RESULTS:
1 - 20 of 30 for "Monique M. van Oers"
Transcriptional dynamics during Heliothis zea nudivirus 1 infection in an ovarian cell line from Helicoverpa zea
Nudiviruses (family Nudiviridae) are double-stranded DNA viruses that infect various insects and crustaceans. Among them Heliothis zea nudivirus 1 (HzNV-1) represents the rare case of a lepidopteran nudivirus inducing a sexual pathology. Studies about molecular pathological dynamics of HzNV-1 or other nudiviruses are scarce. Hence this study aims to provide a transcriptomic profile of HzNV-1 in an ovary-derived cell line of Helicoverpa zea (HZ-AM1) during early (3 6 and 9 h post-infection) and advanced (12 and 24 h post-infection) stages of infection. Total RNA was extracted from both virus- and mock-infected cells and RNA-seq analysis was performed to examine both virus and host transcriptional dynamics. Hierarchical clustering was used to categorize viral genes while differential gene expression analysis was utilized to pinpoint host genes that are significantly affected by the infection. Hierarchical clustering classified the 154 HzNV-1 genes into four temporal phases with early phases mainly involving transcription and replication genes and later phases including genes for virion assembly. In addition a novel viral promoter motif was identified in the upstream region of early-expressed genes. Host gene analysis revealed significant upregulation of heat shock protein genes and downregulation of histone genes. The identification of temporal patterns in viral gene expression enhances the molecular understanding of nudivirus pathology while the identified differentially expressed host genes highlight the key pathways most hijacked by HzNV-1 infection.
Functional analysis of the baculovirus per os infectivity factors 3 and 9 by imaging the interaction between fluorescently labelled virions and isolated midgut cells
Baculovirus occlusion-derived viruses (ODVs) contain ten known per os infectivity factors (PIFs). These PIFs are crucial for midgut infection of insect larvae and form with the exception of PIF5 an ODV entry complex. Previously R18-dequenching assays have shown that PIF3 is dispensable for binding and fusion with midgut epithelial cells. Oral infection nevertheless fails in the absence of PIF3. PIF9 has not been analysed in much depth yet. Here the biological role of these two PIFs in midgut infection was examined by monitoring the fate of fluorescently labelled ODVs when incubated with isolated midgut cells from Spodoptera exigua larvae. Confocal microscopy showed that in the absence of either PIF3 or PIF9 the ODVs bound to the brush borders but the nucleocapsids failed to enter the cells. Finally we discuss how the results obtained for PIF3 with dequenching assays and confocal microscopy can be explained by a two-phase fusion process.
The C-termini of the baculovirus per os infectivity factors 1 and 2 mediate ODV oral infectivity by facilitating the binding of PIF0 and PIF8 to the core of the entry complex
Oral infection of caterpillars by baculoviruses is initiated by occlusion-derived virus particles (ODVs) that infect midgut epithelium cells. The ODV envelope therefore contains at least ten different proteins which are called per os infectivity factors (PIFs). Nine of these PIFs form the so-called ODV entry complex that consists of a stable core formed by PIF1 2 3 and 4 to which the other PIFs [PIF0 6 7 8 and 9 (ac108)] bind with lower affinity. PIF1 and 2 are not only essential for complex formation but also mediate ODV-binding to the epithelial brush border probably via the C-termini. To study the involvement of these PIFs during midgut infection in greater detail we assessed the oral infectivity and the ability to form the complex of a series of PIF1 and PIF2 C-terminal truncation mutants of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) which were constructed in this study. Limited truncation of either PIF1 or 2 already severely impaired the ODV oral infectivity but did not affect the formation of the core complex. However the entry complex as a whole was not assembled in these mutants as PIF0 and 8 failed to bind to the core. This suggests that the interactions between the core and the loosely associated PIFs are important for the ODV infectivity and that complex formation complicates the determination of the exact roles of PIF1 and 2 during midgut infection. We also showed that the presence of PIF0 6 and the ZF-domain of PIF8 are crucial for complex formation.
ICTV Virus Taxonomy Profile: Nudiviridae
Members of the family Nudiviridae are large dsDNA viruses with distinctive rod-shaped nucleocapsids and circular genomes of 96–232 kbp. Nudiviruses have been identified from a diverse range of insects and crustaceans and are closely related to baculoviruses. This is a summary of the International Committee on Taxonomy of Viruses Report on the taxonomy of the family Nudiviridae which is available at ictv.global/report/nudiviridae.
The baculovirus Ac108 protein is a per os infectivity factor and a component of the ODV entry complex
Wild-type ODVs (Wt) have an intact ODV entry complex in their envelope and are orally infectious towards insect larvae (left panel). In the absence of Ac108 (mut ac108) the stable core is still present but nevertheless fails to form an entry complex affecting the ODV oral infectivity (right panel). The components of the core complex are depicted in yellow and the loosely associated components are depicted in red. PIF7 is depicted in green as its affinity with the complex is currently not known.
Baculoviruses orally infect insect larvae when they consume viral occlusion bodies (OBs). OBs consist of a crystalline protein matrix in which the infectious virus particles the occlusion-derived viruses (ODVs) are embedded. The protein matrix dissolves in the alkaline environment of the insect’s midgut lumen. The liberated ODVs can then infect midgut endothelial cells through the action of at least nine different ODV-envelope proteins called per os infectivity factors (PIFs). These PIF proteins mediate ODV oral infectivity but are not involved in the systemic spread of the infection by budded viruses (BVs). Eight of the known PIFs form a multimeric complex named the ODV entry complex. In this study we show for Autographa californica multiple nucleopolyhedrovirus that mutation of the ac108ORF abolishes the ODV oral infectivity while production and infectivity of the BVs remains unaffected. Furthermore repair of the ac108 mutant completely recovered oral infectivity. With an HA-tagged repair mutant we were able to demonstrate by Western analysis that the Ac108 protein is a constituent of the ODV entry complex where the formation was abolished in the absence of this protein. Based on these results we conclude that ac108 encodes a per os infectivity factor (PIF9) that is also an essential constituent of the ODV entry complex.
ICTV Virus Taxonomy Profile: Baculoviridae
The family Baculoviridae comprises large viruses with circular dsDNA genomes ranging from 80 to 180 kbp. The virions consist of enveloped rod-shaped nucleocapsids and are embedded in distinctive occlusion bodies measuring 0.15–5 µm. The occlusion bodies consist of a matrix composed of a single viral protein expressed at high levels during infection. Members of this family infect exclusively larvae of the insect orders Lepidoptera Hymenoptera and Diptera. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Baculoviridae which is available at www.ictv.global/report/baculoviridae.
Protein–protein interactions among the structural proteins of Chilo iridescent virus
Chilo iridescent virus (CIV) officially named invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic virus with a ~212-kb linear dsDNA genome that encodes 215 putative open reading frames (ORFs). Proteomic analysis has revealed that the CIV virion consists of 54 virally encoded proteins. In this study we identified the interactions between the structural proteins using the yeast two-hybrid system. We cloned 47 structural genes into both bait and prey vectors and then analysed the interactions in Saccharomyces cerevisiae strain AH109. A total of 159 protein–protein interactions were detected between the CIV structural proteins. Only ORF 179R showed a self-association. Four structural proteins that have homologues in iridoviruses (118L 142R 274L and 295L) showed indirect interactions with each other. Seven proteins (138R 142R 361L 378R 395R 415R and 453R) interacted with the major capsid protein 274L. The putative membrane protein 118L a homologue of the frog virus 3/Ranagrylio virus 53R protein showed direct interactions with nine other proteins (117L 229L 307L 355R 366R 374R 378R 415R and 422L). The interaction between 118L and 415R was confirmed by a GST pull-down assay. These data indicate that 415R is a potential matrix protein connecting the envelope protein 118L with the major capsid protein 274L.
Baculoviruses require an intact ODV entry-complex to resist proteolytic degradation of per os infectivity factors by co-occluded proteases from the larval host
Baculoviruses orally infect caterpillars in the form of occlusion-derived viruses (ODVs). The ODV-envelope contains a number of proteins which are essential for oral infectivity called per os infectivity factors (PIFs). Most of these PIFs are involved in the formation of an ODV-entry complex that consists of a stable core formed by PIF1 PIF2 PIF3 and PIF4 and the more loosely associated PIFs P74 (PIF0) and P95 (PIF8). PIF1 PIF2 and PIF3 are essential for formation of the stable core whereas deletion of the pif4 gene results in the formation of a smaller complex. P74 is not needed for formation of the stable core. We show here in larva-derived ODVs of the Autographa californica multicapsid nucleopolyhedrovirus that PIF-proteins are degraded by host-derived proteases after deletion of a single pif-gene. Constituents of the stable core-complex appeared to be more resistant to proteases as part of the complex than as monomer as in ODVs of a p74 deletion mutant only the stable core was found but no PIF monomers. When the stable core lacks PIF4 it lost its proteolytic resistance as the resulting smaller core complex was degraded in a pif4 deletion mutant. We also identified PIF6 as a loosely associated component of the entry complex that appeared nevertheless important for the proteolytic resistance of the stable core which was degraded after deletion of pif6. We conclude from these results that an intact entry-complex in the ODV-envelope is prerequisite for proteolytic resistance of PIF-proteins under the alkaline conditions of the larval midgut.
Mutational and functional analysis of N-linked glycosylation of envelope fusion protein F of Helicoverpa armigera nucleopolyhedrovirus
The envelope fusion (F) protein of baculoviruses is a heavily N-glycosylated protein that plays a significant role in the virus infection cycle. N-Linked glycosylation of virus envelope glycoprotein is important for virus envelope glycoprotein folding and its function in general. There are six predicted N-glycosylation sites in the F (HaF) protein of Helicoverpa armigera nucleopolyhedrovirus (HearNPV). The N-glycosylation site located in the F2 subunit (N104) of HaF has been identified and functionally characterized previously (Long et al. 2007). In this study the other five potential N-glycosylation sites located in the HaF1 subunit namely N293 N361 N526 N571 and N595 were analysed extensively to examine their N-glycosylation and relative importance to the function of HaF. The results showed that four of these five potential glycosylation sites in the F1 subunit N293 N361 N526 and N571 were N-glycosylated in F proteins of mature HearNPV budded viruses (BVs) but that N595 was not. In general the conserved site N526 was critical to the functioning of HaF as absence of N-glycosylation of N526 reduced the efficiency of HaF folding and trafficking consequently decreased fusogenicity and modified the subcellular localization of HaF proteins and thus impaired virus production and infectivity. The absence of N-glycosylation at other individual sites was found to have different effects on the fusogenicity and subcelluar distribution of HaF proteins in HzAM1 cells. In summary N-glycosylation plays comprehensive roles in HaF function and virus infectivity which is further discussed.
Temporal proteomic analysis and label-free quantification of viral proteins of an invertebrate iridovirus
Invertebrate iridescent virus 6 (IIV-6) is a nucleocytoplasmic virus with a ~212 kb linear dsDNA genome that encodes 215 putative ORFs. The IIV-6 virion-associated proteins consist of at least 54 virally encoded proteins. One of our previous findings showed that most of these proteins are encoded by genes from the early transcriptional class. This indicated that these structural proteins may not only function in the formation of the virion but also in the initial stage of viral infection. In the current study we followed the protein expression profile of IIV-6 over time in Drosophila S2 cells by label-free quantification using a proteomic approach. A total of 95 virally encoded proteins were detected in infected cells of which 37 were virion proteins. The expressed IIV-6 virion proteins could be categorized into three main clusters based on their expression profiles: proteins with stably low expression levels during infection proteins with exponentially increasing expression levels during infection and proteins that were initially highly abundant but showed slightly reduced levels after 48 h post-infection. We thus provided novel information on the kinetics of virion and infected cell-specific protein levels that assists in our understanding of gene regulation in this lesser-known DNA virus model.
Thirty years of baculovirus–insect cell protein expression: from dark horse to mainstream technology
In December 1983 a seminal paper appeared on the overexpression of human IFN-β in insect cells with a genetically engineered baculovirus. The finding that baculoviruses produced massive amounts of two proteins (polyhedrin and p10) by means of two very strong promoters and that the corresponding genes were dispensable for virus propagation in insect cells was crucial in the development of this expression system. During the next 30 years major improvements were achieved over the original baculovirus expression vector (BEV) system facilitating the engineering of the baculovirus vectors the modification of the sugar moieties of glycoproteins expressed in insect cells and the scale-up of the cell culture process. To date thousands of recombinant proteins have been produced in this successful expression system including several protein-based human and veterinary vaccines that are currently on the market. Viral vectors based on adeno-associated virus are being produced using recombinant baculovirus technology and the first gene therapy treatment based on this method has been registered. Specially adapted BEVs are used to deliver and express heterologous genes in mammalian cells and they may be used for gene therapy and cancer treatment in the future. The purpose of this review is to highlight the thirtieth ‘anniversary’ of this expression system by summarizing the fundamental research and major technological advances that allowed its development whilst noting challenges for further improvements.
Live imaging of baculovirus infection of midgut epithelium cells: a functional assay of per os infectivity factors
The occlusion-derived viruses (ODVs) of baculoviruses are responsible for oral infection of insect hosts whereas budded viruses (BVs) are responsible for systemic infection within the host. The ODV membrane proteins play crucial roles in mediating virus entry into midgut epithelium cells to initiate infection and are important factors in host-range determination. For Autographa californica multiple nucleopolyhedrovirus (AcMNPV) seven conserved ODV membrane proteins have been shown to be essential for oral infectivity and are called per os infectivity factors (PIFs). Information on the function of the individual PIF proteins in virus entry is limited partly due to the lack of a good in vitro system for monitoring ODV entry. Here we constructed a baculovirus with EGFP fused to the nucleocapsid to monitor virus entry into primary midgut epithelium cells ex vivo using confocal fluorescence microscopy. The EGFP-labelled virus showed similar BV virulence and ODV infectivity as WT virus. The ability to bind and enter host cells was then visualized for WT AcMNPV and viruses with mutations in P74 (PIF0) PIF1 or PIF2 showing that P74 is required for ODV binding whilst PIF1 and PIF2 play important roles in the entry of ODV after binding to midgut cells. This is the first live imaging of ODV entry into midgut cells and complements the genetic and biochemical evidence for the role of PIFs in the oral infection process.
Temporal classification and mapping of non-polyadenylated transcripts of an invertebrate iridovirus
The temporal expression of the 54 Chilo iridescent virus (CIV) virion protein genes was investigated by combining drug treatments that inhibit protein or DNA synthesis and an RT-PCR strategy particularly suitable for non-polyadenylated mRNAs. This method generates a uniform 3′ terminus by ligation of a 5′-phosphorylated oligonucleotide to the 3′ end of the transcript that is recognized by a complementary primer during RT-PCR. This analysis showed that CIV virion proteins are encoded by genes in all three predetermined temporal classes: 23 immediate-early 11 delayed-early and seven late virion gene transcripts were identified and assigned to ORFs. Early transcription of many virion protein genes supports the notion that virion proteins may also play essential roles in the initial stages of infection. In addition some of the early gene products present in the virion may reflect the intracellular path that the virus follows during infection.
Correlation between structure, protein composition, morphogenesis and cytopathology of Glossina pallidipes salivary gland hypertrophy virus
The Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) is a dsDNA virus with rod-shaped enveloped virions. Its 190 kb genome contains 160 putative protein-coding ORFs. Here the structural components protein composition and associated aspects of GpSGHV morphogenesis and cytopathology were investigated. Four morphologically distinct structures: the nucleocapsid tegument envelope and helical surface projections were observed in purified GpSGHV virions by electron microscopy. Nucleocapsids were present in virogenic stroma within the nuclei of infected salivary gland cells whereas enveloped virions were located in the cytoplasm. The cytoplasm of infected cells appeared disordered and the plasma membranes disintegrated. Treatment of virions with 1 % NP-40 efficiently partitioned the virions into envelope and nucleocapsid fractions. The fractions were separated by SDS-PAGE followed by in-gel trypsin digestion and analysis of the tryptic peptides by liquid chromatography coupled to electrospray and tandem mass spectrometry. Using the MaxQuant program with Andromeda as a database search engine a total of 45 viral proteins were identified. Of these ten and 15 were associated with the envelope and the nucleocapsid fractions respectively whilst 20 were detected in both fractions most likely representing tegument proteins. In addition 51 host-derived proteins were identified in the proteome of the virus particle 13 of which were verified to be incorporated into the mature virion using a proteinase K protection assay. This study provides important information about GpSGHV biology and suggests options for the development of future anti-GpSGHV strategies by interfering with virus–host interactions.
Proteomic analysis of Glossina pallidipes salivary gland hypertrophy virus virions for immune intervention in tsetse fly colonies
Many species of tsetse flies (Diptera: Glossinidae) can be infected by a virus that causes salivary gland hypertrophy (SGH). The genomes of viruses isolated from Glossina pallidipes (GpSGHV) and Musca domestica (MdSGHV) have recently been sequenced. Tsetse flies with SGH have reduced fecundity and fertility which cause a serious problem for mass rearing in the frame of sterile insect technique (SIT) programmes to control and eradicate tsetse populations in the wild. A potential intervention strategy to mitigate viral infections in fly colonies is neutralizing of the GpSGHV infection with specific antibodies against virion proteins. Two major GpSGHV virion proteins of about 130 and 50 kDa respectively were identified by Western analysis using a polyclonal rabbit antibody raised against whole GpSHGV virions. The proteome of GpSGHV containing the antigens responsible for the immune-response was investigated by liquid chromatography tandem mass spectrometry and 61 virion proteins were identified by comparison with the genome sequence. Specific antibodies were produced in rabbits against seven candidate proteins including the ORF10/C-terminal fragment ORF47 and ORF96 as well as proteins involved in peroral infectivity PIF-1 (ORF102) PIF-2 (ORF53) PIF-3 (ORF76) and P74 (ORF1). Antiserum against ORF10 specifically reacted to the 130 kDa protein in a Western blot analysis and to the envelope protein of GpSGHV detected by using immunogold-electron microscopy. This result suggests that immune intervention of viral infections in colonies of G. pallidipes is a realistic option.
DNA photolyases of Chrysodeixis chalcites nucleopolyhedrovirus are targeted to the nucleus and interact with chromosomes and mitotic spindle structures
Cyclobutane pyrimidine dimer (CPD) photolyases convert UV-induced CPDs in DNA into monomers using visible light as the energy source. Two phr genes encoding class II CPD photolyases PHR1 and PHR2 have been identified in Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV). Transient expression assays in insect cells showed that PHR1–EGFP fusion protein was localized in the nucleus. Early after transfection PHR2–EGFP was distributed over the cytoplasm and nucleus but over time it became localized predominantly in the nucleus. Immunofluorescence analysis with anti-PHR2 antiserum showed that early after transfection non-fused PHR2 was already present mainly in the nucleus suggesting that the fusion of PHR2 to EGFP hindered its nuclear import. Both PHR–EGFP fusion proteins strongly colocalized with chromosomes and spindle aster and midbody structures during host-cell mitosis. When PHR2–EGFP-transfected cells were superinfected with Autographa californica multiple-nucleocapsid NPV (AcMNPV) the protein colocalized with virogenic stroma the replication factories of baculovirus DNA. The collective data support the supposition that the PHR2 protein plays a role in baculovirus DNA repair.
Host-range expansion of Spodoptera exigua multiple nucleopolyhedrovirus to Agrotis segetum larvae when the midgut is bypassed
Given the high similarity in genome content and organization between Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and Agrotis segetum nucleopolyhedrovirus (AgseNPV) as well as the high percentages of similarity found between their 30 core genes the specificity of these NPVs was analysed for the respective insect hosts S. exigua and A. segetum. The LD50 for AgseNPV in second-instar A. segetum larvae was 83 occlusion bodies per larva and the LT50 was 8.1 days. AgseNPV was orally infectious for S. exigua but the LD50 was 10 000-fold higher than for SeMNPV. SeMNPV was not infectious for A. segetum larvae when administered orally but an infection was established by injection into the haemocoel. Bypassing midgut entry by intrahaemocoelic inoculation suggested that the midgut is the major barrier in A. segetum larvae for infection by SeMNPV. Delayed-early genes of SeMNPV are expressed in the midgut of A. segetum larvae after oral infections indicating that the virus is able to enter midgut epithelial cells and that it proceeds through the first phases of the infection process. The possible mechanisms of A. segetum resistance to SeMNPV in per os infections are discussed.
Two viruses that cause salivary gland hypertrophy in Glossina pallidipes and Musca domestica are related and form a distinct phylogenetic clade
Glossina pallidipes and Musca domestica salivary gland hypertrophy viruses (GpSGHV and MdSGHV) replicate in the nucleus of salivary gland cells causing distinct tissue hypertrophy and reduction of host fertility. They share general characteristics with the non-occluded insect nudiviruses such as being insect-pathogenic having enveloped rod-shaped virions and large circular double-stranded DNA genomes. MdSGHV measures 65×550 nm and contains a 124 279 bp genome (∼44 mol% G+C content) that codes for 108 putative open reading frames (ORFs). GpSGHV measuring 50×1000 nm contains a 190 032 bp genome (28 mol% G+C content) with 160 putative ORFs. Comparative genomic analysis demonstrates that 37 MdSGHV ORFs have homology to 42 GpSGHV ORFs as some MdSGHV ORFs have homology to two different GpSGHV ORFs. Nine genes with known functions (dnapol ts pif-1 pif-2 pif-3 mmp p74 odv-e66 and helicase-2) a homologue of the conserved baculovirus gene Ac81 and at least 13 virion proteins are present in both SGHVs. The amino acid identity ranged from 19 to 39 % among ORFs. An (A/T/G)TAAG motif similar to the baculovirus late promoter motif was enriched 100 bp upstream of the ORF transcription initiation sites of both viruses. Six and seven putative microRNA sequences were found in MdSGHV and GpSGHV genomes respectively. There was genome. Collinearity between the two SGHVs but not between the SGHVs and the nudiviruses. Phylogenetic analysis of conserved genes clustered both SGHVs in a single clade separated from the nudiviruses and baculoviruses. Although MdSGHV and GpSGHV are different viruses their pathology host range and genome composition indicate that they are related.
Low multiplicity of infection in vivo results in purifying selection against baculovirus deletion mutants
The in vivo fate of Autographa californica multiple nucleopolyhedrovirus deletion mutants originating from serial passage in cell culture was investigated by passaging a population enriched in these mutants in insect larvae. The infectivity of polyhedra and occlusion-derived virion content per polyhedron were restored within two passages in vivo. The frequency of occurrence of deletion mutants was determined by real-time PCR. The frequency of the non-homologous region origin (non-HR ori) of DNA replication was reduced to wild-type levels within two passages. The frequency of the polyhedrin gene did not increase and remained below wild-type levels. A low m.o.i. during the initial infection in insect larvae causing strong purifying selection for autonomously replicating viruses could explain these observations. The same virus population used in vivo was also passaged once at a different m.o.i. in cell culture. A similar effect (i.e. lower non-HR ori frequency) was observed at low m.o.i. only indicating that m.o.i. was the key selective condition.
The Chilo iridescent virus DNA polymerase promoter contains an essential AAAAT motif
The delayed-early DNA polymerase promoter of Chilo iridescent virus (CIV) officially known as Invertebrate iridescent virus was fine mapped by constructing a series of increasing deletions and by introducing point mutations. The effects of these mutations were examined in a luciferase reporter gene system using Bombyx mori cells transfected with promoter constructs and infected with CIV. When the size of the upstream element was reduced from position −19 to −15 relative to the transcriptional start site the luciferase activity was reduced to almost zero. Point mutations showed that each of the 5 nt (AAAAT) located between –19 and –15 were equally essential for promoter activity. Mutations at individual bases around the transcription initiation site showed that the promoter extended until position −2 upstream of the transcription start site. South-Western analysis showed that a protein of approximately 100 kDa interacted with the −19 nt promoter fragment in CIV-infected cells. This binding did not occur with a point mutant that lacked promoter activity. The AAAAT motif was also found in the DNA polymerase promoter region of other iridoviruses and in other putative CIV delayed-early genes.