Autographa californica multiple nucleopolyhedrovirus ac53 plays a role in nucleocapsid assembly

c Baculovirus VP1054 protein is a structural component of both of the virion types budded virus (BV) and occlusion-derived virus (ODV), but its exact role in virion morphogenesis is poorly defined. In this paper, we reveal sequence and functional similarity between the baculovirus protein VP1054 and the cellular purine-rich element binding protein PUR-alpha (PUR␣). The data strongly suggest that gene transfer has occurred from a host to an ancestral baculovirus. Deletion of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vp1054 gene completely prevented viral cell-to-cell spread. Electron microscopy data showed that assembly of progeny nucleocapsids is dramatically reduced in the absence of VP1054. More precisely, VP1054 is required for proper viral DNA encapsidation, as deduced from the formation of numerous electron-lucent capsid-like tubules. Complementary searching identified the presence of genetic elements composed of repeated GGN trinucleotide motifs in baculovirus genomes, the target sequence for PUR␣ proteins. Interestingly, these GGN-rich sequences are disproportionally distributed in baculoviral genomes and mostly occurred in proximity to the gene for the major occlusion body protein polyhedrin. We further demonstrate that the VP1054 protein specifically recognizes these GGN-rich islands, which at the same time encode crucial proline-rich domains in p78/83, an essential gene adjacent to the polyhedrin gene in the AcMNPV genome. While some viruses, like human immunodeficiency virus type 1 (HIV-1) and human JC virus (JCV), utilize host PUR␣ protein, baculoviruses encode the PUR␣-like protein VP1054, which is crucial for viral progeny production.

Section: Discussionsupporting

confidence: 84%

Baculovirus VP1054 Is an Acquired Cellular PURα, a Nucleic Acid-Binding Protein Specific for GGN Repeats

Marek

Romier

Galibert

et al. 2013

“…However, the capsid structures resulting from loss of the aforementioned genes were located not only in the RZ but also in the VS (interspace of the electron-dense mats) (16,(18)(19)(20)22), whereas in vAc54KO-transfected cells, no capsid structures were observed in the VS (Fig. 2B).…”

Section: Resultsmentioning

confidence: 99%

“…In addition, abnormal electron-dense bodies were found in the VS, and empty capsid structures were found in the RZ in cells transfected with vAc54KO, indicating that ablation of ac54 disrupted the normal assembly of nucleocapsids. In addition to ac54, some other AcMNPV genes (i.e., 38K, ac53, ac83, pk-1, and p6.9) have also been implicated in nucleocapsid assembly, and their individual deletion results in the cessation of assembly and the appearance of empty capsid structures in both the RZ and VS (16)(17)(18)(19)(20)22). However, in the present study, neither capsid structures nor VP39 was observed inside the VS in Sf9 cells transfected with vAc54KO, implying a special role of VP1054 in the baculovirus life cycle besides nucleocapsid assembly.…”

Section: Discussionmentioning

confidence: 99%

“…Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most intensively studied baculovirus. In addition to the major capsid protein VP39 (11,12), AcMNPV nucleocapsids contain several minor components (6,13), such as the following: P6.9, a protamine-like protein that is responsible for the condensation of viral DNA to form the nucleocapsid core (14)(15)(16); PK-1, a virus-encoded protein kinase that is required for the hyperphosphorylation of P6.9, which facilitates the release of viral DNA from the nucleocapsid (17); and 38K, Ac53, VP91 (encoded by ac83), and VLF-1, which are also classified as minor capsid proteins although their exact functions are poorly understood (18)(19)(20)(21). Single deletion of the genes encoding each of the six minor capsid proteins mentioned above results in the failure of nucleocapsid assembly and the distribution of abnormally long electron-lucent tubular structures at the electrondense edges of the stroma, indicating that the packaging of DNA and nucleoproteins into capsids is interrupted (16,(18)(19)(20)(21)(22).…”

mentioning

confidence: 99%

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The Autographa californica Multiple Nucleopolyhedrovirus ac54 Gene Is Crucial for Localization of the Major Capsid Protein VP39 at the Site of Nucleocapsid Assembly

Guan

Zhong

et al. 2016

Self Cite

Baculovirus DNAs are synthesized and inserted into preformed capsids to form nucleocapsids at a site in the infected cell nucleus, termed the virogenic stroma. Nucleocapsid assembly of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) requires the major capsid protein VP39 and nine minor capsid proteins, including VP1054. However, how VP1054 participates in nucleocapsid assembly remains elusive. In this study, the VP1054-encoding gene (ac54) was deleted to generate the ac54-knockout AcMNPV (vAc54KO). In vAc54KO-transfected cells, nucleocapsid assembly was disrupted, leading to the formation of abnormally elongated capsid structures. Interestingly, unlike cells transfected with AcMNPV mutants lacking other minor capsid proteins, in which capsid structures were distributed within the virogenic stroma, ac54 ablation resulted in a distinctive location of capsid structures and VP39 at the periphery of the nucleus. The altered distribution pattern of capsid structures was also observed in cells transfected with AcMNPV lacking BV/ODV-C42 or in cytochalasin D-treated AcMNPV-infected cells. BV/ODV-C42, along with PP78/83, has been shown to promote nuclear filamentous actin (F-actin) formation, which is another requisite for nucleocapsid assembly. Immunofluorescence using phalloidin indicated that the formation and distribution of nuclear F-actin were not affected by ac54 deletion. However, immunoelectron microscopy revealed that BV/ODV-C42, PP78/83, and 38K failed to integrate into capsid structures in the absence of VP1054, and immunoprecipitation further demonstrated that in transient expression assays, VP1054 interacted with BV/ODV-C42 and VP80 but not VP39. Our findings suggest that VP1054 plays an important role in the transport of capsid proteins to the nucleocapsid assembly site prior to the process of nucleocapsid assembly. IMPORTANCEBaculoviruses are large DNA viruses whose replication occurs within the host nucleus. The localization of capsids into the capsid assembly site requires virus-induced nuclear F-actin; the inhibition of nuclear F-actin formation results in the retention of capsid structures at the periphery of the nucleus. In this paper, we note that the minor capsid protein VP1054 is essential for the localization of capsid structures, the major capsid protein VP39, and the minor capsid protein 38K into the capsid assembly site. Moreover, VP1054 is crucial for correct targeting of the nuclear F-actin factors BV/ODV-C42 and PP78/83 for capsid maturation. However, the formation and distribution of nuclear F-actin are not affected by the lack of VP1054. We further reveal that VP1054 interacts with BV/ODV-C42 and a capsid transport-related protein, VP80. Taken together, our findings suggest that VP1054 plays a unique role in the pathway(s) for transport of capsid proteins. The family Baculoviridae includes a diverse group of insect-specific viruses that contain circular double-stranded DNA within a rod-shaped protein capsid enclosed by a lipid envelope (1, 2). Two forms of virions (bu...

“…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.…”

mentioning

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 ...