ABSTRACT
Introduction: Ectopic foreign bodies in the maxillary sinus occur rarely. Ectopic tooth eruption rarely occurs in the orbit, nasal cavity, maxillary sinus, and elsewhere. Ectopic eruption of teeth in the maxillary sinus is most commonly associated with wisdom teeth and is rarely associated with supernumerary teeth. This rare phenomenon may be accompanied by chronic recurrent sinusitis with headaches and facial pain. However, fibro-osseous lesions in the paranasal sinuses are discovered incidentally on X-ray images and are often asymptomatic. Osteoma is the most common fibro-osseous lesion that develops in the paranasal and nasal sinuses. Osteomas rarely cause serious symptoms such as orbital lesions and intracranial invasion. Case Presentation: We report a rare case of exostosis containing supernumerary teeth within the maxillary sinus. A characteristic pedicled bone lesion with a clear border on computed tomography was the undefined orthopantomogram radiopacity in the maxillary sinus, and the lesion contained supernumerary teeth. As the patient had chronic nasal congestion, the tumor was surgically removed. Pathologically, the surgical specimen revealed an osteoma. The patient's symptoms of chronic sinusitis disappeared. Because the patient had no history of midface trauma or surgery, the supernumerary teeth were speculated to have migrated during a reactive osteogenic process caused by chronic sinusitis. Conclusions: A foreign body in the maxillary sinus can be easily diagnosed by computed tomography. Surgical removal is recommended if the foreign body is symptomatic or occupies more than half of the maxillary sinus. This can help resolve chronic sinusitis symptoms and prevent serious complications in the future.
ABSTRACT
Patients with advanced cancer are frequently burdened with a severe sensation of fatigue called cancer-related fatigue (CRF). CRF is induced at various stages and treatments, such as cachexia and chemotherapy, and reduces the overall survival of patients. Objective and quantitative assessment of CRF could contribute to the diagnosis and prediction of treatment efficacy. However, such studies have not been intensively performed, particularly regarding metabolic profiles. Here, we conducted plasma metabolomics of 15 patients with urological cancer. The patients with and without fatigue, including those with cachexia or chemotherapy-induced fatigue, were compared. Significantly lower concentrations of valine and tryptophan were observed in fatigued patients than in non-fatigued patients. In addition, significantly higher concentrations of polyamine pathway metabolites were observed in patients with fatigue and cachexia than in those without cachexia. Patients with exacerbated fatigue due to chemotherapy showed significantly decreased cysteine and methionine metabolism before chemotherapy compared with those without fatigue exacerbation. These findings suggest that plasma metabolic profiles could help improve the diagnosis and monitoring of CRF.
Subject(s)
Cachexia , Neoplasms , Humans , Cachexia/etiology , Cachexia/diagnosis , Neoplasms/complications , Neoplasms/drug therapy , Metabolomics , Metabolome , Fatigue/etiologyABSTRACT
Globins have been studied as model proteins to elucidate the principles of protein evolution. This was achieved by understanding the relationship between amino acid sequence, three-dimensional structure, physicochemical properties, and physiological function. Previous molecular phylogenies of chordate globin genes revealed the monophyletic evolution of urochordate globins and suggested convergent evolution. However, to provide evidence of convergent evolution, it is necessary to determine the physicochemical and functional similarities between vertebrates and urochordate globins. In this study, we determined the expression patterns of Ciona globin genes using real-time RT-PCR. Two genes (Gb-1 and Gb-2) were predominantly expressed in the branchial sac, heart, and hemocytes and were induced under hypoxia. Combined with the sequence analysis, our findings suggest that Gb-1/-2 correspond to vertebrate hemoglobin-α/-ß. However, we did not find a robust similarity between Gb-3, Gb-4, and vertebrate globins. These results suggested that, even though Ciona globins obtained their unique functions differently from vertebrate globins, the two of them shared some physicochemical features and physiological functions. Our findings offer a good example for understanding the molecular mechanisms underlying gene co-option and convergence, which could lead to evolutionary innovations.
Subject(s)
Ciona intestinalis , Lancelets , Animals , Humans , Globins/genetics , Ciona intestinalis/genetics , Lancelets/genetics , Vertebrates/genetics , Amino Acid Sequence , Multigene Family , Phylogeny , Evolution, MolecularABSTRACT
Tumor suppressor p53 plays a central role in response to DNA damage. DNA-damaging agents modulate nuclear actin dynamics, influencing cell behaviors; however, whether p53 affects the formation of nuclear actin filaments remains unclear. In this study, we found that p53 depletion promoted the formation of nuclear actin filaments in response to DNA-damaging agents, such as doxorubicin (DOXO) and etoposide (VP16). Even though the genetic probes used for the detection of nuclear actin filaments exerted a promotive effect on actin polymerization, the detected formation of nuclear actin filaments was highly dependent on both p53 depletion and DNA damage. Whilst active p53 is known to promote caspase-1 expression, the overexpression of caspase-1 reduced DNA damage-induced formation of nuclear actin filaments in p53-depleted cells. In contrast, co-treatment with DOXO and the pan-caspase inhibitor Q-VD-OPh or the caspase-1 inhibitor Z-YVAD-FMK induced the formation of nuclear actin filament formation even in cells bearing wild-type p53. These results suggest that the p53-caspase-1 axis suppresses DNA damage-induced formation of nuclear actin filaments. In addition, we found that the expression of nLifeact-GFP, the filamentous-actin-binding peptide Lifeact fused with the nuclear localization signal (NLS) and GFP, modulated the structure of nuclear actin filaments to be phalloidin-stainable in p53-depleted cells treated with the DNA-damaging agent, altering the chromatin structure and reducing the transcriptional activity. The level of phosphorylated H2AX (γH2AX), a marker of DNA damage, in these cells also reduced upon nLifeact-GFP expression, whilst details of the functional relationship between the formation of nLifeact-GFP-decorated nuclear actin filaments and DNA repair remained to be elucidated. Considering that the loss of p53 is associated with cancer progression, the results of this study raise a possibility that the artificial reinforcement of nuclear actin filaments by nLifeact-GFP may enhance the cytotoxic effect of DNA-damaging agents in aggressive cancer cells through a reduction in gene transcription.
Subject(s)
Actins , Tumor Suppressor Protein p53 , Actins/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Actin Cytoskeleton/metabolism , DNA Damage , Caspases/metabolism , DNA/metabolismABSTRACT
Although the phagocytic activity of macrophages has long been studied, the involvement of microtubules in the process is not well understood. In this study, we improved the fixation protocol and revealed a dynamically rearranging microtubule network in macrophages, consisting of a basal meshwork, thick bundles at the cell edge, and astral microtubules. Some astral microtubules extended beneath the cell cortex and continued to form bundles at the cell edge. These microtubule assemblies were mutually exclusive of actin accumulation during membrane ruffling. Although the stabilization of microtubules with paclitaxel did not affect the resting stage of the macrophages, it reduced the phagocytic activity and membrane ruffling of macrophages activated with serum-MAF, which induced rapid phagocytosis. In contrast, the destabilization of microtubules with nocodazole enhanced membrane ruffling and the internalization of phagocytic targets suggesting an inhibitory effect of the microtubule network on the remodeling of the actin network. Meanwhile, the microtubule network was necessary for phagosome maturation. Our detailed analyses of cytoskeletal filaments suggest a phagocytosis control system involving Ca2+ influx, the destabilization of microtubules, and activation of actin network remodeling, followed by the translocation and acidification of phagosomes on the microtubule bundles.
Subject(s)
Actins , Phagocytosis , Actins/metabolism , Phagocytosis/physiology , Macrophages/metabolism , Microtubules/metabolism , Cytoskeleton/metabolismABSTRACT
In many animal species, the body axis is determined by the relocalization of maternal determinants, organelles, or unique cell populations in a cytoskeleton-dependent manner. In the ascidian first cell cycle, the myoplasm, including mitochondria, endoplasmic reticulum (ER), and maternal mRNAs, move to the future posterior side concomitantly (called ooplasmic segregation or cytoplasmic and cortical reorganization). This translocation consists of first and second phases depending on the actin and microtubule, respectively. However, the transition from first to second phase, that is, translocation of myoplasmic components from microfilaments to microtubules, has been poorly investigated. In this study, we analyzed the relationship between these cytoskeletons and myoplasmic components during the first cell cycle and their role in morphogenesis by inhibitor experiments. Owing to our improved visualization techniques, there was unexpected F-actin accumulation at the vegetal pole during this transition period. When this F-actin was depolymerized, the microtubule structure was strongly affected, the myoplasmic components, including maternal mRNA, were mislocalized, and the anteroposterior axis formation was disordered. These results suggested the importance of F-actin during the first cell cycle and the existence of interactions between microfilaments and microtubules, implying the enigmatic mechanism of ooplasmic segregation. Solving this mystery leads us to an improved understanding of ascidian early development.
ABSTRACT
Axis formation is one of the most important events occurring at the beginning of animal development. In the ascidian egg, the antero-posterior axis is established at this time owing to a dynamic cytoplasmic movement called cytoplasmic and cortical reorganisation. During this movement, mitochondria, endoplasmic reticulum (ER), and maternal mRNAs (postplasmic/PEM RNAs) are translocated to the future posterior side. Although accumulating evidence indicates the crucial roles played by the asymmetrical localisation of these organelles and the translational regulation of postplasmic/PEM RNAs, the organisation of ER has not been described in sufficient detail to date owing to technical difficulties. In this study, we developed three different multiple staining protocols for visualising the ER in combination with mitochondria, microtubules, or mRNAs in whole-mount specimens. We defined the internally expanded "dense ER" using these protocols and described cisterna-like structures of the dense ER using focused ion beam-scanning electron microscopy. Most importantly, we described the dynamic changes in the colocalisation of postplasmic/PEM mRNAs and dense ER; for example, macho-1 mRNA was detached and excluded from the dense ER during the second phase of ooplasmic movements. These detailed descriptions of the association between maternal mRNA and ER can provide clues for understanding the translational regulation mechanisms underlying axis determination during ascidian early embryogenesis.
Subject(s)
RNA, Messenger, Stored , Urochordata , Animals , Embryonic Development/genetics , Endoplasmic Reticulum , Oocytes , RNA, Messenger/genetics , RNA, Messenger, Stored/genetics , Urochordata/geneticsABSTRACT
In the first ascidian cell cycle, cytoplasmic and cortical reorganization is required for distributing maternal factors to their appropriate positions, resulting in the formation of the embryonic axis. This cytoplasmic reorganization is considered to depend on the cortical microfilament network in the first phase and on the sperm astral microtubule (MT) in the second phase. Recently, we described three novel MT structures: a deeply extended MT meshwork (DEM) in the entire subcortical region of the unfertilized egg, transiently accumulated MT fragments (TAF) in the vegetal pole, and a cortical MT array in the posterior vegetal cortex (CAMP). Particularly, our previous study showed CAMP to contribute to the movement of myoplasm. In addition, it is very similar to the parallel MT array, which appears during cortical rotation in Xenopus eggs. However, how these MT structures are organized is still unclear. Here, we investigated the relationship between the egg activation pathway and MT structures during the first ascidian cell cycle. First, we carefully analyzed cell cycle progression through meiosis I and II and the first mitosis, and successfully established a standard time table of cell cycle events. Using this time table as a reference, we precisely described the behavior of novel MT structures and revealed that it was closely correlated with cell cycle events. Moreover, pharmacological experiments supported the relationship between these MT structures and the signal transduction mechanisms that begin after fertilization, including Ca2+ signaling, MPF signaling, and MEK/MAPK signaling. Especially, CAMP formation was directed by activities of cyclin-dependent kinases. As these MT structures are conserved, at least, within chordate group, we emphasize the importance of understanding the controlling mechanisms of MT dynamics, which is important for embryonic axis determination in the ascidian egg.
Subject(s)
Centrosome/metabolism , Microtubules/metabolism , Ovum/metabolism , Signal Transduction , Urochordata/cytology , Urochordata/metabolism , Animals , Butadienes/pharmacology , Calcium Signaling/drug effects , Cell Cycle/drug effects , Centrosome/drug effects , Cyclin-Dependent Kinases/metabolism , Male , Meiosis/drug effects , Microtubules/drug effects , Models, Biological , Nitriles/pharmacology , Ovum/cytology , Ovum/drug effects , Roscovitine/pharmacology , Signal Transduction/drug effects , Spermatozoa/drug effects , Spermatozoa/metabolism , Urochordata/drug effectsABSTRACT
Eggs have developed their own strategies for early development. Amphibian, teleost fish, and ascidian eggs show cortical rotation and an accompanying structure, a cortical parallel microtubule (MT) array, during the one-cell embryonic stage. Cortical rotation is thought to relocate maternal deposits to a certain compartment of the egg and to polarize the embryo. The common features and differences among chordate eggs as well as localized maternal proteins and mRNAs that are related to the organization of MT structures are described in this review. Furthermore, recent studies report progress in elucidating the molecular nature and functions of the noncentrosomal MT organizing center (ncMTOC). The parallel array of MT bundles is presumably organized by ncMTOCs; therefore, the mechanism of ncMTOC control is likely inevitable for these species. Thus, the molecules related to the ncMTOC provide clues for understanding the mechanisms of early developmental systems, which ultimately determine the embryonic axis.
Subject(s)
Chordata/metabolism , Microtubules/metabolism , Zygote/metabolism , Animals , Biological Transport , Centrosome/metabolism , Chordata/embryology , Embryonic DevelopmentABSTRACT
Body axis formation during embryogenesis results from asymmetric localization of maternal factors in the egg. Shortly before the first cleavage in ascidian eggs, cell polarity along the anteroposterior (A-P) axis is established and the cytoplasmic domain (myoplasm) relocates from the vegetal to the posterior region in a microtubule-dependent manner. Through immunostaining, tubulin accumulation during this reorganization is observable on the myoplasm cortex. However, more detailed morphological features of microtubules remain relatively unknown. In this study, we invented a new reagent that improves the immunostaining of cortical microtubules and successfully visualized a parallel array of thick microtubules. During reorganization, they covered nearly the entire myoplasm cortical region, beneath the posterior-vegetal cortex. We designated this microtubule array as CAMP (cortical array of microtubules in posterior vegetal region). During the late phase of reorganization, CAMP shrank and the myoplasm formed a crescent-like cytoplasmic domain. When the CAMP formation was inhibited by sodium azide, myoplasmic reorganization and A-P axis formation were both abolished, suggesting that CAMP is important for these two processes.
Subject(s)
Cell Polarity/physiology , Ciona intestinalis/metabolism , Cytoplasm/metabolism , Microtubules/metabolism , Oocytes/metabolism , Animals , Ciona intestinalis/cytology , Embryonic Development/physiology , Oocytes/cytologyABSTRACT
Gastrointestinal stromal tumors (GISTs) are a type of sarcoma, and the most common mesenchymal tumor of the gastrointestinal tract. Systemic chemotherapy is recommended for unresectable or metastatic GISTs. Imatinib is an oral multitargeted receptor tyrosine kinase inhibitor that is effective as adjuvant chemotherapy for primary high-risk cases, and as palliative chemotherapy for unresectable or metastatic cases. For imatinib-resistant cases, second-line chemotherapy with sunitinib is recommended due to significantly longer median progression-free survival and higher response rates compared with a placebo. A 54-year-old woman presented with persistent upper abdominal pain and anorexia. An upper gastrointestinal endoscopy and computed tomography revealed a submucosal tumor of the stomach with no apparent metastases. The patient underwent total radical gastrectomy, and was diagnosed histologically with high-risk GIST for recurrence, therefore, the patient received adjuvant chemotherapy with imatinib. However, multiple liver and lymph node metastases were detected, and the patient received sunitinib therapy. After four cycles of sunitinib, the liver and lymph node metastases disappeared, and a complete response (CR) was achieved. To date, there have been no cases of CR in the prospective clinical trials examining the effects of sunitinib, or in case reports worldwide. Therefore, this is a very rare case report of a patient with metastatic GISTs who achieved CR with sunitinib as second-line chemotherapy.
ABSTRACT
OBJECTIVE: Disseminated intravascular coagulation (DIC) is a clinical condition with high mortality that is characterized by the systemic activation of coagulation pathways resulting in multiple organ failure. Although no standard treatment for DIC has been established, recent reports have indicated that recombinant human soluble thrombomodulin (rTM) is effective against DIC. METHODS: To elucidate the clinical characteristics and outcomes of DIC, we retrospectively analyzed 92 DIC patients who were treated with rTM at Miyazaki Prefectural Hospital over a 4-year period (62 patients had infectious diseases and 30 patients had hematological diseases). A diagnosis of DIC was made based on the diagnostic criteria of the Japanese Association for Acute Medicine (JAAM) and Japanese Ministry of Health and Welfare (JMHW) for infectious diseases and hematological diseases, respectively. In addition to treating the underlying disease, rTM was administered for six consecutive days. RESULTS: In this study, 49 of the 92 DIC patients (53.3%) experienced resolution of DIC seven days after administration (46.8% patients with infectious disease and 66.7% with hematological disease). A higher survival rate was observed after a 28-day observation period in 69 of the 92 patients (75.0%) (72.6% of the patients with infectious disease and 80.0% of the patients with hematological disease). A lower DIC score at the initiation of rTM treatment was closely related to a higher rate of resolution of DIC. CONCLUSION: Our findings indicate that rTM therapy is an effective, safe and feasible treatment for DIC patients. Furthermore, making an accurate and early diagnosis of DIC and providing subsequent immediate treatment with rTM may improve the resolution of DIC.
Subject(s)
Communicable Diseases/drug therapy , Disseminated Intravascular Coagulation/drug therapy , Hematologic Diseases/drug therapy , Thrombomodulin/therapeutic use , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Communicable Diseases/diagnosis , Communicable Diseases/mortality , Disseminated Intravascular Coagulation/diagnosis , Disseminated Intravascular Coagulation/mortality , Female , Hematologic Diseases/diagnosis , Hematologic Diseases/mortality , Humans , Male , Middle Aged , Mortality/trends , Recombinant Proteins/therapeutic use , Retrospective Studies , Treatment Outcome , Young AdultABSTRACT
In Japan, recent trends have seen wild silk preferred over cultivated silk because of its texture. Some cases of fraud have occurred where cultivated silk garments are sold as wild silk. Samples from these cases, morphological observation using light microscope and polarized microscope have been conducted in forensic science laboratories. Sometimes scanning electron microscopy was also carried out. However, the morphology of silk shows quite wide variation, which makes it difficult to discriminate wild and cultivated silks by this method. In this report, silk discrimination was investigated using conventional instrumental analyses commonly available in forensic laboratories, such as Fourier-transfer infrared spectrometry (FT-IR), pyrolysis-gas chromatography/mass spectrometry (pyr-GC/MS) and differential thermal analysis (DTA). By FT-IR, cultivated and wild silk gave similar infrared spectra, but wild silk had a characteristic peak at 965 cm(-1) from the deformation vibration of the carbon-carbon double bond of the indole ring. Comparison of the pyrograms of cultivated and wild silk showed that wild silk had large indole and skatole peaks that cultivated silk did not, and these peaks might arise from tryptophan. The results of thermogravimetry/DTA showed that the endothermic peak was about 40 °C higher for wild silk than for cultivated silk. Using a combination of these results, cultivated and wild silk could be discriminated by common forensic instrumental techniques.
Subject(s)
Fraud , Silk , Animals , Bombyx , Differential Thermal Analysis , Gas Chromatography-Mass Spectrometry , Humans , Spectroscopy, Fourier Transform Infrared , ThermogravimetryABSTRACT
BACKGROUND: Yeast is recognized as a generally safe microorganism and is utilized for the production of pharmaceutical products, including vaccines. We previously showed that expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts released Gag virus-like particles (VLPs) extracellularly, suggesting that the production system could be used in vaccine development. In this study, we further establish HIV-1 genome packaging into Gag VLPs in a yeast cell system. RESULTS: The nearly full-length HIV-1 genome containing the entire 5' long terminal repeat, U3-R-U5, did not transcribe gag mRNA in yeast. Co-expression of HIV-1 Tat, a transcription activator, did not support the transcription. When the HIV-1 promoter U3 was replaced with the promoter for the yeast glyceraldehyde-3-phosphate dehydrogenase gene, gag mRNA transcription was restored, but no Gag protein expression was observed. Co-expression of HIV-1 Rev, a factor that facilitates nuclear export of gag mRNA, did not support the protein synthesis. Progressive deletions of R-U5 and its downstream stem-loop-rich region (SL) to the gag start ATG codon restored Gag protein expression, suggesting that a highly structured noncoding RNA generated from the R-U5-SL region had an inhibitory effect on gag mRNA translation. When a plasmid containing the HIV-1 genome with the R-U5-SL region was coexpressed with an expression plasmid for Gag protein, the HIV-1 genomic RNA was transcribed and incorporated into Gag VLPs formed by Gag protein assembly, indicative of the trans-packaging of HIV-1 genomic RNA into Gag VLPs in a yeast cell system. The concentration of HIV-1 genomic RNA in Gag VLPs released from yeast was approximately 500-fold higher than that in yeast cytoplasm. The deletion of R-U5 to the gag gene resulted in the failure of HIV-1 RNA packaging into Gag VLPs, indicating that the packaging signal of HIV-1 genomic RNA present in the R-U5 to gag region functions similarly in yeast cells. CONCLUSIONS: Our data indicate that selective trans-packaging of HIV-1 genomic RNA into Gag VLPs occurs in a yeast cell system, analogous to a mammalian cell system, suggesting that yeast may provide an alternative packaging system for lentiviral RNA.
Subject(s)
Gene Products, gag/metabolism , Genome, Viral , HIV-1/genetics , Saccharomyces cerevisiae/metabolism , Vaccines, Virus-Like Particle/metabolism , Gene Products, gag/genetics , HIV Long Terminal Repeat , HIV Reverse Transcriptase/genetics , HIV Reverse Transcriptase/metabolism , HeLa Cells , Humans , Plasmids/genetics , Plasmids/metabolism , Promoter Regions, Genetic , Protein Biosynthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Ribonucleoproteins, Small Nucleolar/genetics , Transcription, Genetic , Vaccines, Virus-Like Particle/genetics , Vaccines, Virus-Like Particle/immunology , tat Gene Products, Human Immunodeficiency Virus/genetics , tat Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
BACKGROUND: It has been accepted that HIV buds from the cell surface in T lymphocytes, whereas in macrophages it buds into intracellular endosomes. Recent studies, on the other hand, suggest that HIV preferentially buds from the cell surface even in monocytic cells. However, most studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Such studies would provide a better understanding of a reservoir for HIV. RESULTS: We observed HIV budding in latently infected T lymphocytic and monocytic cell lines following TNF-alpha stimulation and examined the upregulation of host factors that may be involved in particle production. Electron microscopy analysis revealed that reactivation of latently infected J1.1 cells (latently infected Jurkat cells with HIV-1) and U1 cells (latently infected U937 cells with HIV-1) displayed HIV particle budding predominantly at the plasma membrane, a morphology that is similar to particle budding in acutely infected Jurkat and U937 cells. When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation. This upregulation was similarly observed when Jurkat and U937 cells were acutely infected with HIV-1 but not when just stimulated with TNF-alpha, suggesting that CD44 upregulation was linked with HIV production but not with cell stimulation. The molecules in endocytic pathways such as CD63 and HRS were also upregulated when U1 cells were reactivated and U937 cells were acutely infected with HIV-1. Confocal microscopy revealed that these upregulated host molecules were recruited to and accumulated at the sites where mature particles were formed at the plasma membrane. CONCLUSION: Our study indicates that HIV particles are budded at the plasma membrane upon reactivation from latency, a morphology that is similar to particle budding in acute infection. Our data also suggest that HIV expression may lead to the upregulation of certain host cell molecules that are recruited to sites of particle assembly, possibly coordinating particle production.
Subject(s)
Cell Membrane/chemistry , Cell Membrane/virology , HIV Infections/virology , HIV-1/physiology , Hyaluronan Receptors/biosynthesis , Virus Assembly , Antigens, CD/biosynthesis , Cell Line , Cell Membrane/ultrastructure , Endosomal Sorting Complexes Required for Transport , Gene Expression Profiling , Humans , Microscopy, Confocal , Microscopy, Electron, Transmission , Monocytes/ultrastructure , Monocytes/virology , Phosphoproteins/biosynthesis , Platelet Membrane Glycoproteins/biosynthesis , T-Lymphocytes/ultrastructure , T-Lymphocytes/virology , Tetraspanin 30 , Up-RegulationABSTRACT
Superinfection rates of human immunodeficiency virus type 1 (HIV-1) have increasingly been leading to more variation in HIV-1, as evidenced by the emergence of circulating recombinant forms (CRFs). We recently reported complementation in a persistently replication-defective subtype B-infected cell clone, L-2, by superinfection with CRF15_01B. The L-2 cells continuously produce immature particles due to a one-base insertion at pol protease. Proviruses in the superinfected cells carried both subtypes and produced particles with a mature morphology. In this study, we examined possible recombination following complementation to generate replication-competent variants by using three cell clones prepared from superinfected L-2 cells. The individual clones predominantly expressed the initial subtype B-derived mature Gag proteins. However, the viral particles carried both subtype B with the mutation and wild-type CRF15_01B at pol, suggesting the generation of virions with heterozygous RNAs. Interestingly, with cell-free passages of the progeny, defective particles disappeared, and were replaced with heterogeneous recombinants in the pol region with sequences derived from CRF15_01B that expressed subtype B phenotype. Thus, even a defective form of persistent HIV-1 can become replication-competent through superinfection-mediated complementation followed by recombination. These findings suggest the significance of long-lived infected cells as recipients for superinfection.
Subject(s)
HIV Infections/virology , HIV-1/genetics , HIV-1/pathogenicity , Recombination, Genetic , Superinfection/virology , B-Lymphocytes/ultrastructure , B-Lymphocytes/virology , CD4-Positive T-Lymphocytes/virology , Fluorescent Antibody Technique, Indirect , Genetic Variation , HIV-1/physiology , Humans , Microscopy, Electron , Phenotype , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, RNA , Virus Integration , Virus Replication , gag Gene Products, Human Immunodeficiency Virus/analysis , pol Gene Products, Human Immunodeficiency Virus/analysisABSTRACT
Myristoylation of human immunodeficiency virus (HIV) Gag protein is essential for membrane targeting of Gag and production of viral particles. We show here that coexpression of wild-type and nonmyristoylated forms of HIV Gag resulted in severe inhibition of viral particle production, indicating that the nonmyristoylated counterpart had a dominant negative effect on particle release. When coexpressed, the nonmyristoylated Gag partially incorporated into membrane and lipid raft fractions, likely through coassembly with the wild-type Gag. The membrane and raft associations of the wild-type Gag appeared unaffected, and yet particle production was severely impaired. When viral particles produced from the coexpressing cells were analyzed, the wild-type Gag was more abundant than the nonmyristoylated Gag. Confocal microscopy showed that both forms of Gag were diffusely distributed in the cytoplasm of coexpressing cells but that a portion of the wild-type Gag population was accumulated in EEA1- and CD63-positive endosomes. The intracellular accumulation of Gag was more frequently observed at late time points. The Gag accumulation was also observed on the cell surface protrusion. Electron microscopy of the coexpressing cells revealed budding arrest phenotypes, including the occurrence of interconnected virions on the plasma membrane, and intracellular budding. We also show that the inhibition of particle production and the Gag accumulation to endosomes were suppressed when the nucleocapsid (NC) domain was deleted from the nonmyristoylated Gag, although the NC-deleted Gag was still capable of coassembly. Overall, our data indicate that coassembly with the nonmyristoylated Gag impairs HIV particle release, a phenomenon that may involve NC-mediated Gag-Gag interaction.
Subject(s)
Gene Products, gag/pharmacology , HIV/drug effects , Myristic Acid , Sequence Deletion , Virion/drug effects , Cell Membrane , Endosomes , Gene Products, gag/genetics , Gene Products, gag/pharmacokinetics , HeLa Cells , Humans , Microscopy, Electron , Virion/physiologyABSTRACT
We have previously shown that the expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts produces Gag virus-like particles (VLPs) at the plasma membrane, indicating that yeast has all the host factors necessary for HIV-1 Gag assembly. Here we expand the study by using diverse primate lentiviral Gags and show that yeast does not support the production of HIV-2 or simian immunodeficiency virus SIVmac Gag VLPs but allows the production of SIVagm and SIVmnd Gag VLPs. Particle budding was observed at the surfaces of cells expressing SIVagm and SIVmnd Gags, but cells expressing HIV-2 and SIVmac Gags showed only membrane-ruffling structures, although they were accompanied with electron-dense submembrane layers, suggesting arrest at an early stage of particle budding. Comparison of HIV-1 and HIV-2 Gag expression revealed broadly equivalent levels of intracellular Gag expression and Gag N-terminal myristoylation in yeast. Both Gags showed the same membrane-binding ability and were incorporated into lipid raft fractions at a physiological concentration of salt. HIV-2 Gag, however, failed to form a high-order multimer and easily dissociated from the membrane, phenomena which were not observed in higher eukaryotic cells. A series of chimeric Gags between HIV-1 and HIV-2 and Gag mutants with amino acid substitutions revealed that a defined region in helix 2 of HIV-2 MA (located on the membrane-binding surface of MA) affects higher-order Gag assembly and particle production in yeast. Together, these data suggest that yeast may lack a host factor(s) for HIV-2 and SIVmac Gag assembly.
Subject(s)
Gene Products, gag/metabolism , HIV-1/metabolism , HIV-2/metabolism , Saccharomyces cerevisiae/metabolism , Spheroplasts/metabolism , Virus Assembly , Amino Acid Substitution , Gene Expression , Gene Products, gag/genetics , HIV-1/genetics , HIV-1/ultrastructure , HIV-2/genetics , HIV-2/ultrastructure , Humans , Membrane Microdomains/genetics , Membrane Microdomains/metabolism , Membrane Microdomains/ultrastructure , Mutation, Missense , Myristic Acid/metabolism , Protein Binding/genetics , Protein Processing, Post-Translational , Protein Structure, Secondary/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae/virology , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/metabolism , Spheroplasts/genetics , Spheroplasts/ultrastructureABSTRACT
Superinfection with human immunodeficiency virus type 1 (HIV-1) in human subjects, defined as reinfection with a heterologous strain of HIV-1, has become a topic of great interest. To illustrate the significance of this occurrence, we performed HIV-1 superinfection of L-2 cells, which were isolated from MT-4 cells persistently infected with subtype B HIV-1 as a cell clone continuously producing defective HIV-1 particles. L-2 cells carrying provirus with a one-base insertion in the pol protease were superinfected with HIV-1 derived from primary isolates of subtype B or CRF01_AE. The kinetics of the superinfection in L-2 were very slow compared with those of primary infections in MT-4. Interestingly, L-2 shifted after superinfection to become a producer of highly cytopathogenic HIV-1. Molecular characterization revealed that superinfection occurred in only about 10% of the CRF01_AE-superinfected L-2, which carried provirus of both subtypes and produced viral particles containing genomic RNA of both subtypes. Surprisingly, such cytopathogenic HIV-1 showed predominantly the original subtype B phenotype. Thus, the mechanism of the production of cytopathic HIV-1 seemed to be mediated by trans complementation with pol products of superinfected CRF01_AE. These findings suggest the significance of long-lived infected cells as recipients for superinfection that could result in the generation of new HIV-1 variants with high virulence in patients who are off therapy or do not adhere to treatment, and may indicate the need for precautions against such superinfection.
Subject(s)
Cytopathogenic Effect, Viral , Defective Viruses/growth & development , Defective Viruses/pathogenicity , HIV-1/growth & development , HIV-1/pathogenicity , T-Lymphocytes/virology , Cell Line , Defective Viruses/genetics , Genes, pol , HIV-1/genetics , Humans , Proviruses/genetics , Proviruses/pathogenicity , RNA, Viral/geneticsABSTRACT
We previously reported that a mutant full-sized plasmid DNA vaccine regime in macaques was effective against a homologous challenge [Akahata W, Ido E, Shimada T, Katsuyama K, Yamamoto H, Uesaka H, et al. DNA vaccination of macaques by a full genome HIV-1 plasmid which produces non-infectious virus particles. Virology 2000;275:116-24; Akahata W, Ido E, Akiyama H, Uesaka H, Enose Y, Horiuchi R, et al. DNA vaccination of macaques by a full genome SHIV-1 plasmid that produces non-infectious virus particles. J Gen Virol 2003;84:2237-44]. In this study, to evaluate the DNA vaccination regime against a heterologous challenge, a novel plasmid named pSHIV-ZF1*IL-2 was constructed. Four monkeys were intramuscularly and intradermally injected four times with the pSHIV-ZF1*IL-2. Vaccinated monkeys were intravenously challenged with a highly pathogenic, heterologous SHIV at 11 weeks post vaccination. All the vaccinated monkeys suppressed the challenge virus rapidly under the detectable level by 16 weeks post challenge. One vaccinated monkey was protected from a loss of CD4+ T cells. These results suggest pSHIV-ZF1*IL-2 alone seems partially effective even against a challenge with a heterologous, pathogenic virus.