Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.

38K (ac98) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene whose function is unknown. To determine the role of 38K in the baculovirus life cycle, a 38K knockout bacmid containing the AcMNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a 38K repair bacmid was constructed by transposing the 38K open reading frame with its native promoter region into the polyhedrin locus of the 38K knockout bacmid. After transfection of these viruses into Spodoptera frugiperda cells, the 38K knockout bacmid led to a defect in production of infectious budded virus, while the 38K repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Slot blot analysis indicated that 38K deletion did not affect the levels of viral DNA replication. Subsequent immunoelectron-microscopic analysis revealed that masses of electron-lucent tubular structures containing the capsid protein VP39 were present in cells transfected with 38K knockout bacmids, suggesting that nucleocapsid assembly was interrupted. In contrast, the production of normal nucleocapsids was restored when the 38K knockout bacmid was rescued with a copy of 38K. Recombinant virus that expresses 38K fused to green fluorescent protein as a visual marker was constructed to monitor protein transport and localization within the nucleus during infection. Fluorescence was first detected along the cytoplasmic periphery of the nucleus and subsequently localized to the center of the nucleus. These results demonstrate that 38K plays a role in nucleocapsid assembly and is essential for viral replication in the AcMNPV life cycle.

The Autographa californica multiple nucleopolyhedrovirus orf92 (p33), ac92, is one of 31 genes carried in all sequenced baculovirus genomes, thus suggesting an essential function. Ac92 has homology to the family of flavin adenine dinucleotide-linked sulfhydryl oxidases and is related to the ERV/ALR family of sulfhydryl oxidases. The role of ac92 during virus replication is unknown. Ac92 was associated with the envelope of both budded and occlusion-derived virus (ODV). To investigate the role of Ac92 during virus replication, an ac92-knockout bacmid was generated through homologous recombination in Escherichia coli. Titration and plaque assays showed no virus spread in ac92-knockout bacmid DNA-transfected insect cells. Deletion of ac92 did not affect viral DNA replication. However, ac92-knockout bacmid DNAtransfected cells lacked multiply enveloped occlusion-derived nucleocapsids; instead, singly enveloped nucleocapsids were detected. To gain insight into the requirement for sulfhydryl oxidation during virus replication, a virus was constructed in which the Ac92 C 155 XXC 158 amino acids, important for sulfhydryl oxidase activity, were mutated to A 155 XXA 158 . The mutant virus exhibited a phenotype similar to that of the knockout virus, suggesting that the C-X-X-C motif was essential for sulfhydryl oxidase activity and responsible for the altered ODV phenotype.Baculoviruses infect insects and are characterized by a circular double-stranded DNA genome ranging from 80 to 180 kbp (10), which is packaged within a rod-shaped capsid and surrounded by an envelope. Two types of virions are produced during the baculovirus replication cycle: the budded virus (BV) and the occlusion-derived virus (ODV). Although the nucleocapsids are similar in the two, the envelopes differ in composition, reflecting their different functions during infection (4). The virions have distinct tissue tropism: ODVs infect midgut epithelial cells up to 10,000-fold more efficiently than do BVs, whereas BVs infect cultured cells 1,000-fold more efficiently than do ODVs (24).Alphabaculovirus and Betabaculovirus are two genera of Baculoviridae, based primarily on occlusion body morphology and phylogeny (11). Alphabaculoviruses have been divided into single nucleopolyhedrovirus (SNPV) and multiple nucleopolyhedrovirus (MNPV) occlusions. The M or S refers to whether the ODVs contain multiply or singly enveloped nucleocapsids, respectively (33). The MNPVs have been isolated only from Lepidoptera (23). It has been proposed that the M phenotype has an advantage over the S phenotype. Although the establishment of primary infections may be the same, the presence of multiple virions in MNPVs accelerated the onset of systemic infections (32, 33).The Autographa californica MNPV (AcMNPV) open reading frame 92 (orf92) (ac92, p33) is one of 31 core genes present in all sequenced baculovirus genomes (24). Previous studies showed that Ac92 and its homologs, Culex nigripalpus NPV orf14 and Helicoverpa armigera SNPV orf80, are associated with the ODV (3, 8, 18). Pre...

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p48 (ac103) is a highly conserved baculovirus gene of unknown function. In the present study, we generated a knockout of the p48 gene in an AcMNPV bacmid and investigated the role of P48 in baculovirus life cycle. The p48-null Bacmid vAc(P48-KO-PH-GFP) was unable to propagate in cell culture, while a 'repair' Bacmid vAc(P48-REP-PH-GFP) was able to replicate in a manner similar to a wild-type Bacmid vAc(PH-GFP). Titration assays and Western blotting confirmed that vAc(P48-KO-PH-GFP) was unable to produce budded viruses (BVs). qPCR analysis showed that p48 deletion did not affect viral DNA replication. Electron microscopy indicated that P48 was required for nucleocapsid envelopment to form occlusion-derived viruses (ODVs) and their subsequent occlusion. Confocal analysis showed that P48 prominently condensed in the centre of the nucleus. Our results demonstrate that P48 plays an essential role in BV production and ODV envelopment in the AcMNPV life cycle.

Several mammalian viruses have been shown to induce a cellular DNA damage response during replication, and in some cases, this response is required for optimal virus replication. However, nothing is known about whether a DNA damage response is stimulated by DNA viruses in invertebrates. Cell cycle arrest and apoptosis are two of the downstream effects of the DNA damage response, and both are stimulated by baculovirus infection, suggesting a possible relationship between baculoviruses and the DNA damage response. In the study described in this report, we found that replication of the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) in the cell line Sf9, derived from the lepidopteran insect Spodoptera frugiperda, stimulated a DNA damage response, as indicated by an increased abundance of the S. frugiperda P53 protein (SfP53) and phosphorylation of the histone variant protein H2AX. Stimulation of the DNA damage response was dependent on viral DNA replication. Inhibition of the DNA damage response prevented both the increase in SfP53 accumulation and H2AX phosphorylation and also caused a 10-to 100-fold reduction in virus production, along with decreased viral DNA replication and late gene expression. However, silencing of Sfp53 expression by RNA interference did not significantly affect AcMNPV replication or induction of apoptosis by a mutant of AcMNPV lacking the antiapoptotic gene p35, indicating that these processes are not dependent on SfP53 in Sf9 cells.Eukaryotic cells constantly monitor the integrity of their genomes and rapidly respond to the presence of damaged DNA by activating DNA damage response pathways, which are initiated largely through the activation of two members of the phosphatidylinositol 3-kinase superfamily, ataxia telangiectasia mutated (ATM) and ATM and rad-3 related (ATR) (for a recent review, see reference 50). ATM is activated mainly in response to double-strand breaks in DNA, while ATR responds mainly to single-strand breaks and stalled replication forks. However, there is overlap in the downstream substrates of these kinases. ATM and ATR phosphorylate numerous effector proteins which function in cell cycle checkpoints, DNA repair, and stimulation of apoptosis. One of the many important substrates of ATM and ATR is the transcription factor P53, which regulates the expression of numerous genes that function in all three of these processes. Studies in Drosophila melanogaster have demonstrated that DNA damage responses, including the roles of ATM, ATR, and P53, share many similarities in insects and mammals (53).There is increasing evidence that the ability to manipulate the DNA damage response and associated downstream pathways is crucial for the replication of many viruses. Several mammalian viruses stimulate DNA damage response pathways as a consequence of infection, and these viruses have in turn evolved mechanisms to manipulate these pathways for their own benefit by exploiting or actively inhibiting different parts of the pathways (reviewed in reference 7). For exam...

It has been shown that the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) 38K (ac98) is required for nucleocapsid assembly. However, the exact role of 38K in nucleocapsid assembly remains unknown. In the present study, we investigated the relationship between 38K and the nucleocapsid. Western blotting using polyclonal antibodies raised against 38K revealed that 38K was expressed in the late phase of infection in AcMNPV-infected Spodoptera frugiperda cells and copurified with budded virus (BV) and occlusionderived virus (ODV). Biochemical fractionation of BV and ODV into the nucleocapsid and envelope components followed by Western blotting showed that 38K was associated with the nucleocapsids. Immunoelectron microscopic analysis revealed that 38K was specifically localized to the nucleocapsids in infected cells and appeared to be distributed over the cylindrical capsid sheath of nucleocapsid. Yeast two-hybrid assays were performed to examine potential interactions between 38K and nine known nucleocapsid shell-associated proteins (PP78/83, PCNA, VP1054, FP25, VLF-1, VP39, BV/ODV-C42, VP80, and P24), three non-nucleocapsid shell-associated proteins (P6.9, PP31, and BV/ODV-E26), and itself. The results revealed that 38K interacted with the nucleocapsid proteins VP1054, VP39, VP80, and 38K itself. These interactions were confirmed by coimmunoprecipitation assays in vivo. These data demonstrate that 38K is a novel nucleocapsid protein and provide a rationale for why 38K is essential for nucleocapsid assembly.

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 californica multiple nucleopolyhedrovirus (AcMNPV) orf53 (ac53) is a highly conserved gene existing in all sequenced Lepidoptera and Hymenoptera baculoviruses, but its function remains unknown. To investigate its role in the baculovirus life cycle, an ac53 deletion virus (vAc(ac53KO-PH-GFP)) was generated through homologous recombination in Escherichia coli. Fluorescence and light microscopy and titration analysis revealed that vAc(ac53KO-PH-GFP) could not produce infectious budded virus in infected Sf9 cells. Real-time PCR demonstrated that the ac53 deletion did not affect the levels of viral DNA replication. Electron microscopy showed that many lucent tubular shells devoid of the nucleoprotein core are present in the virogenic stroma and ring zone, indicating that the ac53 knockout affected nucleocapsid assembly. With a recombinant virus expressing an Ac53-GFP fusion protein, we observed that Ac53 was distributed within the cytoplasm and nucleus at 24 h post-infection, but afterwards accumulated predominantly near the nucleus-cytoplasm boundary. These data demonstrate that ac53 is involved in nucleocapsid assembly and is an essential gene for virus production.