into the supernatant and enhanced the extracellular VLPs yield in Sf-9 cells, but gave poor VLPs production in High Five™ (Hi-5) cells. By optimizing the process parameters including host cells, cell density, culture mode and dissolved oxygen (DO), the best extracellular VLPs yield was achieved by infecting Sf-9 cells (4 × 10 6 cells/mL) cultured in the bioreactor (DO = 30%) with Bac-P1-C3CD, which approached ≈64.3 mg/L and represented a ≈43-fold increase over the yield (1.5 mg/L) attained using the old process (Bac-P1-3CD infection of Sf-9 cells in the spinner flasks). The resultant VLPs not only resembled the VLPs produced from Bac-P1-3CD infection in density, size and shape, but also induced potent antibody responses in mouse models. The antibodies neutralized EV71 strains of homologous and heterologous genogroups, implicating the potential of the VLPs to confer cross-protection for the prevention of future epidemics. Altogether, Bac-P1-C3CD and the bioprocess render mass production more economical, obviate the need for cell lysis and hold promise for future industrial vaccine production.

Baculovirus expression of cloned porcine arterivirus generates infectious particles in both insect and mammalian cells

Studies on several viral pathogens have been hampered by the lack of appropriate in vitro systems for their propagation and amplification. Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus containing a single-stranded positive-sense RNA genome (∼15 kb), was served as a model virus and its genomic cDNA was recombinated into baculovirus. We investigated whether infectious virus particles could be generated by expression of the full-length cloned genome from the modified baculovirus vector. The recombinant baculovirus, AcAPRRS, was used to infect sf9 cells. Immunofluorescence assay demonstrated the presence of PRRSV nonstructural protein (nsp) 2 and nucleocapsid (N) protein and electron microscopy revealed PRRSV particles in the culture supernatant. Infectious PRRSV particles were also produced in susceptible MARC-145 cells inoculated with AcAPRRS, and the growth characteristics of the PRRSV generated were similar to those of the parental PRRSV strain. Infectious PRRSV particles were also generated following AcAPRRS transduction of BHK-21 cells and Vero cells that are not sensitive to PRRSV. Titers of PRRSV obtained from BHK-21 and Vero cells were up to 10 4.05 TCID 50/ml. These findings open a new route to the propagation of the virus in vitro and will be of utility in vaccine development.

Deletion of AcMNPV AC16 and AC17 results in delayed viral gene expression in budded virus infected cells but not transfected cells

This study investigated the combined function of the Autographa californica multiple nucleopolyhedrovirus overlapping genes ac16 (BV/ODV-E26, DA26) and ac17. Ac17 is a late gene and the promoter is within the ac16 open reading frame. A double ac16 – ac17 knockout virus was generated to assess the function of each gene independently or together. Loss of ac17 did not affect viral DNA synthesis but budded virus (BV) production was reduced. Deletion of both ac16 – ac17 resulted in reduced viral DNA synthesis and a further reduction in BV production. In BV infected Sf9 cells, viral gene expression was delayed up to 12 h in the absence of both AC16 and AC17 but not if either gene was present. Cells infected by transfecting viral DNA, by-passing the BV particle, exhibited no delay in gene expression from the double knockout virus. AC16 and AC17 are therefore required for rapid viral gene expression in cells infected by BV.

Phosphorylation of eIF2 alpha in Sf9 cells: a stress, survival and suicidal signal

An analysis of the stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF2alpha) involved in translation regulation, in the ovarian cells of Spodoptera frugiperda (Sf9) for its role in cell survival and death reveals that it stimulates casapase activation and cell death in the absence of BiP, a chaperone and stress marker of the endoplasmic reticulum (ER). While Phospho-JNK and GADD-153 levels are elevated in non-ER stress-induced eIF2alpha phosphorylation-mediated cell death, ATF4 levels are elevated both in response to ER and non-ER stress-induced eIF2alpha phosphorylation. Infection of Sf9 cells by wt and a mutant Deltapk2 baculovirus that harbor the anti-apoptotic p35 gene induces BiP expression. However, UV-induced eIF2alpha phosphorylation and caspase activation are mitigated more efficiently by wt, but not by Deltapk2 baculovirus that lacks pk2, an inhibitor of eIF2alpha kinase. z-VAD-fmk, a caspase inhibitor reduces the late stages, but not the initial stages of non-ER stress-induced eIF2alpha phosphorylation, thereby suggesting that eIF2alpha phosphorylation is a cause and consequence of caspase activation. The importance of BiP affecting the delicate balance between eIF2alpha phosphorylation-mediated cell survival and death is further supported by the findings that tunicamycin-treated cells expressing BiP resist eIF2alpha phosphorylation-mediated cell death and addition of a purified recombinant mutant phosphomimetic form, but not wt eIF2alpha, stimulates caspase activation in cell extracts devoid of BiP. These findings therefore suggest that eIF2alpha phosphorylation is primarily a stress signal and evokes adaptive or apoptotic responses depending on its cellular location, changes in gene expression, coincident signaling activities, and inter-protein interactions.

Heterologous expression, purification, and biochemical characterization of a greenbug (Schizaphis graminum) acetylcholinesterase encoded by a paralogous gene (ace‐1)

Acetylcholinesterase is a critical enzyme in the regulation of cholinergic neurotransmission in insects. To produce Schizaphis graminum acetylcholinesterase-1 for structure-function analysis, we constructed a recombinant baculovirus to infect Sf9 cells, which secreted the soluble protein at a final concentration of 4.0 mg/L. The purified enzyme had an apparent M(r) of 70 and 130 kDa in the reducing and nonreducing SDS-polyacrylamide gels, respectively, indicating that it formed a dimer via an intermolecular disulfide bond. The fresh enzyme had a specific activity of 245 U/mg, which stabilized at a lower level (115 U/mg) in storage. The Michaelis constant and maximum velocity were 88.3 +/- 9.6 microM and 133.2 +/- 1.6 U/mg for acetylthiocholine iodide, 113.9 +/- 12.5 muM and 106.4 +/- 3.0 U/mg for acetyl(beta-methyl)thiocholine iodide, 68.9 +/- 7.8 microM and 76.7 +/- 1.0 U/mg for propionylthiocholine iodide, and 201.1 +/- 21.0 microM and 4.4 +/- 0.1 U/mg for S-butyrylthiocholine iodide, respectively. The IC(50) values (5 min, room temperature) of ethopropazine, BW284C51, carbaryl, eserine, malaoxon, and paraoxon were 102, 1.66, 0.94, 0.20, 0.061, 0.016 microM, respectively. The bimolecular reaction constants (k(i)) were (6.50 +/- 0.40) x 10(4) for carbaryl, (1.00 +/- 0.16) x 10(5) for eserine, (4.70 +/- 0.13) x 10(5) for malaoxon, and (9.06 +/- 0.23) x 10(5) M(-1) min(-1) for paraoxon. The enzyme was also inhibited by one of its products, choline, at concentrations higher than 20 mM, suggesting that choline bound to an anionic site and regulated the enzymatic activity.

Autographa californica Multiple Nucleopolyhedrovirus Core Gene ac96 Encodes a Per Os Infectivity Factor ( pif-4 )

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac96 is a core gene, but its role in virus replication is still unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac96-null virus (vAc(96)(null)). Our analyses showed that the absence of ac96 does not affect budded virus (BV) production or viral DNA replication in infected Sf9 cells. Western blotting and confocal immunofluorescence analysis showed that AC96 is expressed in both the cytoplasm and the nucleus throughout infection. In addition, AC96 was detected in the envelope fractions of both BV and occlusion-derived virus. Injection of vAc(96)(null) BV into the hemocoel killed Trichoplusia ni larvae as efficiently as repaired and control viruses; however, vAc(96)(null) was unable to infect the midgut tissue of Trichoplusia ni larvae when inoculated per os. Therefore, the results of this study show that ac96 encodes a new per os infectivity factor (PIF-4).

The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells

Many viruses activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, thereby modulating diverse downstream signaling pathways associated with antiapoptosis, proliferation, cell cycling, protein synthesis and glucose metabolism, in order to augment their replication. To date, the role of the PI3K-Akt pathway in Baculovirus replication has not been defined. In the present study, we demonstrate that infection of Sf9 cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) elevated cellular Akt phosphorylation at 1 h post-infection. The maximum Akt phosphorylation occurred at 6 h post-infection and remained unchanged until 18 h post-infection. The PI3K-speci fi c inhibitor, LY294002, suppressed Akt phosphorylation in a dose-dependent manner, suggesting that AcMNPV-induced Akt phosphorylation is PI3K-dependent. The inhibition of PI3K-Akt activation by LY294002 significantly reduced the viral yield, including a reduction in budded viruses and occlusion bodies. The virus production was reduced only when the inhibitor was added within 24 h of infection, implying that activation of PI3K occurred early in infection. Correspondingly, both viral DNA replication and late (VP39) and very late (POLH) viral protein expression were impaired by LY294002 treatment; LY294002 had no effect on immediate-early (IE1) and early-late (GP64) protein expression. These results demonstrate that the PI3K-Akt pathway is required for efficient Baculovirus replication.

An Alternatively Spliced Isoform of Non-muscle Myosin II-C Is Not Regulated by Myosin Light Chain Phosphorylation

We report a novel isoform of non-muscle myosin II-C (NM II-C), NM II-C2, that is generated by alternative splicing of an exon, C2, encoding 41 amino acids in mice (33 in humans). The 41 amino acids are inserted into loop 2 of the NM II-C heavy chain within the actin binding region. Unlike most vertebrate non-muscle and smooth muscle myosin IIs, baculovirus-expressed mouse heavy meromyosin (HMM) II-C2 demonstrates no requirement for regulatory myosin light chain (MLC(20)) phosphorylation for maximum actin-activated MgATPase activity or maximum in vitro motility as measured by the sliding actin filament assay. In contrast, noninserted HMM II-C0 and another alternatively spliced isoform HMM II-C1, which contains 8 amino acids inserted into loop 1, are dependent on MLC(20) phosphorylation for both actin-activated MgATPase activity and in vitro motility ( Kim, K. Y., Kovacs, M., Kawamoto, S., Sellers, J. R., and Adelstein, R. S. (2005) J. Biol. Chem. 280, 22769-22775 ). HMM II-C1C2, which contains both the C1 and C2 inserts, does not require MLC(20) phosphorylation for full activity similar to HMM II-C2. These constitutively active C2-inserted isoforms of NM II-C are expressed only in neuronal tissue. This is in contrast to NM II-C1 and NM II-C0, both of which are ubiquitously expressed. Full-length NM II-C2-GFP expressed in COS-7 cells localizes to filaments in interphase cells and to the cytokinetic ring in dividing cells.

Autographa californica multiple nucleopolyhedrovirus ORF 23 null mutant produces occlusion-derived virions with fewer nucleocapsids

Two envelope fusion protein gene homologues have been identified in the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). AcMNPV GP64 protein is fusogenic and essential for propagation and pathogenicity. The F homologue (Ac23) is not essential, is fusion-incompetent in standard assays, but contributes to faster host death. Here, we show that occlusion bodies (OBs) from Ac23null mutants and control viruses do not differ significantly in size and the number of occlusion-derived virions (ODVs) contained; however, Ac23null OBs had a much higher percentage of ODVs with a single nucleocapsid (44.6%) than the near-isogenic control (11.3%). Infection of Sf9 cells with Ac23-green fluorescent protein (gfp)-expressing recombinant viruses showed Ac23-gfp fluorescence overlapping perinuclear DAPI staining at later times, a pattern not observed with GP64. These results suggest that F proteins have evolved functions beyond envelope fusion and play a different role from that of GP64 in viruses that contain both proteins.

Localization of human (pro)renin receptor lacking the transmembrane domain on budded baculovirus of Autographa californica multiple nucleopolyhedrovirus

Human (pro)renin receptor (hPRR), a construct with native transmembrane and cytoplasmic domains (hPRR-wTM), and hPRR lacking both (hPRR-w/oTM) were expressed using insect cells. The hPRR-wTM was expressed in the peripheral domains of the nucleus in infected Sf-9 cells, and its localization was observed in endoplasmic reticulum (ER). However, it could not be extracted from recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by Triton X-100 treatment at 4 degrees C. In contrast, hPRR-w/oTM was observed in punctate domains in the cytoplasm of infected Sf-9 cells, but intracellular hPRR-w/oTM did not co-localize in the Golgi apparatus and lysosomes. This indicates that hPRR-wTM and hPRR-w/oTM is localized in the ER and cytoplasmic organelles of Sf-9 cell, respectively. Moreover, the localization of hPRR-w/oTM in budded baculovirus of recombinant AcMNPV was confirmed by Western blotting. This is the first finding of the association of a foreign protein lacking a transmembrane domain with a baculovirus. If this finding is available for double displaying system, being capable of expression on the envelope and the capsid of baculovirus, it will lead to new methodology of baculovirus display system for tissue- and cell-specific targeting and intracellular targeting.

Reconstruction of the P2X2 Receptor Reveals a Vase-Shaped Structure with Lateral Tunnels above the Membrane

In response to the intercellular messenger ATP, P2X receptors transfer various sensory information, including pain. Here we have reconstructed the structure of the P2X(2) receptor at 15 A resolution from more than 90,000 particle images, taken with a cryo-electron microscope equipped with a helium-cooled stage. This three-dimensional depiction, presumably in a closed state, revealed an elongated vase-shaped structure 202 A in height and 160 A in major diameter. The extracellular and transmembrane domains present a two-layered structure, in which a sparse outer layer surrounds a pore-forming inner density. The decreased diameter of a putative ion-conducting pathway at the middle of the membrane was considered to be the narrowest part of the pore, which has been predicted from electrophysiological studies. The sparse, extended structure of the P2X(2) receptor indicates a loose assembly of subunits, which could be a basis for the activation-dependent pore dilation of P2X receptors.

AcMNPV EXON0 (AC141) which is required for the efficient egress of budded virus nucleocapsids interacts with β-tubulin

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encoded protein, EXON0 (AC141), is required for the efficient transport of nucleocapsids out of the nucleus for the production of budded virus (BV). To further elucidate the molecular mechanisms by which EXON0 regulates BV production, EXON0 was tagged at the N-terminus with 3× FLAG–6× His. Protein complexes were isolated by tandem affinity purification and potential EXON0 specific interacting protein partners were gel purified and identified by LC–MS/MS. This analysis showed that the cellular protein, β-tubulin, co-purified with EXON0 which was confirmed by co-immunoprecipitation. In addition, immunofluorescence showed that EXON0 and β-tubulin co-localized during virus infection. The microtubule inhibitors colchicine and nocodazole were used to treat AcMNPV infected Sf9 cells and results showed that BV production was reduced by over 85%. These data suggest that the egress of AcMNPV budded virus may be facilitated by the interaction of EXON0 with β-tubulin and microtubules.

AcMNPV AC16 (DA26, BV/ODV-E26) regulates the levels of IE0 and IE1 and binds to both proteins via a domain located within the acidic transcriptional activation domain

IE0 and IE1 are the primary viral regulatory proteins of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) involved in the transactivation of early genes, stimulation of late gene expression, and viral DNA replication. The protein interactions required for IE0 or IE1 to achieve these varied roles are not well defined, so to identify proteins that interact with IE0 and IE1, tandem affinity purification (TAP) and LC-MS/MS was used. Analysis of purified proteins identified AC16 (DA26, BV/ODV-E26) from TAP tagged IE0 virus infected Sf9 cells. Co-immunoprecipitation confirmed that AC16 interacts with both IE0 and IE1 and yeast 2-hybrid analysis mapped the domain required for interaction with AC16. Mutation of the AC16 binding domain enhanced BV production by viruses expressing only IE0 but had no effect if only IE1 is expressed. An ac16 deletion virus was constructed and was shown not to affect the temporal expression of IE0 and IE1; however the relative level of IE0 to IE1 was significantly increased.

Crystal Structures of the Lyn Protein Tyrosine Kinase Domain in Its Apo- and Inhibitor-bound State

The Src-family protein-tyrosine kinase (PTK) Lyn is the most important Src-family kinase in B cells, having both inhibitory and stimulatory activity that is dependent on the receptor, ligand, and developmental context of the B cell. An important role for Lyn has been reported in acute myeloid leukemia and chronic myeloid leukemia, as well as certain solid tumors. Although several Src-family inhibitors are available, the development of Lyn-specific inhibitors, or inhibitors with reduced off-target activity to Lyn, has been hampered by the lack of structural data on the Lyn kinase. Here we report the crystal structure of the non-liganded form of Lyn kinase domain, as well as in complex with three different inhibitors: the ATP analogue AMP-PNP; the pan Src kinase inhibitor PP2; and the BCR-Abl/Src-family inhibitor Dasatinib. The Lyn kinase domain was determined in its "active" conformation, but in the unphosphorylated state. All three inhibitors are bound at the ATP-binding site, with PP2 and Dasatinib extending into a hydrophobic pocket deep in the substrate cleft, thereby providing a basis for the Src-specific inhibition. Analysis of sequence and structural differences around the active site region of the Src-family PTKs were evident. Accordingly, our data provide valuable information for the further development of therapeutics targeting Lyn and the important Src-family of kinases.