Search

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a large double-stranded DNA (dsDNA) virus that encodes approximately 156 genes and is highly pathogenic to a variety of larval lepidopteran insects in nature. Oral infection of larval midgut cells is initiated by the occlusion-derived virus (ODV), while secondary infection of other tissues is mediated by the budded virus (BV). Global viral gene expression has been studied in detail in BV-infected cell cultures, but studies of ODV infection in the larval midgut are limited. In this study, we examined expression of the ∼156 AcMNPV genes in Trichoplusia ni midgut tissue using a transcriptomic approach. We analyzed expression profiles of viral genes in the midgut and compared them with profiles from a T. ni cell line (Tnms42). Several viral genes ( p6.9 , orf76 , orf75 , pp31 , Ac-bro , odv-e25 , and odv-ec27 ) had high expression levels in the midgut throughout the infection. Also, the expression of genes associated with occlusion bodies ( polh and p10 ) appeared to be delayed in the midgut in comparison with the cell line. Comparisons of viral gene expression profiles revealed remarkable similarities between the midgut and cell line for most genes, although substantial differences were observed for some viral genes. These included genes associated with high level BV production ( fp-25k ), acceleration of systemic infection ( v-fgf ), and enhancement of viral movement ( arif-1/orf20 ). These differential expression patterns appear to represent specific adaptations for virus infection and transmission through the polarized cells of the lepidopteran midgut. IMPORTANCE Baculoviruses such as AcMNPV are pathogens that are natural regulators of certain insect populations. Baculovirus infections are biphasic, with a primary phase initiated by oral infection of midgut epithelial cells by occlusion-derived virus (ODV) virions and a secondary phase in which other tissues are infected by budded-virus (BV) virions. While AcMNPV infections in cultured cells have been studied extensively, comparatively little is known regarding primary infection in the midgut. In these studies, we identified gene expression patterns associated with ODV-mediated infection of the midgut in Trichoplusia ni and compared those results with prior results from BV-infected cultured cells, which simulate secondary infection. These studies provide a detailed analysis of viral gene expression patterns in the midgut, which likely represent specific viral strategies to (i) overcome or avoid host defenses in the gut and (ii) rapidly move infection from the midgut, into the hemocoel to facilitate systemic infection., (Copyright © 2018 American Society for Microbiology.)

Baculoviruses are important insect pathogens that have been developed as protein expression vectors in insect cells and as transduction vectors for mammalian cells. They have large double-stranded DNA genomes containing approximately 156 tightly spaced genes, and they present significant challenges for transcriptome analysis. In this study, we report the first comprehensive analysis of AcMNPV transcription over the course of infection in Trichoplusia ni cells, by a combination of strand-specific RNA sequencing (RNA-Seq) and deep sequencing of 5' capped transcription start sites and 3' polyadenylation sites. We identified four clusters of genes associated with distinctive patterns of mRNA accumulation through the AcMNPV infection cycle. A total of 218 transcription start sites (TSS) and 120 polyadenylation sites (PAS) were mapped. Only 29 TSS were associated with a canonical TATA box, and 14 initiated within or near the previously identified CAGT initiator motif. The majority of viral transcripts (126) initiated within the baculovirus late promoter motif (TAAG), and late transcripts initiated precisely at the second position of the motif. Analysis of 3' ends showed that 92 (77%) of the 3' PAS were located within 30 nucleotides (nt) downstream of a consensus termination signal (AAUAAA or AUUAAA). A conserved U-rich region was found approximately 2 to 10 nt downstream of the PAS for 58 transcripts. Twelve splicing events and an unexpectedly large number of antisense RNAs were identified, revealing new details of possible regulatory mechanisms controlling AcMNPV gene expression. Combined, these data provide an emerging global picture of the organization and regulation of AcMNPV transcription through the infection cycle.

To examine the role of the AcMNPV lef-5 gene in the context of the infection cycle, we generated an AcMNPV lef-5 knockout virus (vAc(lef5ko)) and a complementing cell line that supports viral replication. We examined AcMNPV DNA replication, early and late gene expression, and production of infectious viral progeny in the absence of lef-5. While early gene expression and DNA replication were not reduced by the lef-5 knockout, expression of a late reporter was disrupted and representative late transcripts were dramatically reduced. Progeny virus production was not detected after transfection of Sf9 cells with the lef-5 knockout bacmid, but was rescued by insertion of an egfp- or myc-tagged lef-5 gene into the vAc(lef5ko) genome. An egfp-tagged lef-5 gene from SeMNPV was used to generate a stable Sf9 cell line that supported replication of the vAc(lef5ko) virus. The LEF-5 protein was also found to co-localize with IE-1 in infected cell nuclei., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The pp31 gene of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes a phosphorylated DNA binding protein that associates with virogenic stroma in the nuclei of infected cells. Prior studies of pp31 by transient late expression assays suggested that pp31 may play an important role in transcription of AcMNPV late genes [Todd, J. W., Passarelli, A. L., and Miller, L. K. (1995). Eighteen baculovirus genes, including lef-11, p35, 39K, and p47, support late gene expression. J. Virol. 69, 968-974] although genetic studies of the closely related BmNPV pp31 gene suggested that pp31 may be dispensable [Gomi, S., Zhou, C. E., Yih, W., Majima, K., and Maeda, S. (1997). Deletion analysis of four of eighteen late gene expression factor gene homologues of the baculovirus, BmNPV. Virology 230 (1), 35-47]. In the current study, we examined the role of the pp31 gene in the context of the AcMNPV genome during infection. We used a BACmid-based system to generate a pp31 knockout in the AcMNPV genome. The pp31 knockout was subsequently rescued by reinserting the pp31 gene into the polyhedrin locus of the same virus genome. We found that pp31 was not essential for viral replication although the absence of pp31 resulted in a lower viral titer. Analysis of viral DNA replication in the absence of pp31 showed that the kinetics of viral DNA replication were unaffected. An AcMNPV oligonucleotide microarray was used to compare gene expression from all AcMNPV genes in the presence or absence of pp31. In the absence of pp31, a modest reduction in transcripts was detected for many viral genes (99 genes) while no substantial increase or decrease was observed for 43 genes. Transcripts from 6 genes (p6.9, ORF 97, ORF 60, ORF 98, ORF 102 and chitinase) were reduced by 66% or more compared to the levels detected from the control virus. Microarray results were further examined by qPCR analysis of selected genes. In combination, these data show that deletion of the pp31 gene was not lethal and did not appear to affect viral DNA replication but resulted in an apparent modest down-regulation of a subset of AcMNPV genes that included both early and late genes.

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-6 gene was previously shown to be necessary for optimal transcription from an AcMNPV late promoter in transient late expression assays. In the present study, we examined the expression and cellular localization of lef-6 during the AcMNPV infection cycle and generated a lef-6-null virus for studies of the role of lef-6 in the infection cycle. Transcription of lef-6 was detected from 4 to 48 h postinfection, and the LEF-6 protein was identified in dense regions of infected cell nuclei, a finding consistent with its potential role as a late transcription factor. To examine lef-6 in the context of the AcMNPV infection cycle, we deleted the lef-6 gene from an AcMNPV genome propagated as an infectious BACmid in Escherichia coli. Unexpectedly, the resulting AcMNPV lef-6-null BACmid (vAc(lef6KO)) was able to propagate in cell culture, although virus yields were substantially reduced. Thus, the lef-6 gene is not essential for viral replication in Sf9 cells. Two "repair" AcMNPV BACmids (vAc(lef6KO-REP-P) and vAc(lef6KO-REP-ie1P)) were generated by transposition of the lef-6 gene into the polyhedrin locus of the vAc(lef6KO) BACmid. Virus yields from the two repair viruses were similar to those from wild-type AcMNPV or a control (BACmid-derived) virus. The lef-6-null BACmid (vAc(lef6KO)) was further examined to determine whether the deletion of lef-6 affected DNA replication or late gene transcription in the context of an infection. The lef-6 deletion did not appear to affect viral DNA replication. Using Northern blot analysis, we found that although early transcription was apparently unaffected, both late and very late transcription were delayed in cells infected with the lef-6-null BACmid. This phenotype was rescued in viruses containing the lef-6 gene reinserted into the polyhedrin locus. Thus, the lef-6 gene was not essential for either viral DNA replication or late gene transcription, but the absence of lef-6 resulted in a substantial delay in the onset of late transcription. Therefore, lef-6 appears to accelerate the infection cycle of AcMNPV.

The Autographa californica nucleopolyhedrovirus (AcMNPV) lef-11 gene was previously identified by transient late expression assays as a gene important for viral late gene expression. The lef-11 gene was not previously identified as necessary for DNA replication in transient origin-dependent plasmid DNA replication assays. To examine the role of lef-11 in the context of the infection cycle, we generated a deletion of the lef-11 gene by recombination in an AcMNPV genome propagated as a BACmid in Escherichia coli. The resulting AcMNPV lef-11-null BACmid (vAc(lef11KO)) was unable to propagate in cell culture, although a "repair" AcMNPV BACmid (vAc(lef11KO-REP)), which was generated by transposition of the lef-11 gene into the polyhedrin locus of the vAc(lef11KO) BACmid, was able to replicate in a manner similar to wild-type or control AcMNPV viruses. Thus, the lef-11 gene is essential for viral replication in Sf9 cells. The vAc(lef11KO) BACmid was examined to determine if the defect in viral replication resulted from a defect in DNA replication or from a defect in late transcription. The lef-11-null BACmid and control BACmids were transfected into Sf9 cells, and viral DNA replication was monitored. The viral DNA genome of the lef-11-null BACmid (vAc(lef11KO)) was not amplified, whereas replication and amplification of the genomes of the repair BACmid (vAc(lef11KO-REP)), wild-type AcMNPV, and a nonpropagating gp64-null control BACmid (vAc(GUSgp64KO)) were readily detected. Northern blot analysis of transcripts from selected early, late, and very late genes showed that late and very late transcription was absent in cells transfected with the lef-11-null BACmid. Thus, in contrast to prior studies using transient replication and late expression assays, studies of a lef-11-null BACmid indicate that LEF-11 is required for viral DNA replication during the infection cycle.

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-11 gene was found previously to be necessary to support optimal levels of transient expression from an AcMNPV late promoter. The lef-11 gene is unusual in that it overlaps both upstream (orf38) and downstream (pp31) genes. In this study, the expression and cellular localization of LEF-11 were examined. The lef-11 transcripts were detected from 4 to 36 h post-infection (p.i.). The 1.5 kb lef-11 mRNA initiates 196 nt upstream of the lef-11 translation initiation codon, within the upstream orf38 gene. This relatively long 5' upstream region encodes a potential small upstream open reading frame (ORF) of 58 amino acids that overlaps the lef-11 ORF. The 3' end of the lef-11 mRNA was mapped as co-terminal with mRNAs from the downstream pp31 gene. Using affinity purified anti-LEF-11 antibodies, levels of LEF-11 expression were found to be maximal between approximately 8 and 24 h p.i., although LEF-11 could be detected as late as 72 h p.i. Using immunofluorescence microscopy, it was determined that LEF-11 localized to dense regions of infected cell nuclei, consistent with its role as a possible late transcription factor.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) glycoprotein GP64 is the major envelope protein of the budded virus (BV). GP64 is a class III fusion protein that mediates BV attachment to the cell surface and low-pH-triggered membrane fusion between the BV envelope and the endosome membrane during entry. Class III fusion proteins contain terminal looped structures that are believed to interact with membranes. To examine the functions of 3 loops found at the apex of the GP64 postfusion structure, we generated 2-alanine substitutions that scanned the two so-called fusion loops (loop 1 and loop 2) plus an adjacent loop structure (loop 3) that is closely attached to loop 2 and is also found at the apex of the GP64 postfusion structure. We identified essential residues from Y75 to T86 (loop 1) and N149 to H156 (loop 2) that are required for fusion activity, but no essential residues in loop 3. Further analysis revealed that critical fusion loop residues fall within two groups that are associated with either membrane merger (hemifusion) or fusion pore expansion. We next examined the interactions of soluble GP64 proteins and BV with membranes composed of various phospholipids. BV interacted directly with small unilamellar vesicles (SUVs) comprised of phospholipids phosphatidylcholine and phosphatidic acid (PC/PA) or phosphatidylcholine and phosphatidylserine (PC/PS) under neutral and acidic pH. We also examined the interactions of soluble GP64 constructs containing substitutions of the most hydrophobic residues within each of the two fusion loops. We found that a 2-residue substitution in either single loop (loop 1 [positions 81 and 82] or loop 2 [positions 153 and 154]) was not sufficient to substantially reduce the GP64-liposome interaction, but the same substitutions in both fusion loops severely reduced the GP64-liposome association at neutral pH. These results suggest that critical hydrophobic residues in both fusion loops may be involved in the interaction of GP64 with host cellular membranes and direct GP64-membrane interactions may represent a receptor-binding step prior to a low-pH-triggered conformational change. [ABSTRACT FROM AUTHOR]

Baculovirus F (fusion) proteins are found in the envelopes of budded virions. Recently a Drosophila melanogaster gene (CG4715) that encodes a protein with sequence similarity to baculovirus F proteins was discovered. To examine similarities and differences with baculovirus F proteins, we cloned the D. melanogaster cellular F (Dm-cF) protein gene and analyzed Dm-cF expression and localization. The predicted Dm-cF protein sequence lacks a furin cleavage site, and transiently expressed Dm-cF showed no protein cleavage and no detectable membrane fusion activity. In cell localization studies, transiently expressed Dm-cF was localized to intracellular organdies in D. melanogaster S2 cells, unlike baculovirus F proteins, which localize to cellular plasma membranes. Using reverse transcriptase PCR and Western blot analysis to examine Dm-cF expression in animals, we detected Dm-cF expression in both larval and adult D. melanogaster cells. However, Dm-cF expression was detected only in third instar larvae and adults, suggesting that Dm-cF expression may be developmentally regulated. We also identified genes related to Dm-cF in the genomes of two other Drosophila species, Drosophila yakuba and Drosophila pseudoobscura, and the mosquito Anopheles gambiae. These observations suggest that f genes may be present in the genomes of many insects. Conservation within and between 22 baculovirus and 4 insect F proteins was examined in detail. These studies demonstrate that Dm-cF is expressed in D. melanogaster and suggest that if baculovirus f genes are derived from a host cellular f gene, the function appears to have changed substantially upon adaptation to the baculovirus infection cycle. [ABSTRACT FROM AUTHOR]

The baculovirus envelope protein GP64 is an essential component of the budded virus and is necessary for efficient virion assembly. Little is known regarding intracellular trafficking of GP64 to the plasma membrane, where it is incorporated into budding virions during egress. To identify host proteins and potential cellular trafficking pathways that are involved in delivery of GP64 to the plasma membrane, we developed and characterized a stable Drosophila cell line that inducibly expresses the AcMNPV GP64 protein and used that cell line in combination with a targeted RNA interference (RNAi) screen of vesicular protein trafficking pathway genes. Of the 37 initial hits from the screen, we validated and examined six host genes that were important for trafficking of GP64 to the cell surface. Validated hits included Rab GTPases Rab1 and Rab4, Clathrin heavy chain, clathrin adaptor protein genes AP-1-2b and AP-2m, and Snap29. Two gene knockdowns (Rab5 and Exo84) caused substantial increases (up to 2.5-fold) of GP64 on the plasma membrane. We found that a small amount of GP64 is released from cells in exosomes and that some portion of cell surface GP64 is endocytosed, suggesting that recycling helps to maintain GP64 at the cell surface. IMPORTANCE While much is known regarding trafficking of viral envelope proteins in mammalian cells, little is known about this process in insect cells. To begin to understand which factors and pathways are needed for trafficking of insect virus envelope proteins, we engineered a Drosophila melanogaster cell line and implemented an RNAi screen to identify cellular proteins that aid transport of the model baculovirus envelope protein (GP64) to the cell surface. For this we developed an experimental system that leverages the large array of tools available for Drosophila and performed a targeted RNAi screen to identify cellular proteins involved in GP64 trafficking to the cell surface. Since viral envelope proteins are often critical for production of infectious progeny virions, these studies lay the foundation for understanding how either pathogenic insect viruses (baculoviruses) or insect-vectored viruses (e.g., flaviviruses, alphaviruses) egress from cells in tissues such as the midgut to enable systemic virus infection. [ABSTRACT FROM AUTHOR]

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) GP64 envelope glycoprotein is essential for virus entry and plays an important role in virion budding. An AcMNPV construct that contains a deletion of the gp64 gene is unable to propagate infection from cell to cell, and this defect results from both a severe reduction in the production of budded virions and the absence of GP64 on virions. In the current study, we examined GP64 proteins containing N- and C-terminal truncations of the ectodomain and identified a minimal construct capable of targeting the truncated GP64 to budded virions. The minimal budding and targeting construct of GP64 contained 38 amino acids from the mature N terminus of the GP64 ectodomain and 52 amino acids from the C terminus of GP64. Because the vesicular stomatitis virus (VSV) G protein was previously found to rescue infectivity of a gp64null AcMNPV, we also examined a small C-terminal construct of the VSV G protein. We found that a construct containing 91 amino acids from the C terminus of VSV G (termed G-stem) was capable of rescuing AcMNPV gp64null virion budding to wild-type (wt) or nearly wt levels. We also examined the display of chimeric proteins on the gp64null AcMNPV virion. By generating viruses that expressed chimeric influenza virus hemagglutinin (HA) proteins containing the GP64 targeting domain and coinfecting those viruses with a virus expressing the G-stem construct, we demonstrated enhanced display of the HA protein on gp64null AcMNPV budded virions. The combined use of gp64null virions, VSV G-stem-enhanced budding, and GP64 domains for targeting heterologous proteins to virions should be valuable for biotechnological applications ranging from targeted transduction of mammalian cells to vaccine production. [ABSTRACT FROM AUTHOR]

Viral envelope fusion proteins are important structural proteins that mediate viral entry and may affect or determine the host range of a virus. The acquisition, exchange, and evolution of such envelope proteins may dramatically affect the success and evolutionary divergence of viruses. In the family Baculoviridae, two very different envelope fusion proteins have been identified. Budded virions of group I nucleopolyhedroviruses (NPVs) such as the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), contain the essential GP64 envelope fusion protein. In contrast group II NPVs and granuloviruses have no gp64 gene but instead encode a different envelope protein called F. F proteins from group II NPVs can functionally substitute for GP64 in gp64null AcMNPV viruses, indicating that GP64 and these F proteins serve a similar functional role. Interestingly, AcMNPV (and other gp64-containing group I NPVs) also contain an F gene homolog (Ac23) but the AcMNPV F homolog cannot compensate for the loss of gp64. In the present study, we show that Ac23 is expressed and is found in budded virions. To examine the function of F protein homologs from the gp64containing baculoviruses, we generated an Ac23null AcMNPV genome by homologous recombination in E. coli. We found that Ac23 was not required for viral replication or pathogenesis in cell culture or infected animals. However, Ac23 accelerated the mortality of infected insect hosts by approximately 28% or 26 h. Thus, Ac23 represents an important viral pathogenicity factor in larvae infected with AcMNPV. [ABSTRACT FROM AUTHOR]

IE0 and IE1 of the baculovirus Autographa californica multiple nucleopolyhedrovirus are essential transregulatory proteins required for both viral DNA replication and transcriptional transactivation. IE0 is identical to IE1 except for 54 amino acids at the N-terminus but the functional differences between these two proteins remain unclear. The purpose of this study was to determine the separate roles of these critical proteins in the virus life cycle. Unlike prior studies, IE0 and IE1 were analyzed using viruses that expressed ie0 and ie1 from an identical promoter so that the timing and levels of expression were comparable. IE0 and IE1 were found to equally support viral DNA replication and budded virus (BV) production. However, specific viral promoters were selectively transactivated by IE0 relative to IE1 but only when expressed at low levels. These results indicate that IE0 preferentially transactivates specific viral genes at very early times post-infection enabling accelerated replication and BV production. [ABSTRACT FROM AUTHOR]