Autographa californica Nucleopolyhedrovirus AC141 (Exon0), a Potential E3 Ubiquitin Ligase, Interacts with Viral Ubiquitin and AC66 To Facilitate Nucleocapsid Egress.

During the infection cycle of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), two forms of virions are produced, budded virus (BV) and occlusion-derived virus (ODV). Nucleocapsids that form BV have to egress from the nucleus, whereas nucleocapsids that form ODV remain inside the nucleus. The molecular mechanism that determines whether nucleocapsids remain inside or egress from the nucleus is unknown. AC141 (a predicted E3 ubiquitin ligase) and viral ubiquitin (vUbi) have both been shown to be required for efficient BV production. In this study, it was hypothesized that vUbi interacts with AC141, and in addition, that this interaction was required for BV production. Deletion of both ac141 and vubi restricted viral infection to a single cell, and BV production was completely eliminated. AC141 was ubiquitinated by either vUbi or cellular Ubi, and this interaction was required for optimal BV production. Nucleocapsids in BV, but not ODV, were shown to be specifically ubiquitinated by vUbi, including a 100-kDa protein, as well as high-molecular-weight conjugates. The viral ubiquitinated 100-kDa BV-specific nucleocapsid protein was identified as AC66, which is known to be required for BV production and was shown by coimmunoprecipitation and mass spectrometry to interact with AC141. Confocal microscopy also showed that AC141, AC66, and vUbi interact at the nuclear periphery. These results suggest that ubiquitination of nucleocapsid proteins by vUbi functions as a signal to determine if a nucleocapsid will egress from the nucleus and form BV or remain in the nucleus to form ODV.IMPORTANCE Baculoviruses produce two types of virions called occlusion-derived virus (ODV) and budded virus (BV). ODVs are required for oral infection, whereas BV enables the systemic spread of virus to all host tissues, which is critical for killing insects. One of the important steps for BV production is the export of nucleocapsids out of the nucleus. This study investigated the molecular mechanisms that enable the selection of nucleocapsids for nuclear export instead of being retained within the nucleus, where they would become ODV. Our data show that ubiquitination, a universal cellular process, specifically tags nucleocapsids of BV, but not those found in ODV, using a virus-encoded ubiquitin (vUbi). Therefore, ubiquitination may be the molecular signal that determines if a nucleocapsid is destined to form a BV, thus ensuring lethal infection of the host.

Autographa californica Multiple Nucleopolyhedrovirus AC83 is a Per Os Infectivity Factor (PIF) Protein Required for Occlusion-Derived Virus (ODV) and Budded Virus Nucleocapsid Assembly as well as Assembly of the PIF Complex in ODV Envelopes.

Baculovirus occlusion-derived virus (ODV) initiates infection of lepidopteran larval hosts by binding to the midgut epithelia, which is mediated by per os infectivity factors (PIFs). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes seven PIF proteins, of which PIF1 to PIF4 form a core complex in ODV envelopes to which PIF0 and PIF6 loosely associate. Deletion of any pif gene results in ODV being unable to bind or enter midgut cells. AC83 also associates with the PIF complex, and this study further analyzed its role in oral infectivity to determine if it is a PIF protein. It had been proposed that AC83 possesses a chitin binding domain that enables transit through the peritrophic matrix; however, no chitin binding activity has ever been demonstrated. AC83 has been reported to be found only in the ODV envelopes, but in contrast, the Orgyia pseudotsugata MNPV AC83 homolog is associated with both ODV nucleocapsids and envelopes. In addition, unlike known pif genes, deletion of ac83 eliminates nucleocapsid formation. We propose a new model for AC83 function and show AC83 is associated with both ODV nucleocapsids and envelopes. We also further define the domain required for nucleocapsid assembly. The cysteine-rich region of AC83 is also shown not to be a chitin binding domain but a zinc finger domain required for the recruitment or assembly of the PIF complex to ODV envelopes. As such, AC83 has all the properties of a PIF protein and should be considered PIF8. In addition, pif7 (ac110) is reported as the 38th baculovirus core gene.IMPORTANCE ODV is essential for the per os infectivity of the baculovirus AcMNPV. To initiate infection, ODV binds to microvilli of lepidopteran midgut cells, a process which requires a group of seven virion envelope proteins called PIFs. In this study, we reexamined the function of AC83, a protein that copurifies with the ODV PIFs, to determine its role in the oral infection process. A zinc finger domain was identified and a new model for AC83 function was proposed. In contrast to previous studies, AC83 was found to be physically located in both the envelope and nucleocapsid of ODV. By deletion analysis, the AC83 domain required for nucleocapsid assembly was more finely delineated. We show that AC83 is required for PIF complex formation and conclude that it is a true per os infectivity factor and should be called PIF8.

Microscopic investigation of AcMNPV infection in the Trichoplusia ni midgut.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the type species for the genus Alphabaculovirus in the family Baculoviridae. In nature, AcMNPV infection begins with ingestion of viral occlusion bodies (OBs) from which occlusion-derived viruses (ODV) are released to infect midgut cells. This study explored the early stages of Trichoplusia ni midgut infection using recombinant viruses expressing green fluorescent protein (GFP) and/or a VP39-mCherry fusion protein under the control of early and late promoters, respectively. Using a recombinant ie1:GFP virus, the anterior midgut region was identified as the predominant site for primary infection. Infection of midguts using the GFP-VP39mCherry-dual labelled recombinant virus revealed that active viral replication and cell-to-cell spread was required for the formation of infection foci and the subsequent spread to uninfected midgut cells and tracheoblasts. The spread of the infection from primary infected cells to secondary cells within the midgut was shown to be dependent upon the membrane fusion protein GP64.

Defining the roles of the baculovirus regulatory proteins IE0 and IE1 in genome replication and early gene transactivation.

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.

Deletion of AcMNPV ac146 eliminates the production of budded virus.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac146 is a highly conserved gene in the Alpha- and Betabaculovirus genera that has an unknown function. Northern blot analysis and transcript mapping showed that ac146 is transcribed at late times post infection as a 1.2 kb mRNA. To determine the role of ac146 in the baculovirus life cycle ac146 knock out viruses were constructed. Transfection and plaque assays showed that all the ac146 deletions produced a single cell phenotype indicating that no infectious budded virus (BV) was produced, however occlusion bodies were formed. The lack of BV production was confirmed by viral titration utilizing both qPCR and TCID50. Analysis of BV and occlusion derived virus (ODV) revealed that AC146 is associated with both forms of the virus and is modified specifically in ODV. This study therefore demonstrates that AC146 is a late virion associated protein and is essential for the viral life cycle.

Analysis of the temporal expression of Trichoplusia ni single nucleopolyhedrovirus genes following transfection of BT1-Tn-5B1-4 cells.

Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), a 134,394-bp double-stranded DNA group II Nucleopolyhedrovirus, is pathogenic to the lepidopteran T. ni. TnSNPV transcription is temporally regulated and divided into three promoter sequence-dependent classes (early, late and very late genes). A viral oligonucleotide DNA microarray containing all potential (144) viral genes of TnSNPV was designed to investigate global viral gene expression during cell infection. Total BT1-Tn-5B1-4 cellular mRNAs extracted between 0 and 72 h posttransfection with TnSNPV genomic DNA were hybridized to the microarray. Initial average expression of early genes was detected between 12 and 24 h posttransfection while late genes were mainly detected between 24 and 72 h posttransfection. The microarray expression profiling data verified many computer predicted promoter assignments. K-means clustering was used to sort the 144 genes based on their temporal expression pattern similarities. This clustering resulted in the confirmation and temporal class assignment of previously unidentified genes and promoters.

Sequence analysis of the complete genome of Trichoplusia ni single nucleopolyhedrovirus and the identification of a baculoviral photolyase gene.

The genome of the Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), a group II NPV which infects the cabbage looper (T. ni), has been completely sequenced and analyzed. The TnSNPV DNA genome consists of 134,394 bp and has an overall G + C content of 39%. Gene analysis predicted 144 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. Comparisons with previously sequenced baculoviruses indicate that 119 TnSNPV ORFs were homologues of previously reported viral gene sequences. Ninety-four TnSNPV ORFs returned an Autographa californica multiple NPV (AcMNPV) homologue while 25 ORFs returned poor or no sequence matches with the current databases. A putative photolyase gene was also identified that had highest amino acid identity to the photolyase genes of Chrysodeixis chalcites NPV (ChchNPV) (47%) and Danio rerio (zebrafish) (40%). In addition unlike all other baculoviruses no obvious homologous repeat (hr) sequences were identified. Comparison of the TnSNPV and AcMNPV genomes provides a unique opportunity to examine two baculoviruses that are highly virulent for a common insect host (T. ni) yet belong to diverse baculovirus taxonomic groups and possess distinct biological features. In vitro fusion assays demonstrated that the TnSNPV F protein induces membrane fusion and syncytia formation and were compared to syncytia formed by AcMNPV GP64.

Tight transcriptional regulation of foreign genes in insect cells using an ecdysone receptor-based inducible system.

The use of insect cells has been highly successful for the expression of foreign proteins from baculoviruses or plasmid vectors. Here, we describe a tight transcriptional regulation of foreign genes in insect cells using an ecdysone receptor-based inducible system. The system includes the DEF domains of the spruce budworm (Choristoneura fumiferana) EcR (CfEcR) fused to the Saccharomyces cerevisiae GAL4 DNA-binding domain and the EF domains of mammalian Mus musculus retinoid X receptor (MmRXR) fused to the acidic activation domains (AADs) of the baculovirus transactivators IE1 and IE0. Using a GAL4 response element in reporter constructs, both transient and stable expression in insect lepidopteran cells showed that the chimeric MmRXR and CfEcR only activated the reporter genes in the presence of inducer; no gene expression was detectable in the absence of inducer. Characterization of heterogenous activation domains in insect cells showed that the AADs from Autographa californica multiple nucleopolyhedrovirus (MNPV) IE1 and Orgyia pseudotsugata MNPV IE0 consistently exhibited higher inducible levels than the archetype AAD from herpesvirus VP16 in insect cells. To confirm the tight regulation of this system the highly toxic protein, diphtheria toxin (DT), was used. In the absence of an inducer no cytotoxic effect was observed in insect cells that had been transiently transformed with DT expressing plasmids. This system will therefore be a very useful tool for biotechnology applications expressing highly toxic proteins in insect cells and for studying the functional genomics of insects and microorganisms that infect them.

The Autographa californica multiple nucleopolyhedrovirus ie0-ie1 gene complex is essential for wild-type virus replication, but either IE0 or IE1 can support virus growth.

The immediate-early ie0-ie1 gene complex expresses the only baculovirus spliced gene that produces an alternate protein product. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) IE1 is a potent transcriptional transactivator that is essential for viral replication in transient assays. IE1 contains 582 amino acids that are arranged into different domains, including an acidic activation domain at the N terminus, a DNA binding domain, and an oligomerization domain at the C terminus. IE0 is a 52-amino-acid N-terminally elongated form of IE1. We investigated the functions of IE0 and IE1 in virus-infected cells by constructing the first ie1 open reading frame knockout virus. An infectious AcMNPV bacmid was used to generate the ie1 knockout, and the resulting virus, AcBacIE1KO, effectively deletes both ie0 and ie1. AcBacIE1KO does not infect Spodoptera frugiperda cells, showing that the ie0-ie1 gene complex is essential for viral infection. Rescue viruses of AcBacIE1KO were constructed that express only IE1, IE1 and IE0, or only IE0. Our results show that both IE0 and IE1 can function independently, but not equivalently, to support replication, producing infectious virus. Viruses expressing predominately, or only, IE0 produced significantly fewer cells with polyhedra than either the IE1 counterpart or wild-type virus. In addition, DNA replication was prolonged and budded virus and late gene expression were delayed. Viruses expressing only IE1 also produced fewer polyhedra, but replication was slightly faster and achieved higher levels than that of the wild-type virus. Both IE0 and IE1 are therefore required and must be expressed in the correct quantitative ratios to achieve a wild-type infection.

Complete comparative genomic analysis of two field isolates of Mamestra configurata nucleopolyhedrovirus-A.

A second genotype of Mamestra configurata nucleopolyhedrovirus-A (MacoNPV-A), variant 90/4 (v90/4), was identified due to its altered restriction endonuclease profile and reduced virulence for the host insect, M. configurata, relative to the archetypal genotype, MacoNPV-A variant 90/2 (v90/2). To investigate the genetic differences between these two variants, the genome of v90/4 was sequenced completely. The MacoNPV-A v90/4 genome is 153 656 bp in size, 1404 bp smaller than the v90/2 genome. Sequence alignment showed that there was 99.5 % nucleotide sequence identity between the genomes of v90/4 and v90/2. However, the v90/4 genome has 521 point mutations and numerous deletions and insertions when compared to the genome of v90/2. Gene content and organization in the genome of v90/4 is identical to that in v90/2, except for an additional bro gene that is found in the v90/2 genome. The region between hr1 and orf31 shows the greatest divergence between the two genomes. This region contains three bro genes, which are among the most variable baculovirus genes. These results, together with other published data, suggest that bro genes may influence baculovirus genome diversity and may be involved in recombination between baculovirus genomes. Many ambiguous residues found in the v90/4 sequence also reveal the presence of 214 sequence polymorphisms. Sequence analysis of cloned HindIII fragments of the original MacoNPV field isolate that the 90/4 variant was derived from indicates that v90/4 is an authentic variant and may represent approximately 25 % of the genotypes in the field isolate. These results provide evidence of extensive sequence variation among the individual genomes comprising a natural baculovirus outbreak in a continuous host population.

Role of AcMNPV IE0 in baculovirus very late gene activation.

IE0 is the only known baculovirus protein that is produced by splicing. In this study, we have explored the role of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) IE0 and its interaction with IE1 in the activation of very late gene expression from the polyhedrin promoter using transient assays. IE0 is co-expressed with IE1 throughout infection up to late times post-infection (p.i.) but shows peak levels of expression at early times. Significant changes in the ratios of the relative levels of IE0 to IE1 were observed throughout the course of infection. To study IE0 in the absence of IE1, we constructed a plasmid pAc-IE0(M-->A) that expressed only IE0. This was due to a mutation of the internal AUG that prevented translation of IE1 from the ie0 mRNA. Both IE0 and IE0(M-->A) were able to replace IE1 in transient assays, showing that IE0 is functional for very late gene activation and should be considered the 20th late gene expression factor (lef). In transient assays, IE0 showed that maximum very late gene expression is achieved at very low relative levels of protein. In contrast, IE1 requires higher levels of protein to obtain maximum very late gene expression. Furthermore, when the levels of IE0 become too high, very late gene expression rapidly declines. Interestingly, co-expression of IE0 and IE1 results in a mutually antagonistic affect on very late gene expression.

The acidic activation domains of the baculovirus transactivators IE1 and IE0 are functional for transcriptional activation in both insect and mammalian cells.

The acidic activation domains (AADs) of the baculovirus transactivators IE1 and IE0 are essential for transcriptional transactivation. To compare the relative transcriptional activation potentials of IE1 and IE0 AADs of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Orgyia pseudotsugata MNPV (OpMNPV), we constructed two ecdysone receptor (EcR)-based inducible expression systems to analyse six baculovirus AADs in two insect cell lines (Ld652Y and Sf9) and two mammalian cell lines (NIH-3T3 and CHO). For insect cell expression, the AADs were fused to the C, D, E and F domains of the spruce budworm Choristoneura fumiferana EcR. For mammalian cell expression the AADs were fused to the E and F domains of mammalian Mus musculus retinoid X receptor. In Ld652Y and Sf9 cells, chimeric proteins containing the AcMNPV AADs activated gene expression to higher levels than those containing the OpMNPV AADs. In NIH-3T3 cells, chimeras containing AcMNPV IE1 and IE0 AADs consistently activated gene expression to higher levels than the archetypal mammalian herpesvirus VP16 AAD. In contrast, OpMNPV AADs only activated expression by 5-15 % relative to the VP16 AAD. In CHO cells, both AcMNPV and OpMNPV AADs exhibited intermediate transactivation levels relative to VP16 AAD. These results show that the baculovirus AADs are functional for transcriptional activation in mammalian cells and that AcMNPV AADs generally appear to be more potent than OpMNPV AADs in both insect and mammalian cells.

Deletion of pe38 attenuates AcMNPV genome replication, budded virus production, and virulence in heliothis virescens.

The pe38 gene product of Autographa californica M nucleopolyhedrovirus (AcMNPV) has been shown to be involved in transcriptionally transactivating viral genes and augmenting viral DNA replication in transient assays. To assess the role of pe38 during infection, we generated a knockout virus, Delta pe38-E9/E9, in which the pe38 open reading frame was replaced with that of the green fluorescent protein. We compared mutant and wild-type (WT) viral replication in insect cell culture and virulence in Heliothis virescens larvae. Compared to WT, Delta pe38-E9/E9 budded virus (BV) production was delayed by at least 3 h, and BV yields were reduced over 99%. Similarly, Delta pe38-E9/E9 DNA synthesis levels were greatly reduced relative to those of WT, but onset of DNA replication was the same for both viruses. In bioassays, nearly sevenfold more Delta pe38-E9/E9 virus than WT virus was required to achieve an LD(50) when administered orally, but not hemocoelically. These results support the hypothesis that the kinetics of AcMNPV BV production greatly impact virulence in larvae infected orally (the natural route of infection) and that PE38 is an important, but not essential, factor in viral DNA synthesis and BV production.

Identification and genomic analysis of a second species of nucleopolyhedrovirus isolated from Mamestra configurata.

MacoNPV-96B is a nucleopolyhedrovirus isolated from naturally infected Mamestra configurata (Lepidoptera: Noctuidae) larvae. It was initially identified due to its completely different restriction endonuclease profile relative to the previously sequenced Mamestra configurata virus MacoNPV-90/2 (Q. Li, C. Donly, L. Li, L. G. Willis, D. A. Theilmann, and M. Erlandson, 2002, Virology 294, 106-121). The MacoNPV-96B host range and virulence were also found to differ significantly from those of the previous isolate. To further understand the complex of viruses infecting M. configurata, the genome of MacoNPV-96B was completely sequenced and analyzed in comparison with the genome of MacoNPV-90/2 and other sequenced baculoviruses. MacoNPV-96B consists of 158,482 bp, and 168 open reading frames (ORFs) of 150 nucleotides or longer with minimal overlap have been identified. The genome of MacoNPV-96B is 3422 bp larger than MacoNPV-90/2 and although gene arrangement is virtually identical, there are 9 ORFs unique to MacoNPV-96B and 10 unique to MacoNPV-90/2. bro genes were found to be associated with nonhomologous regions, suggesting that bro genes may facilitate recombination between genomes. A major difference in the gene content between the two viruses is a 5.4-kb insert in MacoNPV-96B, which is highly homologous to a cluster of Xestia c-nigrum granulovirus (XecnGV) ORFs, suggesting recent recombination events between these two viruses. Nucleotide sequence and amino acid sequence identity between the common ORFs of MacoNPV-96B and MacoNPV-90/2 average 87 and 90%, respectively. The sequence data suggest that MacoNPV-96B and MacoNPV-90/2 are closely related but have diverged and evolved into two separate species. This is the first study to identify highly related but separately evolving viruses in the same insect host and geographic location. A new Identity-GeneParityPlot analysis was developed to perform a comparison of two viral genomes in gene content and arrangement as well as homology level of individual ORFs.

Sequence and organization of the Mamestra configurata nucleopolyhedrovirus genome.

The nucleotide sequence of the genome of the nucleopolyhedrovirus (NPV) from Mamestra configurata (MacoNPV, isolate 90/2), a group II NPV, was determined and analyzed. The MacoNPV DNA genome consists of 155,060 bp and has an overall G+C content of 41.7%. Computer-assisted analysis predicted 169 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. BLAST searches and comparisons with completely sequenced baculoviruses indicated that there were 66 ORFs conserved among the nine baculoviruses compared and an additional 17 ORFs were conserved among the NPVs. The gene content and gene arrangement in MacoNPV were most similar to those of SeMNPV, including two putative odv-e66 and p26 gene homologues. However, in contrast to SeMNPV, 8 ORFs with homology to baculovirus repeat ORFs (bro) and single copies of enhancin and conotoxin-like protein ORFs were found in MacoNPV. The MacoNPV genome contained four homologous regions, each with 10 to 17 repeated sequences. Each repeat was 60 to 86 nucleotides in length and contained an approximately 43-bp-long imperfect palindrome. There were 13 ORFs unique to MacoNPV, ranging from a small ORF of 196 bp to larger ORFs of up to 1047 bp, and many of these contained typical early and late baculovirus consensus promoters.