Leslie G. Willis

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

ABSTRACT 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.

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.

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

ABSTRACT 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.

Analysis of sequences involved in IE2 transactivation of a baculovirus immediate-early gene promoter and identification of a new regulatory motif.

Opep-2 is a unique baculovirus early gene that has only been identified in the Orgyia pseudotsugata multiple capsid nucleopolyhedrovirus (OpMNPV). Previous analyses have shown this gene is expressed at very early times post-infection (p.i.) but is shut down by 36-48 h p.i. The promoter of opep-2 therefore, represents a class of early genes that is temporally regulated. In this study, a detailed analysis of the opep-2 promoter is performed to analyze the role individual motifs play in early gene expression. A new 13 base pair regulatory element was identified and shown to be essential in controlling high-level expression of this gene. In addition, mutational analysis revealed that GATA and CACGTG motifs, which have been shown to bind cellular factors in Sf9 and Ld652Y cells, played minor roles in influencing opep-2 expression in the absence of other viral factors. The OpMNPV transactivator IE2 causes a significant activation of the opep-2 promoter. Cotransfection of an extensive number of promoter deletions and mutations did not show any sequence specificity for IE2 transactivation. This is the first detailed analysis of the sequence requirements for IE2 transactivation, and these results suggest that IE2 does not bind directly to specific elements in the opep-2 promoter.

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.

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.

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.

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

ABSTRACT 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.