IE1 of Autographa californica Multiple Nucleopolyhedrovirus Activates Low Levels of Late Gene Expression in the Absence of Virus RNA Polymerase.

Early and late gene expressions of baculoviruses have been known to rely on host RNA polymerase II and a virus-encoded RNA polymerase, separately. In this study, we found that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) recombinant bacmids with the individual RNA polymerase subunit genes deleted could support low levels of expression of a reporter gene under the control of the promoter of a typical late gene, vp39, in transfected Sf9 cells. Through multistep subcloning of a genomic library of the virus and transient expression assay analysis, ie1 was identified to be the only viral gene that was responsible for activation of late gene expression in the absence of the viral RNA polymerase. Furthermore, IE1 was found to be capable of activating reporter gene expression from the promoters of additional late genes polh, p6.9, odv-e18, odv-e25, and gp41, independent of any additional viral factors. Deletion of ie1 from the virus genome eliminated late gene expression. The IE1-activated late gene expression was enhanced by the viral hr4b. It was shown to be insensitive to inhibition of α-amanitin and did not appear to have stable transcription start sites. It is proposed that IE1 may serve to recruit newly synthesized viral RNA polymerase to viral DNA by activating low levels of pretranscription of the late genes to create an appropriate DNA conformation. IMPORTANCE The late gene expression of baculovirus has been known to depend on the virus-encoded RNA polymerase, which consists of four subunits. The immediate-early gene ie1 was found to be required for viral early gene expression, late gene expression, and DNA replication. How it functions in late gene expression remains unclear. In this study, we found that AcMNPV IE1 could activate low levels of gene expression from late gene promoters independently of any additional viral factors, with nonspecific transcription start sites. This new finding will shed light on the role of IE1 in the regulation of late gene expression and the understanding of the mechanism of late gene transcription initiation.

Comparative genomic analysis of three geographical isolates from China reveals high genetic stability of Plutella xylostella granulovirus.

In this study, the genomes of three Plutella xylostella granulovirus (PlxyGV) isolates, PlxyGV-W and PlxyGV-Wn from near Wuhan and PlxyGV-B from near Beijing, China were completely sequenced and comparatively analyzed to investigate genetic stability and diversity of PlxyGV. PlxyGV-W, PlxyGV-B and PlxyGV-Wn consist of 100,941bp, 100,972bp and 100,999bp in length with G + C compositions of 40.71-40.73%, respectively, and share nucleotide sequence identities of 99.5-99.8%. The three individual isolates contain 118 putative protein-encoding ORFs in common. PlxyGV-W, PlxyGV-B and PlxyGV-Wn have ten, nineteen and six nonsynonymous intra isolate nucleotide polymorphisms (NPs) in six, fourteen and five ORFs, respectively, including homologs of five DNA replication/late expression factors and two per os infectivity factors. There are seventeen nonsynonymous inter isolate NPs in seven ORFs between PlxyGV-W and PlxyGV-B, seventy three nonsynonymous NPs in forty seven ORFs between PlxyGV-W and PlxyGV-Wn, seventy seven nonsynonymous NPs in forty six ORFs between PlxyGV-B and PlxyGV-Wn. Alignment of the genome sequences of nine PlxyGV isolates sequenced up to date shows that the sequence homogeneity between the genomes are over 99.4%, with the exception of the genome of PlxyGV-SA from South Africa, which shares a sequence identity of 98.6-98.7% with the other ones. No events of gene gain/loss or translocations were observed. These results suggest that PlxyGV genome is fairly stable in nature. In addition, the transcription start sites and polyadenylation sites of thirteen PlxyGV-specific ORFs, conserved in all PlxyGV isolates, were identified by RACE analysis using mRNAs purified from larvae infected by PlxyGV-Wn, proving the PlxyGV-specific ORFs are all genuine genes.

Autographa californica multiple nucleopolyhedrovirus ac75 is required for egress of nucleocapsids from the nucleus and formation of de novo intranuclear membrane microvesicles.

In this study, Autographa californica multiple nucleopolyhedrovirus ac75 was functionally characterized. Ac75 has homologs in all sequenced genomes of alphabaculoviruses, betabaculoviruses, and gammabaculoviruses. It was determined to encode a protein that is associated with the nucleocapsid of budded virus and with both envelope and nucleocapsids of occlusion-derived virus. Sf9 cells transfected by an ac75-knockout bacmid resulted in the infection being restricted to single cells. No budded virus were detected although viral DNA replication and late gene expression were unaffected. Electron microscopy revealed that the virogenic stroma, nucleocapsids and occlusion bodies appeared normal in the cells transfected by an ac75-knockout bacmid. However, the nucleocapsids were unenveloped, the occlusion bodies did not contain any virions or nucleocapsids, and no nucleocapsids were found outside the nucleus or spanning the nuclear membrane. In addition, de novo intranuclear membrane microvesicles that are the precursor of occlusion-derived virus envelopes were absent in the nuclei of transfected cells. Confocal microscopy showed that AC75 protein appeared in the cytoplasm as early as 6 hours post infection. It localized to the ring zone at the periphery of the nucleus from 15 to 24 hours post infection and demonstrated light blocky cloud-like distribution in the center of the nucleus. AC75 was found to co-immunoprecipitate with BV and ODV associated envelope protein ODV-E25. The data from this study suggest that ac75 is essential for induction of the intranuclear membrane microvesicles, it appears to be required for the intranuclear envelopment of nucleocapsids, and is also essential for egress of nucleocapsids from the nuclei, in infected cells.

Plutella xylostella granulovirus late gene promoter activity in the context of the Autographa californica multiple nucleopolyhedrovirus genome.

Within Baculoviridae, little is known about the molecular mechanisms of replication in betabaculoviruses, despite extensive studies in alphabaculoviruses. In this study, the promoters of nine late genes of the betabaculovirus Plutella xylostella granulovirus (PlxyGV) were cloned into a transient expression vector and the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome, and compared with homologous late gene promoters of AcMNPV in Sf9 cells. In transient expression assays, all PlxyGV late promoters were activated in cells transfected with the individual reporter plasmids together with an AcMNPV bacmid. In infected cells, reporter gene expression levels with the promoters of PlxyGV e18 and AcMNPV vp39 and gp41 were significantly higher than those of the corresponding AcMNPV or PlxyGV promoters, which had fewer late promoter motifs. Observed expression levels were lower for the PlxyGV p6.9, pk1, gran, p10a, and p10b promoters than for the corresponding AcMNPV promoters, despite equal numbers of late promoter motifs, indicating that species-specific elements contained in some late promoters were favored by the native viral RNA polymerases for optimal transcription. The 8-nt sequence TAAATAAG encompassing the ATAAG motif was conserved in the AcMNPV polh, p10, and pk1 promoters. The 5-nt sequence CAATT located 4 or 5 nt upstream of the T/ATAAG motif was conserved in the promoters of PlxyGV gran, p10c, and pk1. The results of this study demonstrated that PlxyGV late gene promoters could be effectively activated by the RNA polymerase from AcMNPV, implying that late gene expression systems are regulated by similar mechanisms in alphabaculoviruses and betabaculoviruses.

Functional characterization of Autographa californica multiple nucleopolyhedrovirus gp16 (ac130).

To investigate the function of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) gp16, multiple gp16-knockout and repair mutants were constructed and characterized. No obvious difference in productivity of budded virus, DNA synthesis, late gene expression and morphogenesis was observed between gp16-knockout and repair viruses, but gp16 deletion resulted in six hours of lengthening in ST50 to the third instar Spodoptera exigua larvae in bioassays. GP16 was fractionated mainly in the light membrane fraction, by subcellular fractionation. A GP16-EGFP fusion protein was predominantly localized close around the nuclear membrane in infected cells, being coincident with formation of the vesicles associated with the nuclear membrane, which hosted nucleocapsids released from the nucleus. These data suggest that gp16 is not required for viral replication, but may be involved in membrane trafficking associated with the envelopment/de-envelopment of budded viruses when they cross over the nuclear membrane and pass through cytoplasm.

Autographa californica multiple nucleopolyhedrovirus orf132 encodes a nucleocapsid-associated protein required for budded-virus and multiply enveloped occlusion-derived virus production.

UNLABELLED: Autographa californica multiple nucleopolyhedrovirus orf132 (named ac132) has homologs in all genome-sequenced group I nucleopolyhedroviruses. Its role in the viral replication cycle is unknown. In this study, ac132 was shown to express a protein of around 28 kDa, which was determined to be associated with the nucleocapsids of both occlusion-derived virus and budded virus. Confocal microscopy showed that AC132 protein appeared in central region of the nucleus as early as 12 h postinfection with the virus. It formed a ring zone at the periphery of the nucleus by 24 h postinfection. To investigate its role in virus replication, ac132 was deleted from the viral genome by using a bacmid system. In the Sf9 cell culture transfected by the ac132 knockout bacmid, infection was restricted to single cells, and the titer of infectious budded virus was reduced to an undetectable level. However, viral DNA replication and the expression of late genes vp39 and odv-e25 and a reporter gene under the control of the very late gene p10 promoter were unaffected. Electron microscopy showed that nucleocapsids, virions, and occlusion bodies were synthesized in the cells transfected by an ac132 knockout bacmid, but the formation of the virogenic stroma and occlusion bodies was delayed, the numbers of enveloped nucleocapsids were reduced, and the occlusion bodies contained mainly singly enveloped nucleocapsids. AC132 was found to interact with envelope protein ODV-E18 and the viral DNA-binding protein P6.9. The data from this study suggest that ac132 possibly plays an important role in the assembly and envelopment of nucleocapsids. IMPORTANCE: To our knowledge, this is the first report on a functional analysis of ac132. The data presented here demonstrate that ac132 is required for production of the budded virus and multiply enveloped occlusion-derived virus of Autographa californica multiple nucleopolyhedrovirus. This article reveals unique phenotypic changes induced by ac132 deletion on the virus and multiple new findings on ac132.

Homologous region 1 of Plutella xylostella granulovirus functions as an enhancer for early gene expression.

Homologous regions (hrs) contained in genomes of baculoviruses have been shown to function as origins of viral DNA replication in alphabaculoviruses and betabaculoviruses, and as enhancers for early gene expression in alphabaculoviruses. The hr sequences of betabaculoviruses differ substantially from the ones of alphabaculoviruses. The enhancing property of betabaculovirus hrs has not been reported. In this study, transient assays were performed to investigate the effects of Plutella xylostella granulovirus (PlxyGV) hr1 on early gene expression of the virus. It was shown that hr1 stimulated reporter gene expression from the promoters of four early genes--ie1, dnapol, lef1, and lef9--independent of additional viral gene expression. The PlxyGV ie1 was shown to repress reporter gene expression from all four early gene promoters in a Trichoplusia ni cell line, both in the presence and absence of hr.

The p35 and ie1 of Autographa californica multiple nucleopolyhedrovirus could rescue late gene expression of Plutella xylostella granulovirus in nonpermissive cell lines.

There are no stable permissive cell lines available for in vitro replication of PlxyGV. In this study, several PlxyGV bacmids containing egfp and plasmids expressing the luciferase gene (luc) were constructed and used to transfect insect cell lines from Plutella xylostella, Trichoplusia ni (Hi5), and Spodoptera frugiperda. Fluorescence was observed only in the cells transfected with a bacmid with egfp driven by a PlxyGV ie1 promoter, but not by a PlxyGV vp39 or granulin promoter. In transient assays, various levels of LUC activity were detected in the cells transfected with individual reporter plasmids containing the luc driven by the promoters of PlxyGV early genes ie1, exon0, dnapol, lef1, lef9, and orf105, suggesting that the PlxyGV early genes could be activated in the cells independent of virus infection. The addition of a PlxyGV bacmid in the transfections activated luc expression from the promoters of PlxyGV late genes vp39 and granulin only at minimum levels, and caused significant reduction in luc expression from the early promoters, may be due to apoptosis triggered by the PlxyGV bacmid. PlxyGV reporter bacmids containing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genes p35 or p35 and ie1 or p35, ie1 and gp64 expressed LUC from a PlxyGV vp39 promoter at levels of 2.6, 8.3, and 23 times higher than those produced by the basic PlxyGV reporter bacmid, respectively, in transfected Hi5 cells. Green fluorescence was present in the cultures of all three cell lines transfected by a PlxyGV bacmid containing egfp with a vp39 promoter and AcMNPV ie1, p35, and gp64 with their native promoters. The fluorescence was also observed in the culture of Hi5 cells inoculated with the supernatant from the transfection. These results suggest that AcMNPV p35 could rescue late gene expression, and the ie1, p35, and gp64 may cooperatively rescue replication of PlxyGV in the cells.

High-performance large-scale flexible dye-sensitized solar cells based on anodic TiO2 nanotube arrays.

A simple strategy to fabricate flexible dye-sensitized solar cells involves the use of photoanodes based on TiO2 nanotube (TNT) arrays with rear illumination. The TNT films (tube length ∼35 µm) were produced via anodization, and sensitized with N719 dye for photovoltaic characterization. Pt counter electrodes of two types were used: a conventional FTO/glass substrate for a device of rigid type and an ITO/PEN substrate for a device of flexible type. These DSSC devices were fabricated into either a single-cell structure (active area 3.6×0.5 cm2) or a parallel module containing three single cells (total active area 5.4 cm2). The flexible devices exhibit remarkable performance with efficiencies η=5.40% (single cell) and 4.77% (parallel module) of power conversion, which outperformed their rigid counterparts with η=4.87% (single cell) and 4.50% (parallel model) under standard one-sun irradiation. The flexible device had a greater efficiency of conversion of incident photons to current and a broader spectral range than the rigid device; a thinner electrolyte layer for the flexible device than for the rigid device is a key factor to improve the light-harvesting ability for the TNT-DSSC device with rear illumination. Measurements of electrochemical impedance spectra show excellent catalytic activity and superior diffusion characteristics for the flexible device. This technique thus provides a new option to construct flexible photovoltaic devices with large-scale, light-weight, and cost-effective advantages for imminent applications in consumer electronics.

Effects of Early or Overexpression of the Autographa californica Multiple Nucleopolyhedrovirus orf94 (ODV-e25) on Virus Replication.

odv-e25(e25) is one of the core genes of baculoviruses. To investigate how it functions in the replication cycle of a baculovirus, a number of Autographa californica multiple nucleopolyhedrovirus recombinants with e25 under control of the promoter of immediate early gene ie1, or the promoter of the very late hyperexpressed gene p10, were constructed using a bacmid system, and the effects of early expression or overexpression of e25 on replication of the virus were evaluated. Microscopy and titration assays demonstrated that bacmids with e25 under control of ie1 promoter were unable to produce budded viruses; and that the recombinant viruses with e25 under control of p10 promoter generated budded virus normally, but formation of occlusion bodies were dramatically reduced and delayed in the infected cells. Electron microscopy showed that there were no mature virions or intact nucleocapsids present in the cells transfected with a recombinant bacmid with e25 under control of ie1 promoter. Quantitative real-time PCR analysis demonstrated that alteration of the e25 promoter did not affect viral DNA synthesis. The reporter gene expression from the promoter of the major capsid protein gene vp39 was reduced 63% by early expression of e25. Confocal microscopy revealed that E25 was predominantly localized in nuclei by 24 hours post infection with wild-type virus, but it remained in the cytoplasm in the cells transfected with a recombinant bacmid with e25 under control of the ie1 promoter, suggesting that the transport of E25 into nuclei was regulated in a specific and strict time dependent manner.

Computational studies on polynitropurines as potential high energy density materials.

As part of a search for high energy density materials (HEDMs), a series of purine derivatives with nitro groups were designed computationally. The relationship between the structures and the performances of these polynitropurines was studied. Density functional theory (DFT) at the B3LYP/6-311G** level was employed to evaluate the heats of formation (HOFs) of the polynitropurines by designing an isodesmic reaction method. Results indicated that the HOFs were influenced by the number and positions of substituent groups. Detonation properties were evaluated using the Kamlet-Jacobs equations, based on the theoretical densities and heats of formation of the polynitropurines. The relative stabilities of the polynitropurines were studied via the pyrolysis mechanism and the UB3LYP/6-311G** method. Homolysis of the ring-NO2 bond is predicted to be the initial step in the thermal decomposition of these purine derivatives. Considering their detonation properties and relative stabilities, the tetranitropurine (D1) derivatives may be regarded as potential candidates for practical HEDCs. These results may provide useful information for further investigations.

Porphyrin-sensitized solar cells.

Nature has chosen chlorophylls in plants as antennae to harvest light for the conversion of solar energy in complicated photosynthetic processes. Inspired by natural photosynthesis, scientists utilized artificial chlorophylls - the porphyrins - as efficient centres to harvest light for solar cells sensitized with a porphyrin (PSSC). After the first example appeared in 1993 of a porphyrin of type copper chlorophyll as a photosensitizer for PSSC that achieved a power conversion efficiency of 2.6%, no significant advance of PSSC was reported until 2005; beta-linked zinc porphyrins were then reported to show promising device performances with a benchmark efficiency of 7.1% reported in 2007. Meso-linked zinc porphyrin sensitizers in the first series with a push-pull framework appeared in 2009; the best cell performed comparably to that of a N3-based device, and a benchmark 11% was reported for a porphyrin sensitizer of this type in 2010. With a structural design involving long alkoxyl chains to envelop the porphyrin core to suppress the dye aggregation for a push-pull zinc porphyrin, the PSSC achieved a record 12.3% in 2011 with co-sensitization of an organic dye and a cobalt-based electrolyte. The best PSSC system exhibited a panchromatic feature for light harvesting covering the visible spectral region to 700 nm, giving opportunities to many other porphyrins, such as fused and dimeric porphyrins, with near-infrared absorption spectral features, together with the approach of molecular co-sensitization, to enhance the device performance of PSSC. According to this historical trend for the development of prospective porphyrin sensitizers used in PSSC, we review systematically the progress of porphyrins of varied kinds, and their derivatives, applied in PSSC with a focus on reports during 2007-2012 from the point of view of molecular design correlated with photovoltaic performance.

[Effects of mutations in the autographa californica multiple nucleopolyhedrovirus E25 on its trafficking to nucleus and budded virus production].

This study was performed to investigate the effects of different regions of the Autographa califor nica multiple nucleopolyhedrovirus envelope protein E25 on its trafficking into nucleus and nuclear localization in host cells and on virus replication. Fourteen recombinant bacmids, each containing an e25 mutant with substitution or insertion of egfp, in the absence or presence of the native e25, were constructed and used to transfect Sf9 cells. The E25-EGFP fusion proteins and native E25 expressed in the cells transfect ed with individual recombinant bacmid were traced by autofluorescence from EGFP or by immuno-fluorescence assays. Confocal microscopy revealed that the E25-EGFP fusion protein with the N-domain (2-45aa) of E25 substituted by EGFP only distributed in the cytoplasm in transfected cells; and the fusion protein with EGFP inserted at the laa/2aa site of E25 completely remained outside of the nucleus and resided along the nuclear membrane. The E25-EGFPs with 46-118aa of E25 substituted by EGFP or with EGFP inserted at the 118aa/119aa site were present outside, across from the nuclear membrane or in nuclear plasm in dot-like shapes. The fusion proteins with the C-domain substituted by EGFP or with EGFP inserted at the site of 45/46aa or at the C-terminal formed a condensed ring or spread throughout the nucleus, in a similar manner to the E25 distributed in the cells transfected by the e25-knockout repair bacmid. These results prove that the N-terminal domain is critical for nuclear transportation of E25 and possibly to its position on the cytoplasm membrane as well; and the sequence downstream of the N-terminal domain also affects trafficking and nuclear localization of the protein. In cells transfected with bacmids containing both the native e25 and individual e25-egfp mutants, the E25-EGFP fusion proteins co-localized with E25 individually, showing similar patterns of subcellular localization as E25 mutants in the absence of native E25 in most cases, suggesting that the E25 likely exists and functions as dimmers or polymers. Production of infectious BV was dramatically reduced and even completely eliminated in most cases, either in the absence or presence of the native e25.

Theoretical investigation on detonation performances and thermodynamic stabilities of the prismane derivatives.

Based on DFT-B3LYP/6-311G** method, the molecular geometric structures of polynitramineprismanes are fully optimized. The detonation performances, energy gaps, strain energies, as well as their stability were investigated to look for high energy density compounds (HEDCs). Our results show that all polynitramineprismanes have high and positive heat of formation. To construct the relationship between stabilities and structures, energy gaps and bond dissociation energies are calculated, and these results show that the energy gaps of prismane derivatives are much higher than that of TATB (0.1630). In addition, the C-C bonds on cage are confirmed as trigger bond in explosive reaction. All polynitramineprismanes have large strain energies, and the strain energies of all compounds are slightly smaller than prismane, which indicated that the strain energies were somewhat released compared to prismane. Considering the quantitative criteria of HEDCs, hexanitramineprismane is a good candidate of high energy compounds.

Looking for high energy density compounds among polynitraminecubanes.

Based on fully optimized geometric structures at DFT-B3LYP/6-311G** level, we calculated electronic structures, heats of formation, strain energies, bond dissociation energies and detonation performance (detonation velocity and detonation pressure) for a series of polynitraminecubanes. Our results have shown that energy gaps of cubane derivatives are much higher than that of triaminotrinitrobenzene (TATB), which means that cubane derivatives may be more sensitive than TATB. Polynitraminecubanes have high and positive heats of formation, and a good linear relationship between heats of formation and nitramine group numbers was presented. As the number of nitramine groups in the molecule increases, the enthalpies of combustion values are increasingly negative, but the specific enthalpy of combustion values decreases. It is found that all cubane derivatives have high strain energies, which are affected by the number and position of nitramine group. The calculated bond dissociation energies of C-NHNO(2) and C-C bond show that the C-C bond should be the trigger bond in the pyrolysis process. It is found that detonation velocity (D), detonation pressure (P) and molecule density (ρ) have good linear relationship with substituented group numbers. Heptanitraminecubane and octanitraminecubane have good detonation performance over 1,3,5,7-tetranitro-1,3,5,7-tetraazacyclooctane (HMX), and they can be regarded as potential candidates of high energy density compounds (HEDCs). The results have not only shown that these compounds may be used as HEDCs, but also provide some useful information for further investigation.

[Functional analysis of the late expression factor genes of plutella xylostella granulovirus].

Plutella xylostella granulovirus (PlxyGV) contains homologs of 15 Autographa californica MNPV (AcMNPV) late expression factor (lef) genes. The prospective products of 14 PlxyGV lef genes (ie-0 is not included) share 13%-53% amino acid similarity with their corresponding homologs of AcMNPV, among which LEF-9, LEF-8 and P47, three subunits of the virus-encoded RNA polymerase, share 49%, 53% and 46% sequence identity, respectively. In this study, an established transient expression system was used to test the ability of the PlxyGV LEFs to activate an AcMNPV vp39 promoter-driven reporter gene in SF9 cells. It was shown that PlxyGV le f-2 replaced the corresponding AcMNPV gene and exhibited partial activity in the context of the remaining set of AcMNPV le fs. PlxyGV LEF-2 was found to contain additional 100aa and 70aa at the C-terminus in comparison with the LEF-2 of other GVs and lepidopteran NPVs respectively.

Density functional calculations for a high energy density compound of formula C6H 6-n (NO 2) n.

A series of polynitroprismanes, C(6)H(6-n )(NO(2))(n) (n = 1-6) intended for use as high energy density compounds (HEDCs) were designed computationally. Their electronic structures, heats of formation, interactions between nitro groups, specific enthalpies of combustion, bond dissociation energies, and explosive performances (detonation velocities and detonation pressures) were calculated using density functional theory (DFT) with the 6-311 G** basis set. The results showed that all of the polynitroprismanes had high positive heats of formation that increased with the number of substitutions for the prismane derivatives, while the specific enthalpy of combustion decreased as the number of nitro groups increased. In addition, the range of enthalpy of combustion reducing is getting smaller. Interactions between ortho (vicinal) groups deviate from the group additivity rule and decrease as the number of nitro groups increases. In terms of thermodynamic stability, all of the polynitroprismanes had higher bond dissociation energies (BDEs) than RDX and HMX. Detonation velocities and detonation pressures were estimated using modified Kamlet-Jacobs equations based on the heat of detonation (Q) and the theoretical density of the molecule (ρ). It was found that ρ, D, and P are strongly linearly related to the number of nitro groups. Taking both their energetic properties and thermal stabilities into account, pentanitroprismane and hexanitroprismane are potential candidate HEDCs.

[Plutella xylostella granulovirus PP31 interacts with two host proteins].

Protein-protein interactions between viruses and hosts are common during viral infection and replication. In this study, a cDNA library from larvae of Plutella xylostella was constructed and used for screening of genes encoding proteins interacting with Plutella xylostella granulovirus (PlxyGV) proteins. Two cDNA clones containing genes encoding proteins interacting with PlxyGV PP31 were identified by yeast two-hybrid assays. Sequence analysis showed that the genes encoded homologues of receptor for activated protein C kinase (RACK) and methionine aminopeptidase 2 (MetAP2), respectively. The P. xylostella rack gene and the PlxyGV pp31 was expressed in an E. coli strain to produce proteins fused with a 6-His or a GST tag. It was shown that the rack was expressed as a 38kD peptide as prospected. The 38kD His-tagged peptide was co-purified with GST-PP31 by GST-bind resin in GST-pulldown assays, confirming interaction between the PlxyGV PP31 and the RACK protein of P. xylostella.

Theoretical calculation about the valence and Rydberg excited states of hydrogen cyanide.

The singlet and triplet excited states of hydrogen cyanide have been computed by using the complete active space self-consistent field and completed active space second order perturbation methods with the atomic natural orbital (ANO-L) basis set. Through calculations of vertical excitation energies, we have probed the transitions from ground state to valence excited states, and further extensions to the Rydberg states are achieved by adding 1s1p1d Rydberg orbitals into the ANO-L basis set. Four singlet and nine triplet excited states have been optimized. The computed adiabatic energies and the vertical transition energies agree well with the available experimental data and the inconsistencies with the available theoretical reports are discussed in detail.

[Cloning and functional research of Arp2/3-P40/ARPC1 subunit of Sf9 cells].

The baculovirus-induced actin polymerization is mainly associated with the virus nucleocapsid protein P78/83, which is homologous with WASP proteins that can activate Arp2/3 complex and induce the actin polymerization. In order to explore the role of Arp2/3 complex in the baculovirus replication, the P40 subunit of Arp2/3 complex from Sf9 (Spodoptera frugiperda 9) cell line was cloned and characterized. Immunofluorescent microscopy assay indicated that P40 was recruited to the inner-side of nuclear membrane during virus infection, which was in accordance with nuclear F-actin distribution in virus-infected cells as documented in our previous research, suggesting P40 could be used to track Arp2/3 complex subcellular distribution changes during virus infection. In addition, co-immunoprecipitation assay demonstrated that P40 interacted with P78/83 only in virus-infected cells, suggesting that actin polymerization induced by P78/83-Arp2/3 complex during baculovirus infection was regulated by some unidentified virus factors.