Cell Fusion Activity Research Articles

Development and Characterization of an Oncolytic Human Adenovirus-Based Vector Co-Expressing the Adenovirus Death Protein and p14 Fusion-Associated Small Transmembrane Fusogenic Protein.

Human adenovirus (HAdV)-based oncolytic vectors, which are designed to preferentially replicate in and kill cancer cells, have shown modest efficacy in human clinical trials in part due to poor viral distribution throughout the tumor mass. Previously, we showed that expression of the p14 fusion-associated small transmembrane (FAST) fusogenic protein could enhance oncolytic HAdV efficacy and reduce tumor growth rate in a human xenograft mouse model of cancer. We now explore whether co-expression of the adenovirus death protein (ADP) with p14 FAST protein could synergize to further enhance oncolytic vector efficacy. ADP is naturally encoded within the early region 3 (E3) of HAdV, a region which is frequently removed from HAdV-based vectors, and functions to enhance cell lysis and progeny release. We evaluated a variety of approaches to achieve optimal expression of the two proteins, the most efficient method being insertion of an expression cassette within the E3 deletion, consisting of the coding sequences for p14 FAST protein and ADP separated by a self-cleaving peptide derived from the porcine teschovirus-1 (P2A). However, the quantities of p14 FAST protein and ADP produced from this vector were reduced approximately 10-fold compared to a similar vector-expressing only p14 FAST protein and wildtype HAdV, respectively. Compared to our original oncolytic vector-expressing p14 FAST protein alone, reduced expression of p14 FAST protein and ADP from the P2A construct reduced cell-cell fusion, vector spread, and cell-killing activity in human A549 adenocarcinoma cells in culture. These studies show that a self-cleaving peptide can be used to express two different transgenes in an armed oncolytic HAdV vector, but also highlight the challenges in maintaining adequate transgene expression when modifying vector design.

Effects of polystyrene nano- and microplastics on human breast epithelial cells and human breast cancer cells

The continuous littering of the environment with plastic and the resulting nano- and microplastics produced from various processes are ever-increasing problems. These materials also affect humans, as the absorption and accumulation of nano- and microplastics and their effects on health have thus far been rarely researched, which also applies to cancer. In the present study, the absorption of different sizes of polystyrene (PS) nano- and microplastics (PS particles) into human breast epithelial cells and human breast cancer cells was investigated. Subsequently, how the proliferation, colony and mammosphere formation abilities, cell fusion and migration of the cells were influenced by the PS particles were investigated. Our data revealed granularity-, dose- and cell line-dependent absorption of the PS particles, with the highest absorption observed in the MDA-MB-231-DSP1-7 cells and the lowest in the M13SV1_Syn1-DSP8-11 cells. Neither the colony-forming ability nor the cell fusion activity increased with the addition of PS particles. In contrast, slight, partially significant stimulatory effects on both proliferation and cell migration were observed, although these effects depended on the particle quantity and size and the cell line used. In summary, PS particles are absorbed by human breast epithelial and human breast cancer cells and influence cells that may be associated with cancer progression.

Herpes Simplex Virus 1 Glycoprotein B from a Hyperfusogenic Virus Mediates Enhanced Cell-Cell Fusion.

Herpes simplex virus 1 (HSV-1) causes significant morbidity and death in humans worldwide. Herpes simplex virus 1 has a complex fusion mechanism that is incompletely understood. The HSV-1 strain ANG has notable fusion and entry activities that distinguish it from wild type. HSV-1 ANG virions fused with the Vero cell surface at 4 °C and also entered cells more efficiently at 15 °C, relative to wild type HSV-1 strain KOS virions, consistent with a hyperfusogenic phenotype. Understanding the molecular basis for the unique entry and fusion activities of HSV-1 strain ANG will help decipher the HSV fusion reaction and entry process. Sequencing of HSV-1 ANG genes revealed multiple changes in gB, gC, gD, gH, and gL proteins relative to wild type HSV-1 strains. The ANG UL45 gene sequence, which codes for a non-essential envelope protein, was identical to wild type KOS. HSV-1 ANG gB, gD, and gH/gL were necessary and sufficient to mediate cell-cell fusion in a virus-free reporter assay. ANG gB, when expressed with wild type KOS gD and gH/gL, increased membrane fusion, suggesting that ANG gB has hyperfusogenic cell-cell fusion activity. Replacing the KOS gD, gH, or gL with the corresponding ANG alleles did not enhance cell-cell fusion. The novel mutations in the ANG fusion and entry glycoproteins provide a platform for dissecting the cascade of interactions that culminate in HSV fusion and entry.

Histone deacetylase inhibitor boosts anticancer potential of fusogenic oncolytic vaccinia virus by enhancing cell-cell fusion.

Oncolytic viruses have two anticancer functions: direct oncolysis and elicitation of antitumor immunity. We previously developed a novel fusogenic oncolytic vaccinia virus (FUVAC) from a non-fusogenic vaccinia virus (VV) and, by remodeling the tumor immune microenvironment, we demonstrated that FUVAC induced stronger oncolysis and antitumor immune responses compared with non-fusogenic VV. These functions depend strongly on cell-cell fusion induction. However, FUVAC tends to have decreased fusion activity in cells with low virus replication efficacy. Therefore, another combination strategy was required to increase cell-cell fusion in these cells. Histone deacetylase (HDAC) inhibitors suppress the host virus defense response and promote viral replication. Therefore, in this study, we selected an HDAC inhibitor, trichostatin A (TSA), as the combination agent for FUVAC to enhance its fusion-based antitumor potential. TSA was added prior to FUVAC treatment of murine tumor B16-F10 and CT26 cells. TSA increased the replication of both FUVAC and parental non-fusogenic VV. Moreover, TSA enhanced cell-cell fusion and FUVAC cytotoxicity in these tumor cells in a dose-dependent manner. Transcriptome analysis revealed that TSA-treated tumors showed altered expression of cellular component-related genes, which may affect fusion tolerance. In a bilateral tumor-bearing mouse model, combination treatment of TSA and FUVAC significantly prolonged mouse survival compared with either treatment alone or in combination with non-fusogenic VV. Our findings demonstrate that TSA is a potent enhancer of cell-cell fusion efficacy of FUVAC.

Green tea polyphenol EGCg induces cell fusion via reactive oxygen species

BackgroundOsteoclasts are multinucleated cells formed by macrophage cell fusion that are responsible for bone resorption. Previously, we found that treating osteoclastic progenitor cells with (−)-epigallocatechin gallate (EGCg) increased cell fusion. In this study, we aimed to identify factors involved in the cell fusion induced by EGCg. MethodsWe hypothesized that EGCg-induced oxidative stress might be involved in cell fusion, and used macrophage cell line RAW264.7 cells. We evaluated cell fusion activity after adding the antioxidants N-acetyl-l-cysteine (NAC) or catalase in addition to EGCg. The mRNA expressions of genes related to cell fusion and bone resorption were quantified by real-time PCR. Finally, we added hydrogen peroxide and examined its effects on cell fusion and TRAP activity. ResultsEGCg-induced cell fusion was strongly inhibited by the addition of NAC in a dose-dependent manner (EGCg with 5 mM NAC; decreased to 1.5%; p < 0.05), while the inhibitory effect of catalase was limited (EGCg with 500 U/mL catalase; decreased to 27.7%; p < 0.05). DC-STAMP expression was significantly upregulated by EGCg compared with the untreated group, and the upregulation was significantly suppressed by 5 mM NAC. Conversely, Nfatc1 and TRAP expression were not upregulated by EGCg. These results suggest that EGCg induces DC-STAMP expression via reactive oxygen species production, which regulates cell fusion but does not affect the osteoclastic pathway. Although treatment with hydrogen peroxide promoted the formation of multinucleated cells, no increase in TRAP activity was observed, which was similar to EGCg treatment. ConclusionsThis study suggests that the increased cell fusion by EGCg may be induced by oxidative stress due to reactive oxygen species production.

Inhibitors of cannabinoid receptor 1 suppress the cellular entry of Lujo virus

Lujo virus (LUJV), which belongs to Mammarenavirus, family Arenaviridae, has emerged as a pathogen causing severe hemorrhagic fever with high mortality. Currently, there are no effective treatments for arenaviruses, including LUJV. Here, we screened chemical compound libraries of Food and Drug Administration (FDA)-approved drugs and G protein-coupled receptor-associated drugs to identify effective antivirals against LUJV targeting cell entry using a vesicular stomatitis virus-based pseudotyped virus bearing the LUJV envelope glycoprotein (GP). Cannabinoid receptor 1 (CB1) antagonists, such as rimonabant, AM251 and AM281, have been identified as robust inhibitors of LUJV entry. The IC50 of rimonabant was 0.26 and 0.53 μM in Vero and Huh7 cells, respectively. Analysis of the cell fusion activity of the LUJV GP in the presence of CB1 inhibitors revealed that these inhibitors suppressed the fusion activity of the LUJV GP. Moreover, rimonabant, AM251 and AM281 reduced the infectivity of authentic LUJV in vitro, suggesting that the antiviral activity of CB1 antagonists against LUJV is mediated, at least in part, by inhibition of the viral entry, especially, membrane fusion. These findings suggest promising candidates for developing new therapies against LUJV infections.

SARS-CoV-2 Spike Protein Is Capable of Inducing Cell-Cell Fusions Independent from Its Receptor ACE2 and This Activity Can Be Impaired by Furin Inhibitors or a Subset of Monoclonal Antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was responsible for the COVID-19 pandemic, efficiently spreads cell-to-cell through mechanisms facilitated by its membrane glycoprotein spike. We established a dual split protein (DSP) assay based on the complementation of GFP and luciferase to quantify the fusogenic activity of the SARS-CoV-2 spike protein. We provide several lines of evidence that the spike protein of SARS-CoV-2, but not SARS-CoV-1, induced cell-cell fusion even in the absence of its receptor, angiotensin-converting enzyme 2 (ACE2). This poorly described ACE2-independent cell fusion activity of the spike protein was strictly dependent on the proteasomal cleavage of the spike by furin while TMPRSS2 was dispensable. Previous and current variants of concern (VOCs) differed significantly in their fusogenicity. The Delta spike was extremely potent compared to Alpha, Beta, Gamma and Kappa, while the Omicron spike was almost devoid of receptor-independent fusion activity. Nonetheless, for all analyzed variants, cell fusion was dependent on furin cleavage and could be pharmacologically inhibited with CMK. Mapping studies revealed that amino acids 652-1273 conferred the ACE2-independent fusion activity of the spike. Unexpectedly, residues proximal to the furin cleavage site were not of major relevance, whereas residue 655 critically regulated fusion. Finally, we found that the spike's fusion activity in the absence of ACE2 could be inhibited by antibodies directed against its N-terminal domain (NTD) but not by antibodies targeting its receptor-binding domain (RBD). In conclusion, our BSL-1-compatible DSP assay allowed us to screen for inhibitors or antibodies that interfere with the spike's fusogenic activity and may therefore contribute to both rational vaccine design and development of novel treatment options against SARS-CoV-2.

Histochemical assessment on osteoclasts in long bones of toll-like receptor 2 (TLR2) deficient mice

ObjectiveToll-like receptor 2 (TLR2), recognizes a wide variety of pathogen-associated molecular patterns such as lipopolysaccharides, peptidoglycans, and lipopeptides, and is generally believed to be present in monocytes, macrophages, dendritic cells, and vascular endothelial cells. However, no histological examination of osteoclasts, which differentiate from precursors common to macrophages/monocytes, has been performed in a non-infected state of TLR2 deficiency. The objective of this study was to examine the histological properties and function of osteoclasts in the long bones of 8-week-old male TLR2 deficient (TLR2−/−) mice to gain insight into TLR2 function in biological circumstances without microbial infection. MethodsEight-week-old male wild-type and TLR2−/− mice were fixed with paraformaldehyde solution, and their tibiae and femora were used for micro-CT analysis, immunohistochemistry, transmission electron microscopy, and real-time PCR analysis. ResultsTLR2−/− tibiae and femora exhibited increased bone volume of metaphyseal trabeculae and elevated numbers of TRAP-positive osteoclasts. However, the number of multinucleated TRAP-positive osteoclasts was reduced, whereas mononuclear TRAP-positive cells increased, despite the high expression levels of Dc-Stamp and Oc-Stamp. Although TRAP-positive multinucleated and mononuclear osteoclasts showed the immunoreactivity and elevated expression of RANK and siglec-15, they revealed weak cathepsin K-positivity and less incorporation of the mineralized bone matrix, and often missing ruffled borders. It seemed likely that, despite the increased numbers, TLR2−/− osteoclasts reduced cell fusion and bone resorption activity. ConclusionIt seems likely that even without bacterial infection, TLR2 might participate in cell fusion and subsequent bone resorption of osteoclasts.

Potentially reduced fusogenicity of syncytin-2 in New World monkeys.

Syncytin-2 is a membrane fusion protein involved in placenta development that is derived from the endogenous retrovirus envelope gene acquired in the common ancestral lineage of New World and Old World monkeys (OWMs). It is known that syncytin-2 is conserved between apes and OWMs, suggesting its functional importance; however, syncytin-2 of common marmosets (Callithrix jacchus) exhibits lower fusogenic activity than those of humans and OWMs in human cell lines. To obtain insight into the functional diversity of syncytin-2 genes in primates, we examined the syncytin-2 gene in New World monkeys (NWMs). We experimentally evaluated the cell fusion ability of syncytin-2 in humans, C.jacchus, and tufted capuchins (Sapajus apella). We found that the cell fusion ability of S.apella was lower than that of human syncytin-2. Chimeric syncytin-2 constructs revealed that the amino acid differences in the surface unit of S.apella syncytin-2 were responsible for the weak cell fusion activity. In addition, genomic sequence analyses of syncytin-2 revealed that the open reading frames (ORFs) of syncytin-2 were highly conserved in seven apes and 22 OWMs; however, the syncytin-2 ORFs of three of 12 NWM species were truncated. Our results suggest that syncytin-2 in several NWMs may be of less importance than in OWMs and apes, and other syncytin-like genes may be required for placental development in various NWM species.

Broad Tricyclic Ring Inhibitors Block SARS-CoV-2 Spike Function Required for Viral Entry.

The entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells requires binding of the viral spike glycoprotein to the angiotensin-converting enzyme 2 (ACE2) receptor, which triggers subsequent conformational changes to facilitate viral and cellular fusion at the plasma membrane or following endocytosis. Here, we experimentally identified selective and broad inhibitors of SARS-CoV-2 entry that share a tricyclic ring (or similar) structure. The inhibitory effect was restricted to early steps during infection and the entry inhibitors interacted with the receptor binding domain of the SARS-CoV-2 spike but did not significantly interfere with receptor (ACE2) binding. Instead, some of these compounds induced conformational changes or affected spike assembly and blocked SARS-CoV-2 spike cell–cell fusion activity. The broad inhibitors define a highly conserved binding pocket that is present on the spikes of SARS-CoV-1, SARS-CoV-2, and all circulating SARS-CoV-2 variants tested and block SARS-CoV spike activity required for mediating viral entry. These compounds provide new insights into the SARS-CoV-2 spike topography, as well as into critical steps on the entry pathway, and can serve as lead candidates for the development of broad-range entry inhibitors against SARS-CoVs.

Glycan shield of the ebolavirus envelope glycoprotein GP

The envelope glycoprotein GP of the ebolaviruses is essential for host cell entry and the primary target of the host antibody response. GP is heavily glycosylated with up to 17 N-linked sites, numerous O-linked glycans in its disordered mucin-like domain (MLD), and three predicted C-linked mannosylation sites. Glycosylation is important for host cell attachment, GP stability and fusion activity, and shielding from neutralization by serum antibodies. Here, we use glycoproteomics to profile the site-specific glycosylation patterns of ebolavirus GP. We detect up to 16 unique O-linked glycosylation sites in the MLD, and two O-linked sites in the receptor-binding GP1 subunit. Multiple O-linked glycans are observed within N-linked glycosylation sequons, suggesting crosstalk between the two types of modifications. We confirmed C-mannosylation of W288 in full-length trimeric GP. We find complex glycosylation at the majority of N-linked sites, while the conserved sites N257 and especially N563 are enriched in unprocessed glycans, suggesting a role in host-cell attachment via DC-SIGN/L-SIGN. Our findings illustrate how N-, O-, and C-linked glycans together build the heterogeneous glycan shield of GP, guiding future immunological studies and functional interpretation of ebolavirus GP-antibody interactions.

SARS-CoV-2 fusion-inhibitory lipopeptides maintain high potency against divergent variants of concern including Omicron

ABSTRACT The emergence of SARS-CoV-2 Omicron and other variants of concern (VOCs) has brought huge challenges to control the COVID-19 pandemic, calling for urgent development of effective vaccines and therapeutic drugs. In this study, we focused on characterizing the impacts of divergent VOCs on the antiviral activity of lipopeptide-based fusion inhibitors that we previously developed. First, we found that pseudoviruses bearing the S proteins of five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) and one variant of interest (Lambda) exhibited greatly decreased infectivity relative to the wild-type (WT) strain or single D614G mutant, especially the Omicron pseudovirus. Differently, the most of variants exhibited an S protein with significantly enhanced cell fusion activity, whereas the S protein of Omicron still mediated decreased cell–cell fusion. Next, we verified that two lipopeptide-based fusion inhibitors, IPB02V3 and IPB24, maintained the highly potent activities in inhibiting various S proteins-driven cell fusion and pseudovirus infection. Surprisingly, both IPB02V3 and IPB24 lipopeptides displayed greatly increased potencies against the infection of authentic Omicron strain relative to the WT virus. The results suggest that Omicron variant evolves with a reduced cell fusion capacity and is more sensitive to the inhibition of fusion-inhibitory lipopeptides; thus, IPB02V3 and IPB24 can be further developed as potent, broad-spectrum antivirals for combating Omicron and the potential future outbreak of other emerging variants.

RANKL interferes with osteoclastogenesis in PEG-fused U937 cells through LGR4

ABSTRACT Introduction RANKL plays an important role in the differentiation and maturation process of preosteoclast cells. The osteoclast is a multinucleated cell that can have various sizes and a variable number of nuclei. However, there are no models that allow us to understand how successive cell fusions have a limit, or how cell fusion is regulated. Methodology The present investigation was aimed to determine whether fusing U937 cells with PEG to generate osteoclast-like cells expresses LGR4 and whether applying RANKL to these cells modifies osteoclastic activity compared to non-PEG-fused and RANKL-treated cells. Results By fusing U937 cells with PEG, it was found that the LGR4 receptor expression was promoted as early as 24 hours of culture. Applying RANKL before or after fusion inhibits osteoclastic activity. Interfering RANKL interaction with LGR4 in PEG-treated cells recovers and increases cell fusion and osteoclastic activity. PEG-fused U937 cells show osteoclast markers similar to those observed in the classical RANKL-stimulated cell model. Conclusion Our model allows us to understand that RANKL has fusogenic activity during the first days of culture and in fused cells modulates fusion, contributing to differentiate the role of RANKL before and after fusion through LGR4.

The nonstructural p17 protein of a fusogenic bat-borne reovirus regulates viral replication in virus species- and host-specific manners

Nelson Bay orthoreovirus (NBV), a member of the family Reoviridae, genus Orthoreovirus, is a bat-borne virus that causes respiratory diseases in humans. NBV encodes two unique nonstructural proteins, fusion-associated small transmembrane (FAST) protein and p17 protein, in the S1 gene segment. FAST induces cell–cell fusion between infected cells and neighboring cells and the fusogenic activity is required for efficient viral replication. However, the function of p17 in the virus cycle is not fully understood. Here, various p17 mutant viruses including p17-deficient viruses were generated by a reverse genetics system for NBV. The results demonstrated that p17 is not essential for viral replication and does not play an important role in viral pathogenesis. On the other hand, NBV p17 regulated viral replication in a bat cell line but not in other human and animal cell lines. Nuclear localization of p17 is associated with the regulation of NBV replication in bat cells. We also found that p17 dramatically enhances the cell–cell fusion activity of NBV FAST protein for efficient replication in bat cells. Furthermore, we found that a protein homologue of NBV p17 from another bat-borne orthoreovirus, but not those of avian orthoreovirus or baboon orthoreovirus, also supported efficient viral replication in bat cells using a p17-deficient virus-based complementation approach. These results provide critical insights into the functioning of the unique replication machinery of bat-borne viruses in their natural hosts.

SARS-CoV-2 Omicron variant shows less efficient replication and fusion activity when compared with Delta variant in TMPRSS2-expressed cells

ABSTRACT The novel SARS-CoV-2 Omicron variant (B.1.1.529), first found in early November 2021, has sparked considerable global concern and it has >50 mutations, many of which are known to affect transmissibility or cause immune escape. In this study, we sought to investigate the virological characteristics of the Omicron variant and compared it with the Delta variant which has dominated the world since mid-2021. Omicron variant replicated more slowly than the Delta variant in transmembrane serine protease 2 (TMPRSS2)-overexpressing VeroE6 (VeroE6/TMPRSS2) cells. Notably, the Delta variant replicated well in Calu3 cell line which has robust TMPRSS2 expression, while the Omicron variant replicated poorly in this cell line. Competition assay showed that Delta variant outcompeted Omicron variant in VeroE6/TMPRSS2 and Calu3 cells. To confirm the difference in entry pathway between the Omicron and Delta variants, we assessed the antiviral effect of bafilomycin A1, chloroquine (inhibiting endocytic pathway), and camostat (inhibiting TMPRSS2 pathway). Camostat potently inhibited the Delta variant but not the Omicron variant, while bafilomycin A1 and chloroquine could inhibit both Omicron and Delta variants. Moreover, the Omicron variant also showed weaker cell–cell fusion activity when compared with Delta variant in VeroE6/TMPRSS2 cells. Collectively, our results suggest that Omicron variant infection is not enhanced by TMPRSS2 but is largely mediated via the endocytic pathway. The difference in entry pathway between Omicron and Delta variants may have an implication on the clinical manifestations or disease severity.

Weak Electromagnetic Fields Accelerate Fusion of Myoblasts.

Weak electromagnetic fields (WEF) alter Ca2+ handling in skeletal muscle myotubes. Owing to the involvement of Ca2+ in muscle development, we investigated whether WEF affects fusion of myoblasts in culture. Rat primary myoblast cultures were exposed to WEF (1.75 µT, 16 Hz) for up to six days. Under control conditions, cell fusion and creatine kinase (CK) activity increased in parallel and peaked at 4–6 days. WEF enhanced the extent of fusion after one and two days (by ~40%) vs. control, but not thereafter. Exposure to WEF also enhanced CK activity after two days (almost four-fold), but not afterwards. Incorporation of 3H-thymidine into DNA was enhanced by one-day exposure to WEF (~40%), indicating increased cell replication. Using the potentiometric fluorescent dye di-8-ANEPPS, we found that exposure of cells to 150 mM KCl resulted in depolarization of the cell membrane. However, prior exposure of cells to WEF for one day followed by addition of KCl resulted in hyperpolarization of the cell membrane. Acute exposure of cells to WEF also resulted in hyperpolarization of the cell membrane. Twenty-four hour incubation of myoblasts with gambogic acid, an inhibitor of the inward rectifying K+ channel 2.1 (Kir2.1), did not affect cell fusion, WEF-mediated acceleration of fusion or hyperpolarization. These data demonstrate that WEF accelerates fusion of myoblasts, resulting in myotube formation. The WEF effect is associated with hyperpolarization but WEF does not appear to mediate its effects on fusion by activating Kir2.1 channels.

Comparison of Growth Characteristics and Genomics of Two Canine Distemper Virus Strains Isolated From Minks in China.

Canine distemper (CD), caused by the CDV variant strain with HI542N/Y549H, has become an epidemic in fur-bearing animals in China since 2012. To well understand the genomic and replicated characteristics of the CDV variants, we determined the viral growth kinetics and completed the genome sequences of two CDV strains, namely SDZC(17)M2 and LNDL(17)M4, isolated from CDV-infected minks from Shandong and Liaoning province in China, in 2017. SDZC(17)M2 showed higher viral titers and extensive syncytia in BHK-minkSLAM (BMS) cells than LNDL(17)M4. Although both two strains belong to the Asia-1 genotype and clustered an independent clade in the phylogenetic tree, SDZC(17)M2, harboring I542N/Y549H substitutions in the H protein, shared high identity (99.3–99.6% nt) with the other variant strains, whereas LNDL(17)M4, with the only Y549H substitution, shared a lower identity (97.7%–97.9% nt) with the other variant strains. Furthermore, a novel R223K substitution was identified in the conserved cleavage site (RRQRR → RRQKR) of the F protein in the SDZC(17)M2 strain. However, it which did not significantly affect the cell to cell fusion activity when combined with the CDV H/minkSLAM in BHK-21 cells. The key variations in the genome contributed to the virulence and the evolutionary trend need to be determined in the future.

A single mutation in Crimean-Congo hemorrhagic fever virus discovered in ticks impairs infectivity in human cells.

Crimean-Congo hemorrhagic fever (CCHF) is the most widely distributed tick-borne viral infection in the world. Strikingly, reported mortality rates for CCHF are extremely variable, ranging from 5% to 80% (Whitehouse, 2004). CCHF virus (CCHFV, Nairoviridae) exhibits extensive genomic sequence diversity across strains (Deyde et al., 2006; Sherifi et al., 2014). It is currently unknown if genomic diversity is a factor contributing to variation in its pathogenicity. We obtained complete genome sequences of CCHFV directly from the tick reservoir. These new strains belong to a solitary lineage named Europe 2 that is circumstantially reputed to be less pathogenic than the epidemic strains from Europe 1 lineage. We identified a single tick-specific amino acid variant in the viral glycoprotein region that dramatically reduces its fusion activity in human cells, providing evidence that a glycoprotein precursor variant, present in ticks, has severely impaired function in human cells.

Tetraploidization of Immortalized Myoblasts Induced by Cell Fusion Drives Myogenic Sarcoma Development with DMD Deletion.

Whole-genome doubling is the second most frequent genomic event, after TP53 alterations, in advanced solid tumors and is associated with poor prognosis. Tetraploidization step will lead to aneuploidy and chromosomic rearrangements. The mechanism leading to tetraploid cells is important since endoreplication, abortive cytokinesis and cell fusion could have distinct consequences. Unlike processes based on duplication, cell fusion involves the merging of two different genomes, epigenomes and cellular states. Since it is involved in muscle differentiation, we hypothesized that it could play a role in the oncogenesis of myogenic cancers. Spontaneous hybrids, but not their non-fused immortalized myoblast counterparts they are generated from, induced tumors in mice. Unstable upon fusion, the hybrid genome evolved from initial mitosis to tumors with a highly rearranged genome. This genome remodeling finally produced targeted DMD deletions associated with replicative stress, isoform relocalization and metastatic spreading, exactly as observed in human myogenic sarcomas. In conclusion, these results draw a model of myogenic oncogenesis in which cell fusion and oncogene activation combine to produce pleomorphic aggressive sarcomas.

Effects of the SOS (A501C/T605C) and DS (I201C/A433C) Disulfide Bonds on HIV-1 Membrane Envelope Glycoprotein Conformation and Function.

Most broadly neutralizing antibodies and many entry inhibitors target the pretriggered (state 1) conformation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env). Here we examine two previously reported Env mutants designed to be stabilized in this conformation by the introduction of artificial disulfide bonds: A501C/T605C (called SOS) and I201C/A433C (called DS). SOS Env supported virus entry and cell-cell fusion only after exposure to a reducing agent, dithiothreitol (DTT). Deletion of the Env cytoplasmic tail improved the efficiency with which the SOS Env supported virus infection in a reducing environment. The antigenicity of the SOS Env was similar to that of the unmodified Env, except for greater sensitivity to some state 1-preferring ligands. In contrast, viruses with the DS Env were not infectious, even after DTT treatment. The proteolytic maturation of the DS Env on both cell surfaces and virions was severely compromised compared with that of the unmodified Env. The DS Env exhibited detectable cell-fusing activity when DTT was present. However, the profiles of cell-surface Env recognition and cell-cell fusion inhibition by antibodies differed for the DS Env and the unmodified Env. Thus, the DS Env appears to be stabilized in an off-pathway conformation that is nonfunctional on the virus. The SOS change exerted more subtle, context-dependent effects on Env conformation and function.IMPORTANCE The human immunodeficiency virus type 1 (HIV-1) envelope proteins (Envs) bind receptors on the host cell and change shape to allow the virus to enter the cell. Most virus-inhibiting antibodies and drugs recognize a particular shape of Env called state 1. Disulfide bonds formed by cysteine residues have been introduced into soluble forms of the flexible envelope proteins in an attempt to lock them into state 1 for use in vaccines and as research tools. We evaluated the effect of these cysteine substitutions on the ability of the membrane Env to support virus entry and on susceptibility to inhibition by antibodies and small molecules. We found that the conformation of the envelope proteins with the cysteine substitutions differed from that of the unmodified membrane envelope proteins. Awareness of these effects can assist efforts to create stable HIV-1 Env complexes that more closely resemble the state 1 conformation.