Meijin Yuan

Identification of Autographa californica Nucleopolyhedrovirus ac93 as a Core Gene and Its Requirement for Intranuclear Microvesicle Formation and Nuclear Egress of Nucleocapsids

ABSTRACT Autographa californica nucleopolyhedrovirus (AcMNPV) orf93 (ac93) is a highly conserved uncharacterized gene that is found in all of the sequenced baculovirus genomes except for Culex nigripalpus NPV. In this report, using bioinformatics analyses, ac93 and odv-e25 (ac94) were identified as baculovirus core genes and thus p33-ac93-odv-e25 represent a cluster of core genes. To investigate the role of ac93 in the baculovirus life cycle, an ac93 knockout AcMNPV bacmid was constructed via homologous recombination in Escherichia coli. Fluorescence and light microscopy showed that the AcMNPV ac93 knockout did not spread by infection, and titration assays confirmed a defect in budded virus (BV) production. However, deletion of ac93 did not affect viral DNA replication. Electron microscopy indicated that ac93 was required for the egress of nucleocapsids from the nucleus and the formation of intranuclear microvesicles, which are precursor structures of occlusion-derived virus (ODV) envelopes. Immunofluorescence analyses showed that Ac93 was concentrated toward the cytoplasmic membrane in the cytoplasm and in the nuclear ring zone in the nucleus. Western blot analyses showed that Ac93 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV. Our results suggest that ac93, although not previously recognized as a core gene, is one that plays an essential role in the formation of the ODV envelope and the egress of nucleocapsids from the nucleus.

Autographa californica multiple nucleopolyhedrovirus 38K is a novel nucleocapsid protein that interacts with VP1054, VP39, VP80, and itself.

ABSTRACT 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 occlusion-derived 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.

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 occlusion-derived 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.

The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells.

Many viruses activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, thereby modulating diverse downstream signaling pathways associated with antiapoptosis, proliferation, cell cycling, protein synthesis and glucose metabolism, in order to augment their replication. To date, the role of the PI3K-Akt pathway in Baculovirus replication has not been defined. In the present study, we demonstrate that infection of Sf9 cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) elevated cellular Akt phosphorylation at 1 h post-infection. The maximum Akt phosphorylation occurred at 6 h post-infection and remained unchanged until 18 h post-infection. The PI3K-speci fi c inhibitor, LY294002, suppressed Akt phosphorylation in a dose-dependent manner, suggesting that AcMNPV-induced Akt phosphorylation is PI3K-dependent. The inhibition of PI3K-Akt activation by LY294002 significantly reduced the viral yield, including a reduction in budded viruses and occlusion bodies. The virus production was reduced only when the inhibitor was added within 24 h of infection, implying that activation of PI3K occurred early in infection. Correspondingly, both viral DNA replication and late (VP39) and very late (POLH) viral protein expression were impaired by LY294002 treatment; LY294002 had no effect on immediate-early (IE1) and early-late (GP64) protein expression. These results demonstrate that the PI3K-Akt pathway is required for efficient Baculovirus replication.

The Baculovirus Core Gene ac83 Is Required for Nucleocapsid Assembly and Per Os Infectivity of Autographa californica Nucleopolyhedrovirus

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac83 is a baculovirus core gene whose function in the AcMNPV life cycle is unknown. In the present study, an ac83-knockout AcMNPV (vAc83KO) was constructed to investigate the function of ac83 through homologous recombination in Escherichia coli. No budded virions were produced in vAc83KO-transfected Sf9 cells, although viral DNA replication was unaffected. Electron microscopy revealed that nucleocapsid assembly was aborted due to the ac83 deletion. Domain-mapping studies revealed that the expression of Ac83 amino acid residues 451 to 600 partially rescued the ability of AcMNPV to produce infectious budded virions. Bioassays indicated that deletion of the chitin-binding domain of Ac83 resulted in the failure of oral infection of Trichoplusia ni larvae by AcMNPV, but AcMNPV remained infectious following intrahemocoelic injection, suggesting that the domain is involved in the binding of occlusion-derived virions to the peritrophic membrane and/or to other chitin-containing insect tissues. It has been demonstrated that Ac83 is the only component with a chitin-binding domain in the per os infectivity factor complex on the occlusion-derived virion envelope. Interestingly, a functional inner nuclear membrane sorting motif, which may facilitate the localization of Ac83 to the envelopes of occlusion-derived virions, was identified by immunofluorescence analysis. Taken together, these results demonstrate that Ac83 plays an important role in nucleocapsid assembly and the establishment of oral infection.

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

ABSTRACT Our previous study showed that the Autographa californica Nucleopolyhedrovirus (AcMNPV) ac76 gene is essential for both budded virion (BV) and occlusion-derived virion (ODV) development. More importantly, deletion of ac76 affects intranuclear microvesicle formation. However, the exact role by which ac76 affects virion morphogenesis remains unknown. In this report, we characterized the expression, distribution, and topology of Ac76 to further understand the functional role of Ac76 in virion morphogenesis. Ac76 contains an α-helical transmembrane domain, and phase separation showed that it was an integral membrane protein. In AcMNPV-infected cells, Ac76 was detected as a stable dimer that was resistant to SDS and thermal denaturation, and only a trace amount of monomer was detected. A coimmunoprecipitation assay demonstrated the dimerization of Ac76 by high-affinity self-association. Western blot analyses of purified virions and their nucleocapsid and envelope fractions showed that Ac76 was associated with the envelope fractions of both BVs and ODVs. Immunoelectron microscopy revealed that Ac76 was localized to the plasma membrane, endoplasmic reticulum (ER), nuclear membrane, intranuclear microvesicles, and ODV envelope. Amino acids 15 to 48 of Ac76 were identified as an atypical inner nuclear membrane-sorting motif because it was sufficient to target fusion proteins to the ER and nuclear membrane in the absence of viral infection and to the intranuclear microvesicles and ODV envelope during infection. Topology analysis of Ac76 by selective permeabilization showed that Ac76 was a type II integral membrane protein with an N terminus exposed to the cytosol and a C terminus hidden in the ER lumen.

Autographa californica multiple nucleopolyhedrovirus ac53 plays a role in nucleocapsid assembly

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.

An ac34 deletion mutant of Autographa californica nucleopolyhedrovirus exhibits delayed late gene expression and a lack of virulence in vivo

ABSTRACT Ac34 and its homologs are highly conserved in all sequenced alphabaculoviruses. In this paper, we show that ac34 transcripts were detected from 6 to 48 h postinfection (p.i.) in Autographa californica nucleopolyhedrovirus (AcMNPV)-infected Sf9 cells. Ac34 localized to both the cytoplasm and the nuclei of infected cells but was not a viral structural protein. To determine the function of ac34 in the viral life cycle, an ac34 knockout AcMNPV (vAc34KO) was constructed. Compared with wild-type and repair viruses, vAc34KO exhibited an approximately 100-fold reduction in infectious virus production. Further investigations showed that the ac34 deletion did not affect the replication of viral DNA, polyhedron formation, or nucleocapsid assembly but delayed the expression of late genes, such as vp39, 38k, and p6.9. Bioassays revealed that vAc34KO was unable to establish a fatal infection in Trichoplusia ni larvae via per os inoculation. Few infectious progeny viruses were detected in the hemolymph of the infected larvae, indicating that the replication of vAc34KO was attenuated. These results suggest that Ac34 is an activator protein that promotes late gene expression and is essential for the pathogenicity of AcMNPV.

Posttranslational Modifications of Baculovirus Protamine-Like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression.

ABSTRACT Many viruses utilize viral or cellular chromatin machinery for efficient infection. Baculoviruses encode a conserved protamine-like protein, P6.9. This protein plays essential roles in various viral physiological processes during infection. However, the mechanism by which P6.9 regulates transcription remains unknown. In this study, 7 phosphorylated species of P6.9 were resolved in Sf9 cells infected with the baculovirus type species Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Mass spectrometry identified 22 phosphorylation and 10 methylation sites but no acetylation sites in P6.9. Immunofluorescence demonstrated that the P6.9 and virus-encoded serine/threonine kinase PK1 exhibited similar distribution patterns in infected cells, and coimmunoprecipitation confirmed the interaction between them. Upon pk1 deletion, nucleocapsid assembly and polyhedron formation were interrupted and the transcription of viral very late genes was downregulated. Interestingly, we found that the 3 most phosphorylated P6.9 species vanished from Sf9 cells transfected with the pk1 deletion mutant, suggesting that PK1 is involved in the hyperphosphorylation of P6.9. Mass spectrometry suggested that the phosphorylation of the 7 Ser/Thr and 5 Arg residues in P6.9 was PK1 dependent. Replacement of the 7 Ser/Thr residues with Ala resulted in a P6.9 phosphorylation pattern similar to that of the pk1 deletion mutant. Importantly, the decreases in the transcription level of viral very late genes and viral infectivity were consistent. Our findings reveal that P6.9 hyperphosphorylation is a precondition for the maximal hyperexpression of baculovirus very late genes and provide the first experimental insights into the function of the baculovirus protamine-like protein and the related protein kinase in epigenetics. IMPORTANCE Diverse posttranslational modifications (PTMs) of histones constitute a code that creates binding platforms that recruit transcription factors to regulate gene expression. Many viruses also utilize host- or virus-induced chromatin machinery to promote efficient infections. Baculoviruses encode a protamine-like protein, P6.9, which is required for a variety of processes in the infection cycle. Currently, P6.9s PTM sites and its regulating factors remain unknown. Here, we found that P6.9 could be categorized as unphosphorylated, hypophosphorylated, and hyperphosphorylated species and that a virus-encoded serine/threonine kinase, PK1, was essential for P6.9 hyperphosphorylation. Abundant PTM sites on P6.9 were identified, among which 7 Ser/Thr phosphorylated sites were PK1 dependent. Mutation of these Ser/Thr sites reduced very late viral gene transcription and viral infectivity, indicating that the PK1-mediated P6.9 hyperphosphorylation contributes to viral proliferation. These data suggest that a code exists in the sophisticated PTM of viral protamine-like proteins and participates in viral gene transcription.

Functional analysis of Spodoptera litura nucleopolyhedrovirusp49 gene during Autographa californicanucleopolyhedrovirus infection of SpLi-221 cells

The Spodoptera lituranucleopolyhedrovirus (SpltNPV) antiapoptotic gene Splt-p49 is able to rescue replication of a p35-null Autographacalifornicanucleopolyhedrovirus (AcMNPV) in AcMNPV-permissive Sf9 cells. In this study, an AcMNPV p35 knockout bacmid was generated through ET homologous recombination in Escherichia coli and designated as vAcP35-KO. The Splt-p49 gene was transposed into the polyhedrin locus of vAcP35-KO to investigate if Splt-p49 has any antiapoptotic activity in the context of AcMNPV infection of AcMNPV-nonpermissive SpLi-221 cells. Our results demonstrated that Splt-p49 could not inhibit the apoptosis induced by AcMNPV infection of SpLi-221 cells when it was under the control of its native promoter. Western blot analysis showed that Splt-P49 was poorly expressed. When the expression of Splt-P49 was under the control of Drosophilahsp70 promoter, the expression of Splt-P49 was advanced, and a higher level of Splt-P49 was detected. As a result, the apoptosis of SpLi-221 cells was inhibited; however, budded-virus production did not improve in comparison with that in AcMNPV-infected SpLi-221 cells. These data indicated that there are other barrier(s) to AcMNPV replication in the nonpermissive SpLi-221 cells besides apoptosis.