Actin Contributes to the Hyperexpression of Baculovirus Polyhedrin (polh) and p10 as a Component of Transcription Initiation Complex (TIC).
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ABSTRACT:
SUBMITTER: Chen N
PROVIDER: S-EPMC8779803 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:UnlabelledMany 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.ImportanceDiverse 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, P6.9's 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.
| S-EPMC4505634 | biostudies-literature
Project description:Platinum-based anticancer drugs are widely used as a first-line drug for cancers, such as non-small cell lung carcinoma (NSCLC) and bladder cancer. However, the efficacy is limited due to intrinsic or acquired resistance to these drugs. DNA polymerase eta (PolH, Pol?) belongs to the Y-family of DNA polymerases and mediates DNA translesion synthesis, a major mechanism for DNA damage tolerance. Here, we showed that a high level of PolH is associated with cisplatin resistance in lung and bladder cancer. Consistent with this, loss of PolH markedly attenuates cisplatin resistance in both cisplatin-sensitive and cisplatin-resistant lung cancer cells. Interestingly, we found that due to the presence of multiple polyadenylation sites, alternative polyadenylation (APA) produces three major PolH transcripts with various lengths of 3'untranslated region (3'UTR; 427-/2516-/6245-nt). We showed that the short PolH transcript with 427-nt 3'UTR is responsible for high expression of PolH in various cisplatin-resistant lung and bladder cancer cell lines. Importantly, loss of the short PolH transcript significantly sensitizes cancer cells to cisplatin treatment. Moreover, we found that miR-619 selectively inhibits the ability of the long PolH transcript with 6245-nt 3'UTR to produce PolH protein and, subsequently, PolH-dependent cell growth. Together, our data suggest that PolH expression is controlled by APA and that the short PolH transcript produced by APA can escape miR-619-mediated repression and, subsequently, confers PolH-mediated cisplatin resistance. SIGNIFICANCE: A short PolH transcript produced by alternative polyadenylation escapes repression by miR-619 and confers resistance to cisplatin.
| S-EPMC6634999 | biostudies-literature
Project description:Viruses that replicate in the host cell nucleus face challenges in usurping cellular pathways to enable passage through the nuclear envelope [1]. Baculoviruses are enveloped, double-stranded DNA viruses that infect lepidopteran insects and are tools for protein expression, cell transduction, and pest management [2-4]. The type species Autographa californica M nucleopolyhedrovirus (AcMNPV) shares with other pathogens an ability to assemble host actin monomers (G-actin) into actin filaments (F-actin) to drive motility [5]. During early infection, actin-based motility in the cytoplasm speeds AcMNPV transit to the nucleus and passage through nuclear pores, enabling nuclear ingress [6, 7]. During late infection, AcMNPV assembles F-actin within the nucleus [8], which is essential for virus production [9, 10]. However, the function of nuclear F-actin is poorly understood [11], and its mechanistic role in AcMNPV infection was unknown. We show that AcMNPV mobilizes actin within the nucleus to promote egress. AcMNPV nucleocapsids exhibit intranuclear actin-based motility, mediated by the viral protein P78/83 and the host Arp2/3 complex. Viral motility drives transit to the nuclear periphery and is required for viruses to enter protrusions of the nuclear envelope. Moreover, actin polymerization is necessary for viral disruption of nuclear envelope integrity during egress. In the cytoplasm, viruses use actin-based motility to reach the plasma membrane to enable budding. Our results demonstrate that pathogens can harness actin polymerization to disrupt the nuclear envelope. Employing actin for nuclear envelope disruption may reflect viral appropriation of normal functions of nuclear actin in nuclear envelope integrity, stability, and remodeling.
| S-EPMC6137724 | biostudies-literature
Project description:Bundling of rapidly polymerizing actin filaments underlies the dynamics of filopodial protrusions that play an important role in cell migration and cell-cell interaction. Recently, the formation of actin bundles has been reconstituted in vitro, and two scenarios of bundle initiation, involving binding of two filament tips and, alternatively, linking of the tip of one filament to the side of the other, have been discussed. A first theoretical analysis is presented indicating that the two mechanisms can be distinguished experimentally. While both of them result counterintuitively in comparable numbers of bundles, these numbers scale differently with the average bundle length. We propose an experiment for determining which of the two mechanisms is involved in the in vitro bundle formation.
| S-EPMC2668770 | biostudies-literature
Project description:BACKGROUND:Hepatocellular carcinoma (HCC) is one of the most common cancers, whereas the molecular mechanism remains largely unknown. PRAS40 (encoded by AKT1S1) phosphorylation was increased in human melanoma, prostate cancer and lung cancer specimens, which was considered as the results of Akt activation. However the mechanism in detail and its role in HCC stay elusive. METHODS:PRAS40 expression and phosphorylation were analyzed in HCC specimens, and the survival rates of patients were investigated. Functional analyses of PRAS40 in HCC were performed in vivo and in vitro. The miR-124-3p binding sites in PRAS40 were investigated using luciferase assay. MiR-124-3p expression in HCC specimens was examined by In Situ hybridization, and the correlation to PRAS40 level was evaluated. FINDINGS:The phosphorylation, protein and mRNA levels of PRAS40 were increased significantly in HCC specimens from our cohorts and TCGA database, which was positively correlated to the poor prognosis of HCC patients. Compared to Akt1s1+/+ mice, hepatocarcinogenesis was suppressed in Akt1s1-/- mice, and the activation of Akt was impaired. PRAS40 depletion resulted in the inhibition of HCC cellular proliferation. Tumor suppressor miR-124-3p was found to downregulate PRAS40 expression by targeting its 3'UTR. MiR-124-3p levels were inversely correlated to PRAS40 protein and phosphorylation levels in HCC specimens. The proliferation inhibition by miR-124-3p mimics was partially reversed by exogenous PRAS40 introduction in HCC cells. INTERPRETATION:PRAS40 hyperexpression induced by loss of miR-124-3p contributes to PRAS40 hyperphosphorylation and hepatocarcinogenesis. These results could be expected to offer novel clues for understanding hepatocarcinogenesis and developing approaches.
| S-EPMC6950779 | biostudies-literature
Project description:DNA polymerase (pol) η is responsible for error-free translesion DNA synthesis (TLS) opposite ultraviolet light (UV)-induced cis-syn cyclobutane thymine dimers (CTDs) and cisplatin-induced intrastrand guanine crosslinks. POLH deficiency causes one form of the skin cancer-prone disease xeroderma pigmentosum variant (XPV) and cisplatin sensitivity, but the functional impacts of its germline variants remain unclear. We evaluated the functional properties of eight human POLH germline in silico-predicted deleterious missense variants, using biochemical and cell-based assays. In enzymatic assays, utilizing recombinant pol η (residues 1-432) proteins, the C34W, I147N, and R167Q variants showed 4- to 14-fold and 3- to 5-fold decreases in specificity constants (kcat/Km) for dATP insertion opposite the 3'-T and 5'-T of a CTD, respectively, compared to the wild-type, while the other variants displayed 2- to 4-fold increases. A CRISPR/Cas9-mediated POLH knockout increased the sensitivity of human embryonic kidney 293 cells to UV and cisplatin, which was fully reversed by ectopic expression of wild-type pol η, but not by that of an inactive (D115A/E116A) or either of two XPV-pathogenic (R93P and G263V) mutants. Ectopic expression of the C34W, I147N, and R167Q variants, unlike the other variants, did not rescue the UV- and cisplatin-sensitivity in POLH-knockout cells. Our results indicate that the C34W, I147N, and R167Q variants-substantially reduced in TLS activity-failed to rescue the UV- and cisplatin-sensitive phenotype of POLH-deficient cells, which also raises the possibility that such hypoactive germline POLH variants may increase the individual susceptibility to UV irradiation and cisplatin chemotherapy.
| S-EPMC10048814 | biostudies-literature
Project description:Recombinant WT human cardiac actin (WT actin) was expressed using the baculovirus/insect cell expression system, purified, and used to reconstitute the thin-filament of bovine cardiac muscle fibers, together with bovine cardiac tropomyosin (Tm) and troponin (Tn). Effects of [Ca(2+)], [ATP], [phosphate] and [ADP] on tension and tension transients were studied at 25°C by using sinusoidal analysis, and the results were compared with those of native fibers and fibers reconstituted with purified bovine cardiac actin (BVC actin). In actin filament reconstituted fibers (without Tm/Tn), those reconstituted with WT actin showed exactly the same active tension as those reconstituted with purified BVC actin (WT: 0.75±0.06 T0, N=11; BVC: 0.73±0.07 T0, N=12, where T0 is the tension of original fibers before extraction). After Tm/Tn reconstitution, fibers reconstituted with WT actin generated 0.85±0.06 T0 (N=11) compared to 0.98±0.04 T0 (N=12) recovered by those reconstituted with BVC actin. In the presence of Tm/Tn, WT actin reconstituted fibers showed exactly the same Ca(2+) sensitivity as those of the native fibers and BVC actin reconstituted fibers (pCa50: native fibers: 5.69±0.01, N=10; WT: 5.69±0.02, N=11; BVC: 5.68±0.02, N=12). Sinusoidal analysis showed that the cross-bridge kinetics were the same among native fibers, BVC actin reconstituted fibers and WT actin reconstituted fibers, followed by reconstitution of Tm/Tn. These results demonstrate that baculovirus/insect cell expressed actin has no significant differences from tissue purified actin and can be used for thin-filament reconstitution assays. One hypertrophic cardiomyopathy (HCM) causing actin mutant A331P actin was also expressed and studied similarly, and the results were compared to those of the WT actin. In the reconstituted fibers, A331P significantly decreased the tension both in the absence of Tm/Tn (0.55±0.03 T0, N=13) and in their presence (0.65±0.02 T0, N=13) compared to those of the WT (0.75±0.06 T0 and 0.85±0.06 T0, respectively, N=11). A331P also showed decreased pCa50 (5.57±0.03, N=13) compared to that of WT (5.69±0.02, N=11). The cross-bridge kinetics and its distribution were similar between WT and A331P actin reconstituted fibers, indicating that force/cross-bridge was decreased by A331P. In conclusion, A331P causes a weakened cross-bridge force, which leads to a decreased active tension, reduces left-ventricular ejection fraction, and eventually results in the HCM phenotype.
| S-EPMC4264970 | biostudies-literature
Project description:In vitro transcription was used to analyze the promoter specificity of the alpha-amanitin-resistant RNA polymerase that is induced late during infection of Autographa californica multicapsid nuclear polyhedrosis virus. By modifying the preparation of crude nuclear extracts, we have established an assay that permits differentiation between weak late and strong very late viral promoters. The virus-induced RNA polymerase initiates at a TAAG sequence motif in both late and very late promoters. Based on the sensitivity of our in vitro transcription system, we have investigated the sequences responsible for a functional TAAG motif and their putative role with respect to the strength of very late promoters. By constructing hybrid promoters between the early pe38 and the very late polyhedrin promoters, we demonstrated that the replacement of 7 nucleotides upstream of the nonfunctional TAAG sequences in the pe38 promoter with the corresponding sequences of the polyhedrin promoter was sufficient for recognition by the virus-induced RNA polymerase. The strength of the very late polyhedrin promoter was established after replacing the 5' untranslated sequences of the pe38 promoter by those of the polyhedrin promoter in addition to the 7 nucleotides upstream of the TAAG motif.
| S-EPMC109746 | biostudies-literature
Project description:The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a pathogen of lepidopteran insects, has a striking dependence on the host cell actin cytoskeleton. During the delayed-early stage of infection, AcMNPV was shown to induce the accumulation of actin at the cortex of infected cells. However, the dynamics and molecular mechanism of cortical actin assembly remained unknown. Here, we show that AcMNPV induces dynamic cortical clusters of dot-like actin structures that mediate degradation of the underlying extracellular matrix and therefore function similarly to clusters of invadosomes in mammalian cells. Furthermore, we find that the AcMNPV protein actin-rearrangement-inducing factor-1 (ARIF-1), which was previously shown to be necessary and sufficient for cortical actin assembly and efficient viral infection in insect hosts, is both necessary and sufficient for invadosome formation. We mapped the sequences within the C-terminal cytoplasmic region of ARIF-1 that are required for invadosome formation and identified individual tyrosine and proline residues that are required for organizing these structures. Additionally, we found that ARIF-1 and the invadosome-associated proteins cortactin and the Arp2/3 complex localize to invadosomes and Arp2/3 complex is required for their formation. These ARIF-1-induced invadosomes may be important for the function of ARIF-1 in systemic virus spread.
| S-EPMC8351737 | biostudies-literature
Project description:P10 is a small, abundant baculovirus protein that accumulates to high levels in the very late stages of the infection cycle. It is associated with a number of intracellular structures and implicated in diverse processes from occlusion body maturation to nuclear stability and lysis. However, studies have also shown that it is non-essential for virus replication, at least in cell culture. Here, we describe the use of serial block-face scanning electron microscopy to achieve high-resolution 3D characterisation of P10 structures within Trichoplusia ni TN-368 cells infected with Autographa californica multiple nucleopolyhedrovirus. This has enabled unparalleled visualisation of P10 and determined the independent formation of dynamic perinuclear and nuclear vermiform fibrous structures. Our 3D data confirm the sequence of ultrastructural changes that create a perinuclear cage from thin angular fibrils within the cytoplasm. Over the course of infection in cultured cells, the cage remodels to form a large polarised P10 mass and we suggest that these changes are critical for nuclear lysis to release occlusion bodies. In contrast, nuclear P10 forms a discrete vermiform structure that was observed in close spatial association with both electron dense spacers and occlusion bodies; supporting a previously suggested role for P10 and electron dense spacers in the maturation of occlusion bodies. We also demonstrate that P10 hyper-expression is critical for function. Decreasing levels of p10 expression, achieved by manipulation of promoter length, correlated with reduced P10 production, a lack of formation of P10 structures and a concomitant decrease in nuclear lysis.
| S-EPMC6557513 | biostudies-literature