Yingchao Nie

Autographa californica Multiple Nucleopolyhedrovirus Core Gene ac96 Encodes a Per Os Infectivity Factor (pif-4)

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac96 is a core gene, but its role in virus replication is still unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac96-null virus (vAc96null). Our analyses showed that the absence of ac96 does not affect budded virus (BV) production or viral DNA replication in infected Sf9 cells. Western blotting and confocal immunofluorescence analysis showed that AC96 is expressed in both the cytoplasm and the nucleus throughout infection. In addition, AC96 was detected in the envelope fractions of both BV and occlusion-derived virus. Injection of vAc96null BV into the hemocoel killed Trichoplusia ni larvae as efficiently as repaired and control viruses; however, vAc96null was unable to infect the midgut tissue of Trichoplusia ni larvae when inoculated per os. Therefore, the results of this study show that ac96 encodes a new per os infectivity factor (PIF-4).

Analysis of the Autographa californica Multiple Nucleopolyhedrovirus Overlapping Gene Pair lef3 and ac68 Reveals that AC68 Is a Per Os Infectivity Factor and that LEF3 Is Critical, but Not Essential, for Virus Replication

ABSTRACT Autographa californica multiple nucleopolyhedrovirus ac68 is a core gene that overlaps lef3 which encodes the single-stranded DNA binding protein. A knockout (KO) virus lacking both lef3 and ac68 was generated (lef3-ac68 2×KO) to enable the functional study of ac68. To produce an ac68KO virus that did not impact lef3 expression, the lef3-ac68 2×KO virus was repaired with a DNA fragment containing lef3 and ac68, in which ac68 contained point mutations so that only LEF3 was expressed. Repair of lef3-ac68 2×KO with just ac68 generated an lef3KO virus. Analysis of the ac68KO virus showed that viral DNA replication and budded virus (BV) levels were unaffected compared to levels in the double-repair or wild-type (WT) control virus. Bioassay analyses of Trichoplusia ni larvae injected with BV directly into the hemolymph, bypassing the gut, showed no difference in mortality rates between the ac68KO and the WT viruses. However, in oral bioassays the ac68KO occlusion bodies failed to kill larvae. These results show that the core gene ac68 encodes a per os infectivity factor (pif6). The lef3KO virus was also analyzed, and virus replication was drastically reduced compared to WT virus, but very low levels of lef3KO virus DNA replication and BV production could be detected. In addition, in transfected cells P143 was transported to the nucleus in the absence of LEF3. This study therefore shows for the first time that even though the loss of LEF3 severely impairs virus replication, it is not absolutely essential for P143 nuclear import or viral replication.

AcMNPV EXON0 (AC141) which is required for the efficient egress of budded virus nucleocapsids interacts with β-tubulin.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encoded protein, EXON0 (AC141), is required for the efficient transport of nucleocapsids out of the nucleus for the production of budded virus (BV). To further elucidate the molecular mechanisms by which EXON0 regulates BV production, EXON0 was tagged at the N-terminus with 3x FLAG-6x His. Protein complexes were isolated by tandem affinity purification and potential EXON0 specific interacting protein partners were gel purified and identified by LC-MS/MS. This analysis showed that the cellular protein, beta-tubulin, co-purified with EXON0 which was confirmed by co-immunoprecipitation. In addition, immunofluorescence showed that EXON0 and beta-tubulin co-localized during virus infection. The microtubule inhibitors colchicine and nocodazole were used to treat AcMNPV infected Sf9 cells and results showed that BV production was reduced by over 85%. These data suggest that the egress of AcMNPV budded virus may be facilitated by the interaction of EXON0 with beta-tubulin and microtubules.

Deletion of the AcMNPV core gene ac109 results in budded virions that are non-infectious.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac109 is a core gene and its function in the virus life cycle is unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac109 deletion virus (vAc(109KO)). Fluorescence and light microscopy showed that transfection of vAc(109KO) results in a single-cell infection phenotype. Viral DNA replication is unaffected and the development of occlusion bodies in vAc(109KO)-transfected cells evidenced progression to the very late phases of viral infection. Western blot and confocal immunofluorescence analysis showed that AC109 is expressed in the cytoplasm and nucleus throughout infection. In addition, AC109 is a structural protein as it was detected in both budded virus (BV) and occlusion derived virus in both the envelope and nucleocapsid fractions. Titration assays by qPCR and TCID(50) showed that vAc(109KO) produced BV but the virions are non-infectious. The vAc(109KO) BV were indistinguishable from the BV of repaired and wild type control viruses as determined by negative staining and electron microscopy.

AcMNPV AC16 (DA26, BV/ODV-E26) regulates the levels of IE0 and IE1 and binds to both proteins via a domain located within the acidic transcriptional activation domain

IE0 and IE1 are the primary viral regulatory proteins of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) involved in the transactivation of early genes, stimulation of late gene expression, and viral DNA replication. The protein interactions required for IE0 or IE1 to achieve these varied roles are not well defined, so to identify proteins that interact with IE0 and IE1, tandem affinity purification (TAP) and LC-MS/MS was used. Analysis of purified proteins identified AC16 (DA26, BV/ODV-E26) from TAP tagged IE0 virus infected Sf9 cells. Co-immunoprecipitation confirmed that AC16 interacts with both IE0 and IE1 and yeast 2-hybrid analysis mapped the domain required for interaction with AC16. Mutation of the AC16 binding domain enhanced BV production by viruses expressing only IE0 but had no effect if only IE1 is expressed. An ac16 deletion virus was constructed and was shown not to affect the temporal expression of IE0 and IE1; however the relative level of IE0 to IE1 was significantly increased.

Autographa californica multiple nucleopolyhedrovirus core gene ac92 (p33) is required for efficient budded virus production

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac92 is a core gene encoding a protein associated with occlusion derived virus (ODV), binds human P53 and also has flavin adenine dinucleotide linked sulfhydryl oxidase activity but its role in the virus life cycle is not known. To determine ac92 function a deletion virus (vAc(92KO)) was generated and transfected Sf9 cells revealed that vAc(92KO) infection was restricted primarily to single cells and budded virus (BV) titer was reduced over 99.99%. However, viral DNA replication was unaffected and development of occlusion bodies in vAc(92KO)-transfected cells evidenced progression to very late phases of viral infection. AC92 localized to both the cytoplasm and nucleus, and was also associated with BV as well as ODV. In BV AC92 was detected in BV envelope and nucleocapsid fractions. Finally it was shown that the ac92 homologue from the Group II alphabaculovirus Mamestra configurata NPV maco96 could only partially rescue vAc(92KO).

Deletion of AcMNPV AC16 and AC17 results in delayed viral gene expression in budded virus infected cells but not transfected cells

This study investigated the combined function of the Autographa californica multiple nucleopolyhedrovirus overlapping genes ac16 (BV/ODV-E26, DA26) and ac17. Ac17 is a late gene and the promoter is within the ac16 open reading frame. A double ac16-ac17 knockout virus was generated to assess the function of each gene independently or together. Loss of ac17 did not affect viral DNA synthesis but budded virus (BV) production was reduced. Deletion of both ac16-ac17 resulted in reduced viral DNA synthesis and a further reduction in BV production. In BV infected Sf9 cells, viral gene expression was delayed up to 12 h in the absence of both AC16 and AC17 but not if either gene was present. Cells infected by transfecting viral DNA, by-passing the BV particle, exhibited no delay in gene expression from the double knockout virus. AC16 and AC17 are therefore required for rapid viral gene expression in cells infected by BV.

Identification of AcMNPV EXON0 (ac141) domains required for efficient production of budded virus, dimerization and association with BV/ODV-C42 and FP25

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late gene exon0 (ac141) is required for the efficient production of budded virus (BV). EXON0 interacts with nucleopcapsid protein BV/ODV-C42 and FP25 and enables egress of nucleocapsids from the nucleus to the cytoplasm. This study examines the functional domains of EXON0 that play a role in BV production. Six putative domains of the 261 amino acid EXON0 were deleted and examined for functionality by determining their ability to rescue an AcMNPV exon0 knockout bacmid. Domain mapping results showed that all the six domains were required but deletion of the N-terminal acidic region and the leucine zipper domains had the greatest impact on BV production. Yeast 2-hybrid and co-immunoprecipitation demonstrated that EXON0 formed dimers. Point mutation analysis demonstrated that the leucine zipper was required for dimer formation and interaction with BV/ODV-C42 and FP25. The charged domain was also required for BV/ODV-C42 interaction.

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.

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.