Autographa californica Multiple Nucleopolyhedrovirus ac76 Is Involved in Intranuclear Microvesicle Formation

Abstract

In this study, we characterized Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf76 (ac76), which is a highly conserved gene of unknown function in lepidopteran baculoviruses. Transcriptional analysis of ac76 revealed that transcription of multiple overlapping multicistronic transcripts initiates from a canonical TAAG late-transcription start motif but terminates at different 3 ends at 24 h postinfection in AcMNPV-infected Sf9 cells. To investigate the role of ac76 in the baculovirus life cycle, an ac76-knockout virus was constructed using an AcMNPV bacmid system. Microscopy, titration assays, and Western blot analysis demonstrated that the resulting ac76-knockout virus was unable to produce budded viruses. Quantitative real-time PCR analysis demonstrated that ac76 deletion did not affect viral DNA synthesis. Electron microscopy showed that virus-induced intranuclear microvesicles as well as occlusionderived virions were never observed in cells transfected with the ac76-knockout virus. Confocal microscopy analysis revealed that Ac76 was predominantly localized to the ring zone of nuclei during the late phase of infection. This suggests that ac76 plays a role in intranuclear microvesicle formation. To the best of our knowledge, this is the first baculovirus gene identified to be involved in intranuclear microvesicle formation.Baculoviruses are arthropod-specific, rod-shaped, enveloped viruses with circular, supercoiled double-stranded DNA genomes that replicate in the nuclei of host cells (38). The Baculoviridae are divided into four genera: Alphabaculovirus (lepidopteran-specific nucleopolyhedrovirus [NPV]), Betabaculovirus (lepidopteran-specific granuloviruses), Gammabaculovirus (hymenopteran-specific NPV), and Deltabaculovirus (dipteran-specific NPV) (38). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the archetype species of Alphabaculovirus (38). During the AcMNPV life cycle, two virion phenotypes, budded virus (BV) and occlusion-derived virus (ODV), are produced. Both types of virions have similar nucleocapsid structures and identical genetic information, but they differ in the origin and composition of their envelopes and in the roles they play in the baculovirus life cycle (32). During the early phase of infection, viral DNA replicates in the virogenic stroma, and newly synthesized viral genomes are condensed and packaged into rodshaped capsids to form nucleocapsids. Nucleocapsids are transported into the cytoplasm and become enveloped by budding through the GP64-modified plasma membrane, thereby forming BVs. BVs are responsible for spreading infection between susceptible insect tissues and between cells in cell culture. During the late phase of infection, nucleocapsids remain localized within a peristromal ring zone, where they are enveloped in intranuclear microvesicles (whose formation is induced by viral infection) to form ODVs. The ODVs are subsequently embedded into a paracrystalline protein matrix to form occlusion bodies (OBs). ODVs play a role in the horizontal ...

Autographa californicaNucleopolyhedrovirus Ac76: a Dimeric Type II Integral Membrane Protein That Contains an Inner Nuclear Membrane-Sorting Motif

“…An immunoreactive band of ϳ20 kDa was first detected at low levels at 18 hpi, peaked at 48 hpi, and persisted until 72 hpi ( Fig. 2A), demonstrating that ac76 is a late gene, which is consistent with the results of the transcription analyses (13,26). The apparent molecular mass of Ac76:HA was nearly double its predicted molecular mass, indicating that Ac76 might exist as a dimer.…”

“…In addition, electron microscopic analysis showed that cells transfected with vAc76:HA showed characteristics similar to those transfected with vAcWT (data not shown). These results indicated that vAc76:HA was able to rescue the defective phenotype of vAc76KO, as previously described for vAc76 repair (13). The HA-tagged ac76 did not appear to have any effect on viral replication relative to the vAcWT virus; thus, we used HA-tagged ac76 for subsequent analyses.…”

“…Although ac76 is required for intranuclear microvesicle formation, as shown by gene knockout (13), the expression and localization of Ac76 have not been determined because of the lack of a specific antibody. In this study, an HA epitope coding sequence was fused to the 3= end of the ac76 gene in an ac76 repair construct so that Ac76 could be monitored with a commercially available monoclonal antibody to the HA epitope.…”

Autographa californica Multiple Nucleopolyhedrovirus ORF11 Is Essential for Budded-Virus Production and Occlusion-Derived-Virus Envelopment

“…ac25 (dbp) encodes a single DNA binding protein that is essential for the production of nucleocapsids and virogenic stroma (27). ac76 expresses a type II integral membrane protein localized to the ring zone late in infection (28,29). Both ac78, which encodes an envelope protein located in both BVs and ODVs (13), and ac80 (gp41), which encodes a tegument protein located between the virion envelope and capsid (30)(31)(32), are required for the egress of nucleocapsids from the nucleus.…”

“…Recently, several other genes, including ac53, ac76, ac77 (vlf-1), ac78, ac93, ac94, ac98 (38k), ac103 (p48), ac109, and ac142, have been shown to be essential for ODV formation (13,28,33,36,37,(39)(40)(41)(42). Deletion of ac53 (39) or ac98 (38k) (41) leads to defects in nucleocapsid assembly, and deletion of ac76 (28) or ac93 (33) affects intranuclear microvesicle formation, resulting in subsequent nucleocapsid envelopment. The knockout of ac78 (13), ac103 (p48) (43), ac109 (42), or ac142 (37) does not affect nucleocapsid assembly but interferes with nucleocapsid envelopment, which is similar to what was observed with the knockout of ac11 in this study.…”