Deletion of AcMNPV ac146 eliminates the production of budded virus

The endosomal sorting complex required for transport (ESCRT) machinery is necessary for budding of many enveloped viruses. Recently, it was demonstrated that Vps4, the key regulator for recycling of the ESCRT-III complex, is required for efficient infection by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, ESCRT assembly, regulation, and function are complex, and little is known regarding the details of participation of specific ESCRT complexes in AcMNPV infection. In this study, the core components of ESCRT-I (Tsg101 and Vps28) and ESCRT-III (Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60) were cloned from Using a viral complementation system and RNA interference (RNAi) assays, we found that ESCRT-I and ESCRT-III complexes are required for efficient entry of AcMNPV into insect cells. In cells knocking down or overexpressing dominant negative (DN) forms of the components of ESCRT-I and ESCRT-III complexes, entering virions were partially trapped within the cytosol. To examine only egress, cells were transfected with the double-stranded RNA (dsRNA) targeting an individual ESCRT-I or ESCRT-III gene and viral bacmid DNA or viral bacmid DNA that expressed DN forms of ESCRT-I and ESCRT-III components. We found that ESCRT-III components (but not ESCRT-I components) are required for efficient nuclear egress of progeny nucleocapsids. In addition, we found that several baculovirus core or conserved proteins (Ac11, Ac76, Ac78, GP41, Ac93, Ac103, Ac142, and Ac146) interact with Vps4 and components of ESCRT-III. We propose that these viral proteins may form an "egress complex" that is involved in recruiting ESCRT-III components to a virus egress domain on the nuclear membrane. The ESCRT system is hijacked by many enveloped viruses to mediate budding and release. Recently, it was found that Vps4, the key regulator of the cellular ESCRT machinery, is necessary for efficient entry and egress of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, little is known about the roles of specific ESCRT complexes in AcMNPV infection. In this study, we demonstrated that ESCRT-I and ESCRT-III complexes are required for efficient entry of AcMNPV into insect cells. The components of ESCRT-III (but not ESCRT-I) are also necessary for efficient nuclear egress of progeny nucleocapsids. Several baculovirus core or conserved proteins were found to interact with Vps4 and components of ESCRT-III, and these interactions may suggest the formation of an "egress complex" involved in the nuclear release or transport of viral nucleocapsids.

“…12A). In the cases of Ac76-Cm and Cm-Ac146, two bands were detected for each construct, and this has been observed previously (39,54).…”

supporting

confidence: 82%

“…In recent years, numerous gene knockout studies have reported that certain baculovirus core genes (such as those encoding Ac76, Ac78, Ac93, Ac103, Ac142, and Ac146) are required for production of infectious AcMNPV BV. However, it is not clear how these viral proteins are involved in virus infection (34)(35)(36)(37)(38)(39).…”

mentioning

confidence: 99%

Distinct Roles of Cellular ESCRT-I and ESCRT-III Proteins in Efficient Entry and Egress of Budded Virions of Autographa californica Multiple Nucleopolyhedrovirus

Yang

et al. 2018

“…ac93 encodes a BV-and ODV-associated nucleocapsid protein that is present in the BV envelope fraction (33). In addition, ac9 (p78/83) (34), ac54 (vp1054) (35), ac77 (vlf-1) (36), ac98 (38k) (20), ac142 (37), and ac146 (38), which are essential for BV production, have been shown to encode nucleocapsid proteins located in both BV and ODV. Interestingly, all of these genes are not essential for …”

Section: Discussionmentioning

confidence: 99%

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

Tao

Choi

Kim

et al. 2015

ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role of ac11 in the baculovirus life cycle, an ac11 knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5= rapid amplification of cDNA ends (RACE) analyses revealed that ac11 is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in Ac11KO-transfected Sf9 cells. However, quantitative PCR (qPCR) analysis demonstrated that the deletion of ac11 did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of Ac11KO-transfected cells, which demonstrates that the defect in BV production in Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form occlusion-derived viruses (ODVs) and that their subsequent embedding into occlusion bodies (OBs) was also blocked in Ac11KO-transfected cells, demonstrating that ac11 is required for ODV envelopment. These results therefore demonstrate that ac11 is an early gene that is essential for BV production and ODV envelopment. IMPORTANCEBaculoviruses have been extensively used not only as specific, environmentally benign insecticides but also as helper-independent protein expression vectors. Although the function of baculovirus genes in viral replication has been studied by using gene knockout technology, the functions of more than one-third of viral genes, which include some highly conserved genes, are still unknown. In this study, ac11 was proven to play a crucial role in BV production and ODV envelopment. These results will lead to a better understanding of baculovirus infection cycles. The Baculoviridae are a family of insect-specific doublestranded DNA (dsDNA) viruses. Viruses from this family are characterized by rod-shaped, enveloped nucleocapsids with circular, covalently closed, double-stranded DNA genomes of 80 to 180 kbp (1-3). The Baculoviridae family comprises four genera: Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus. Alphabaculoviruses and betabaculoviruses infect lepidopteran larvae, whereas gammabaculoviruses and deltabaculoviruses infect hymenopteran and dipteran larvae, respectively (4). The alphabaculoviruses are further divided into group I and group II on the basis of phylogenetic analysis (5) and the type of envelope fusion protein (GP64 and F, respectively) (6).The infection cycle of baculoviruses includes two distinct viral phenotypes: budded virus (BV) and occlusion-derived virus (ODV). Both BVs and ODVs are identical in terms of nucleocapsid structure and genetic information, but the composition of their envelopes is different to accommodate their respective functions in the infection cycle (7). ODVs, which become embedded ...

“…To determine the titers of infectious and noninfectious BVs released from transfected cells, the genome DNAs contained in BVs in the supernatants from the transfected cell cultures were purified and measured by real-time quantitative PCR (qPCR), as previously described (68). Briefly, 400 l of the supernatant collected from transfected cell culture was mixed with an equal volume of the lysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM EDTA, 0.5% SDS, 20 g/ml RNase A, 80 g/ml protease K) and incubated at 50°C overnight.…”

Section: Methodsmentioning

confidence: 99%

Autographa californica Multiple Nucleopolyhedrovirus orf132 Encodes a Nucleocapsid-Associated Protein Required for Budded-Virus and Multiply Enveloped Occlusion-Derived Virus Production

Yang

Wang

Yue

et al. 2014

Autographa californica multiple nucleopolyhedrovirus orf132 (named ac132) has homologs in all genome-sequenced group I nucleopolyhedroviruses. Its role in the viral replication cycle is unknown. In this study, ac132 was shown to express a protein of around 28 kDa, which was determined to be associated with the nucleocapsids of both occlusion-derived virus and budded virus. Confocal microscopy showed that AC132 protein appeared in central region of the nucleus as early as 12 h postinfection with the virus. It formed a ring zone at the periphery of the nucleus by 24 h postinfection. To investigate its role in virus replication, ac132 was deleted from the viral genome by using a bacmid system. In the Sf9 cell culture transfected by the ac132 knockout bacmid, infection was restricted to single cells, and the titer of infectious budded virus was reduced to an undetectable level. However, viral DNA replication and the expression of late genes vp39 and odv-e25 and a reporter gene under the control of the very late gene p10 promoter were unaffected. Electron microscopy showed that nucleocapsids, virions, and occlusion bodies were synthesized in the cells transfected by an ac132 knockout bacmid, but the formation of the virogenic stroma and occlusion bodies was delayed, the numbers of enveloped nucleocapsids were reduced, and the occlusion bodies contained mainly singly enveloped nucleocapsids. AC132 was found to interact with envelope protein ODV-E18 and the viral DNA-binding protein P6.9. The data from this study suggest that ac132 possibly plays an important role in the assembly and envelopment of nucleocapsids. IMPORTANCETo our knowledge, this is the first report on a functional analysis of ac132. The data presented here demonstrate that ac132 is required for production of the budded virus and multiply enveloped occlusion-derived virus of Autographa californica multiple nucleopolyhedrovirus. This article reveals unique phenotypic changes induced by ac132 deletion on the virus and multiple new findings on ac132.