Journal of Microbiology

Journal Articles

Unexpected Requirement of Small Amino Acids at Position 183 for DNA Binding in the Escherichia coli cAMP Receptor Protein.: Marcus Carranza, Amanda Rea, Daisy Pacheco, Christian Montiel, Jin Park, Hwan Youn; J. Microbiol. 2024;62(10):871-882. Published online September 6, 2024; DOI: https://doi.org/10.1007/s12275-024-00169-2

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Abstract: The Escherichia coli cAMP receptor protein (CRP) relies on the F-helix, the recognition helix of the helix-turn-helix motif, for DNA binding. The importance of the CRP F-helix in DNA binding is well-established, yet there is little information on the roles of its non-base-contacting residues. Here, we show that a CRP F-helix position occupied by a non-base-contacting residue Val183 bears an unexpected importance in DNA binding. Codon randomization and successive in vivo screening selected six amino acids (alanine, cysteine, glycine, serine, threonine, and valine) at CRP position 183 to be compatible with DNA binding. These amino acids are quite different in their amino acid properties (polar, non-polar, hydrophobicity), but one commonality is that they are all relatively small. Larger amino acid substitutions such as histidine, methionine, and tyrosine were made site-directedly and showed to have no detectable DNA binding, further supporting the requirement of small amino acids at CRP position 183. Bioinformatics analysis revealed that small amino acids (92.15% valine and 7.75% alanine) exclusively occupy the position analogous to CRP Val183 in 1,007 core CRP homologs, consistent with our mutant data. However, in extended CRP homologs comprising 3700 proteins, larger amino acids could also occupy the position analogous to CRP Val183 albeit with low occurrence. Another bioinformatics analysis suggested that large amino acids could be tolerated by compensatory small-sized amino acids at their neighboring positions. A full understanding of the unexpected requirement of small amino acids at CRP position 183 for DNA binding entails the verification of the hypothesized compensatory change(s) in CRP.

Genetically Engineered CLDN18.2 CAR-T Cells Expressing Synthetic PD1/CD28 Fusion Receptors Produced Using a Lentiviral Vector.: Heon Ju Lee, Seo Jin Hwang, Eun Hee Jeong, Mi Hee Chang; J. Microbiol. 2024;62(7):555-568. Published online May 3, 2024; DOI: https://doi.org/10.1007/s12275-024-00133-0

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Abstract: This study aimed to develop synthetic Claudin18.2 (CLDN18.2) chimeric antigen receptor (CAR)-T (CAR-T) cells as a treatment for advanced gastric cancer using lentiviral vector genetic engineering technology that targets the CLDN18.2 antigen and simultaneously overcomes the immunosuppressive environment caused by programmed cell death protein 1 (PD-1). Synthetic CAR T cells are a promising approach in cancer immunotherapy but face many challenges in solid tumors. One of the major problems is immunosuppression caused by PD-1. CLDN18.2, a gastric-specific membrane protein, is considered a potential therapeutic target for gastric and other cancers. In our study, CLDN18.2 CAR was a second-generation CAR with inducible T-cell costimulatory (CD278), and CLDN18.2-PD1/CD28 CAR was a third-generation CAR, wherein the synthetic PD1/CD28 chimeric-switch receptor (CSR) was added to the second-generation CAR. In vitro, we detected the secretion levels of different cytokines and the killing ability of CAR-T cells. We found that the secretion of cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) secreted by three types of CAR-T cells was increased, and the killing ability against CLDN18.2-positive GC cells was enhanced. In vivo, we established a xenograft GC model and observed the antitumor effects and off-target toxicity of CAR-T cells. These results support that synthetic anti-CLDN18.2 CAR-T cells have antitumor effect and anti-CLDN18.2-PD1/CD28 CAR could provide a promising design strategy to improve the efficacy of CAR-T cells in advanced gastric cancer.

Enhancement of the solubility of recombinant proteins by fusion with a short-disordered peptide: Jun Ren , Suhee Hwang , Junhao Shen , Hyeongwoo Kim , Hyunjoo Kim , Jieun Kim , Soyoung Ahn , Min-gyun Kim , Seung Ho Lee , Dokyun Na; J. Microbiol. 2022;60(9):960-967. Published online July 14, 2022; DOI: https://doi.org/10.1007/s12275-022-2122-z

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5 Citations

Abstract: In protein biotechnology, large soluble fusion partners are widely utilized for increased yield and solubility of recombinant proteins. However, the production of additional large fusion partners poses an additional burden to the host, leading to a decreased protein yield. In this study, we identified two highly disordered short peptides that were able to increase the solubility of an artificially engineered aggregationprone protein, GFP-GFIL4, from 0.6% to 61% (D3-DP00592) and 46% (D4-DP01038) selected from DisProt database. For further confirmation, the peptides were applied to two insoluble E. coli proteins (YagA and YdiU). The peptides also enhanced solubility from 52% to 90% (YagA) and from 27% to 93% (YdiU). Their ability to solubilize recombinant proteins was comparable with strong solubilizing tags, maltosebinding protein (40 kDa) and TrxA (12 kDa), but much smaller (< 7 kDa) in size. For practical application, the two peptides were fused with a restriction enzyme, I-SceI, and they increased I-SceI solubility from 24% up to 75%. The highly disordered peptides did not affect the activity of I-SceI while I-SceI fused with MBP or TrxA displayed no restriction activity. Despite the small size, the highly disordered peptides were able to solubilize recombinant proteins as efficiently as conventional fusion tags and did not interfere with the function of recombinant proteins. Consequently, the identified two highly disordered peptides would have practical utility in protein biotechnology and industry.

[PROTOCOL] High-throughput cultivation based on dilution-to-extinction with catalase supplementation and a case study of cultivating acI bacteria from Lake Soyang: Suhyun Kim , Miri S. Park , Jaeho Song , Ilnam Kang , Jang-Cheon Cho; J. Microbiol. 2020;58(11):893-905. Published online October 30, 2020; DOI: https://doi.org/10.1007/s12275-020-0452-2

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13 Citations

Abstract: Multi-omics approaches, including metagenomics and single- cell amplified genomics, have revolutionized our understanding of the hidden diversity and function of microbes in nature. Even in the omics age, cultivation is an essential discipline in microbial ecology since microbial cultures are necessary to assess the validity of an in silico prediction about the microbial metabolism and to isolate viruses infecting bacteria and archaea. However, the ecophysiological characteristics of predominant freshwater bacterial lineages remain largely unknown due to the scarcity of cultured representatives. In an ongoing effort to cultivate the uncultured majority of freshwater bacteria, the most abundant freshwater Actinobacteria acI clade has recently been cultivated from Lake Soyang through catalase-supplemented high-throughput cultivation based on dilution-to-extinction. This method involves physical isolation of target microbes from mixed populations, culture media simulating natural habitats, and removal of toxic compounds. In this protocol, we describe detailed procedures for isolating freshwater oligotrophic microbes, as well as the essence of the dilution-to-extinction culturing. As a case study employing the catalase-supplemented dilution-to-extinction protocol, we also report a cultivation trial using a water sample collected from Lake Soyang. Of the 480 cultivation wells inoculated with a single lake-water sample, 75 new acI strains belonging to 8 acI tribes (acI-A1, A2, A4, A5, A6, A7, B1, B4, C1, and C2) were cultivated, and each representative strain per subclade could be revived from glycerol stocks. These cultivation results demonstrate that the protocol described in this study is efficient in isolating freshwater bacterioplankton harboring streamlined genomes.

Long-term continuously monocropped peanut significantly disturbed the balance of soil fungal communities: Mingna Chen , Jiancheng Zhang , Hu Liu , Mian Wang , LiJuan Pan , Na Chen , Tong Wang , Yu Jing , Xiaoyuan Chi , Binghai Du; J. Microbiol. 2020;58(7):563-573. Published online April 22, 2020; DOI: https://doi.org/10.1007/s12275-020-9573-x

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Abstract: Balancing soil microbial diversity and abundance is critical to sustaining soil health, and understanding the dynamics of soil microbes in a monocropping system can help determine how continuous monocropping practices induce soil sickness mediated by microorganisms. This study used previously constructed gradient continuous monocropping plots and four varieties with different monocropping responses were investigated. The feedback responses of their soil fungal communities to short-term and long-term continuous monocropping were tracked using high-throughput sequencing techniques. The analyses indicated that soil samples from 1 and 2 year monocropped plots were grouped into one class, and samples from the 11 and 12 year plots were grouped into another, regardless of variety. At the species level, the F. solani, Fusarium oxysporum, Neocosmospora striata, Acrophialophora levis, Aspergillus niger, Aspergillus corrugatus, Thielavia hyrcaniae, Emericellopsis minima, and Scedosporium aurantiacum taxa showed significantly increased abundances in the long-term monocropping libraries compared to the short-term cropping libraries. In contrast, Talaromyces flavus, Talaromyces purpureogenus, Mortierella alpina, Paranamyces uniporus, and Volutella citrinella decreased in the long-term monocropping libraries compared to the shortterm libraries. This study, combined with our previous study, showed that fungal community structure was significantly affected by the length of the monocropping period, but peanut variety and growth stages were less important. The increase in pathogen abundances and the decrease in beneficial fungi abundances seem to be the main cause for the yield decline and poor growth of long-term monocultured peanut. Simplification of fungal community diversity could also contribute to peanut soil sickness under long-term monocropping. Additionally, the different responses of peanut varieties to monocropping may be related to variations in their microbial community structure.

Pten gene deletion in intestinal epithelial cells enhances susceptibility to Salmonella Typhimurium infection in mice: Cody Howe , Jonathon Mitchell , Su Jin Kim , Eunok Im , Sang Hoon Rhee; J. Microbiol. 2019;57(11):1012-1018. Published online September 25, 2019; DOI: https://doi.org/10.1007/s12275-019-9320-3

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7 Citations

Abstract: Although phosphatase and tensin homolog (PTEN) is typically considered a tumor-suppressor gene, it was recently suggested that PTEN regulates TLR5-induced immune and inflammatory responses in intestinal epithelial cells (IECs), suggesting an immunomodulatory function of PTEN in the gut. However, this alternative function of PTEN has not yet been evaluated in an in vivo context of protection against enteropathogenic bacteria. To address this, we utilized IECrestricted Pten knockout (PtenΔIEC/ΔIEC) and littermate Pten+/+ mice. These mice were subjected to the streptomycin-pretreated mouse model of Salmonella infection, and subsequently given an oral gavage of a low inoculum (2 × 104 CFU) of Salmonella enterica serovar Typhimurium (S. Typhimurium). This bacterial infection not only increased the mortality of PtenΔIEC/ΔIEC mice compared to Pten+/+ mice, but also induced deleterious gastrointestinal inflammation in PtenΔIEC/ΔIEC mice manifested by massive histological damage to the intestinal mucosa. S. Typhimurium infection upregulated pro-inflammatory cytokine production in the intestine of PtenΔIEC/ΔIEC mice compared to controls. Furthermore, bacterial loads were greatly increased in the liver, mesenteric lymph node, and spleen of PtenΔIEC/ΔIEC mice compared to controls. Together, these results suggest that IEC-restricted Pten deficiency renders the host greatly susceptible to Salmonella infection and support an immuneregulatory role of PTEN in the gut.

Lytic KFS-SE2 phage as a novel bio-receptor for Salmonella Enteritidis detection: In Young Choi , Cheonghoon Lee , Won Keun Song , Sung Jae Jang , Mi-Kyung Park; J. Microbiol. 2019;57(2):170-179. Published online January 31, 2019; DOI: https://doi.org/10.1007/s12275-019-8610-0

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Abstract: Since Salmonella Enteritidis is one of the major foodborne pathogens, on-site applicable rapid detection methods have been required for its control. The purpose of this study was to isolate and purify S. Enteritidis-specific phage (KFS-SE2 phage) from an eel farm and to investigate its feasibility as a novel, efficient, and reliable bio-receptor for its employment. KFS-SE2 phage was successfully isolated at a high concentration of (2.31 ± 0.43) × 1011 PFU/ml, and consisted of an icosahedral head of 65.44 ± 10.08 nm with a non-contractile tail of 135.21 ± 12.41 nm. The morphological and phylogenetic analysis confirmed that it belongs to the Pis4avirus genus in the family of Siphoviridae. KFS-SE2 genome consisted of 48,608 bp with 45.7% of GC content. Genome analysis represented KFS-SE2 to have distinctive characteristics as a novel phage. Comparative analysis of KFS-SE2 phage with closely related strains confirmed its novelty by the presence of unique proteins. KFS-SE2 phage exhibited excellent specificity to S. Enteritidis and was stable under the temperature range of 4 to 50°C and pH of 3 to 11 (P < 0.05). The latent time was determined to be 20 min. Overall, a new lytic KFS-SE2 phage was successfully isolated from the environment at a high concentration and the excellent feasibility of KFS-SE2 phage was demonstrated as a new bio-receptor for S. Enteritidis detection.

Water-based extracts of Zizania latifolia inhibit Staphylococcus aureus infection through the induction of human beta-defensin 2 expression in HaCaT cells: Bo Yeon Kang , Seung-Su Lee , Myun-Ho Bang , Hyoik Jeon , Hangeun Kim , Dae Kyun Chung; J. Microbiol. 2018;56(12):910-916. Published online November 27, 2018; DOI: https://doi.org/10.1007/s12275-018-8307-9

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Abstract: Zizania latifolia is a perennial herb belonging to the family Gramineae that has been used as a health food in Asian countries. In this study, we investigated the antimicrobial effect of Z. latifolia, which increased human beta-defensin 2 (hBD2) expression in HaCaT cells. hBD2 expression was further increased in cells treated with Z. latifolia extracts and subsequently infected with Staphylococcus aureus. Inversely, S. aureus infection decreased after treatment. The induction of hBD2 in HaCaT cells was mediated by the Toll-like receptor 2 (TLR2) signaling pathway, including the activation of extracellular signal-regulated kinase (ERK) and activator protein 1 (AP-1). Further study using siRNA revealed that hBD2 played an important role in the inhibition of S. aureus infection in HaCaT cells. Our data suggest that Z. latifolia extracts can be used as an antimicrobial ingredient for skin treatment formulas.

SucA-dependent uptake of sucrose across the outer membrane of Caulobacter crescentus: Samantha K. Modrak , Martha E. Melin , Lisa M. Bowers; J. Microbiol. 2018;56(9):648-655. Published online July 27, 2018; DOI: https://doi.org/10.1007/s12275-018-8225-x

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4 Citations

Abstract: Caulobacter crescentus is an aquatic Gram-negative bacterium that lives in nutrient-poor environments. Like several other aquatic and phytopathogenic bacteria, Caulobacter cells have a relatively large number of genes predicted to encode TonB-dependent receptors (TBDRs). TBDRs transport nutrients across the outer membrane using energy from the proton motive force. We identified one TBDR gene, sucA, which is situated within a cluster of genes predicted to encode a lacIfamily transcription factor (sucR), amylosucrase (sucB), fructokinase (sucC), and an inner membrane transporter (sucD). Given its genomic neighborhood, we proposed that sucA encodes a transporter for sucrose. Using RT-qPCR, we determined that expression of sucABCD is strongly induced by sucrose in the media and repressed by the transcription factor, SucR. Furthermore, cells with a deletion of sucA have a reduced uptake of sucrose. Although cells with a non-polar deletion of sucA can grow with sucrose as the sole carbon source, cells with a polar deletion that eliminates expression of sucABCD cannot grow with sucrose as the sole carbon source. These results show that the suc locus is essential for sucrose utilization while SucA functions as one method of sucrose uptake in Caulobacter crescentus. This work sheds light on a new carbohydrate utilization locus in Caulobacter crescentus.

Research Support, Non-U.S. Gov't

NOTE] Envelope Diversity, Characteristics of V3 Region and Predicted Co-Receptor Usage of Human Immunodeficiency Viruses Infecting North Indians: Raiees Andrabi , Rajesh Kumar , Manju Bala , Ambili Nair , Prakash SS , Vandana Kushwaha , Kalpana Luthra; J. Microbiol. 2012;50(5):869-873. Published online November 4, 2012; DOI: https://doi.org/10.1007/s12275-012-2136-z

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Abstract: Subtypes of human immunodeficiency virus type 1 circulating in 21 north Indian patients were characterized based on the partial sequence of the gp120 envelope protein. A majority of viruses (85.7%, 18/21) were subtype C, while 14.3% (3/21) were subtype A. Sequence analysis revealed that the V3 region was highly conserved compared with V4 and V5. The predicted use of co-receptors indicated exclusive usage of R5, except for two subtype A viruses (AIIMS279 and AIIMS281). Our results demonstrate conservation within the V3 loop of subtype C viruses, and suggest the emergence of non-clade C viruses in the north Indian population.

Journal Article

Regulatory Role of cAMP Receptor Protein over Escherichia coli Fumarase Genes: Yu-Pei Chen , Hsiao-Hsien Lin , Chi-Dung Yang , Shin-Hong Huang , Ching-Ping Tseng; J. Microbiol. 2012;50(3):426-433. Published online June 30, 2012; DOI: https://doi.org/10.1007/s12275-012-1542-6

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Abstract: Escherichia coli expresses three fumarase genes, namely, fumA, fumB, and fumC. In the present study, catabolite repression was observed in the fumA-lacZ and fumC-lacZ fusion strains, but not in the fumB-lacZ fusion strain. The Crp-binding sites in fumA and fumC were identified using an electrophoretic mobility shift assay and footprint analysis. However, the electrophoretic mobility shift assay did not detect band shifts in fumB. Fnr and ArcA serve as transcription regulators of fumarase gene expression. In relation to this, different mutants, including Δcya, Δcrp, Δfnr, and ΔarcA, were used to explore the regulatory role of Crp over fumA and fumC. The results show that Crp is an activator of fumA and fumC gene expression under various oxygen conditions and growth rates. ArcA was identified as the dominant repressor, with the major repression occurring at 0–4% oxygen. In addition, Fnr was confirmed as a repressor of fumC for the first time. This study elucidates the effects of Crp on fumarase gene expression.

Research Support, Non-U.S. Gov'ts

Cyclic AMP-Receptor Protein Activates Aerobactin Receptor IutA Expression in Vibrio vulnificus: Choon-Mee Kim , Seong-Jung Kim , Sung-Heui Shin; J. Microbiol. 2012;50(2):320-325. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-2056-y

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Abstract: The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability.

Ligand-Receptor Recognition for Activation of Quorum Sensing in Staphylococcus aureus: Li-Chun Chen , Li-Tse Tsou , Feng-Jui Chen; J. Microbiol. 2009;47(5):572-581. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0004-2

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13 Citations

Abstract: The accessory gene regulator (agr) locus controls many of the virulence toxins involved in Staphylococcus aureus pathogenesis, and can be divided into four specificity groups. AgrC is the only group-specific receptor to mediate both intra-group activation and inter-group inhibition. We studied the ligand-receptor recognition of the agr system in depth by using a luciferase reporter system to identify the key residues responsible for AgrC activation in two closely related agr groups, AgrC-I, and AgrC-IV. Fusion PCR and site-directed mutagenesis were used to screen for functional residues of AgrC. Our data suggest that for AgrC-IV activation, residue 101 is critical for activating the receptor. In contrast, the key residues for the activation of AgrC-I are located at residues 49~59, 107, and 116. However, three residue changes, T101A, V107S, I116S, are sufficient to convert the AIP recognizing specificity from AgrC-IV to AgrC-I.

Improved Prediction of Coreceptor Usage and Phenotype of HIV-1 Based on Combined Features of V3 Loop Sequence Using Random Forest: Shungao Xu , Xinxiang Huang , Huaxi Xu , Chiyu Zhang; J. Microbiol. 2007;45(5):441-446.; DOI: https://doi.org/2592 [pii]

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Abstract: HIV-1 coreceptor usage and phenotype mainly determined by V3 loop are associated with the disease progression of AIDS. Predicting HIV-1 coreceptor usage and phenotype facilitates the monitoring of R5-to-X4 switch and treatment decision-making. In this study, we employed random forest to predict HIV-1 biological phenotype, based on 37 random features of V3 loop. In comparison with PSSM method, our RF predictor obtained higher prediction accuracy (95.1% for coreceptor usage and 92.1% for phenotype), especially for non-B non-C HIV-1 subtypes (96.6% for coreceptor usage and 95.3% for phenotype). The net charge, polarity of V3 loop and five V3 sites are seven most important features for predicting HIV-1 coreceptor usage or phenotype. Among these features, V3 polarity and four V3 sites (22, 12, 18 and 13) are first reported to have high contribution to HIV-1 biological phenotype prediction.

Journal Article

Role of a Third Extracellular Domain of an Ecotropic Receptor in Moloney Murine Leukemia Virus Infection: Eun Hye Bae , Sung-Han Park , Yong-Tae Jung; J. Microbiol. 2006;44(4):447-452.; DOI: https://doi.org/2407 [pii]

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Abstract: The murine ecotropic retroviral receptor has been demonstrated to function as a mouse cationic amino acid transporter 1 (mCAT1), and is comprised of multiple membranespanning domains. Feral mouse (Mus dunni) cells are not susceptible to infection by the ecotropic Moloney murine leukemia virus (MoMLV), although they can be infected by other ecotropic murine leukemia viruses, including Friend MLV and Rauscher MLV. The relative inability of MoMLV to replicate in M. dunni cells has been attributed to two amino acids (V214 and G236) located within the third extracellular loop of the M. dunni CAT1 receptor (dCAT1). Via the exchange of the third extracellular loop of the mCAT1 cDNA encoding receptor from the permissive mouse and the corresponding portion of cDNA encoding for the nonpermissive M. dunni receptor, we have identified the most critical amino acid residue, which is a glycine located at position 236 within the third extracellular loop of dCAT1. We also attempted to determine the role of the third extracellular loop of the M. dunni CAT1 receptor with regard to the formation of the syncytium. The relationship between dCAT1 and virus-induced syncytia was suggested initially by our previous identification of two MLV isolates (S82F in Moloney and S84A in Friend MLV), both of which are uniquely cytopathic in M. dunni cells. In an attempt to determine the relationship existing between dCAT1 and the virally-induced syncytia, we infected 293-dCAT1 or chimeric dCAT1 cells with the S82F pseudotype virus. The S82F pseudotype virus did not induce the formation of syncytia, but did show increased susceptibility to 293 cells expressing dCAT1. The results of our study indicate that S82F-induced syncytium formation may be the result of cell-cell fusion, but not virus-cell fusion.

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