Phenol Derivatives Obtained from Grape Seed Extract Show Virucidal Activity against Murine Norovirus.

Human noroviruses are the most common pathogens known to cause acute gastroenteritis, a condition that can lead to severe illness among immunocompromised individuals such as organ transplant recipients and the elderly. To date, no safe and effective vaccines or therapeutic agents have been approved for treating norovirus infections. Therefore, we aimed to demonstrate the virucidal activity of grape seed extract (GSE), which contains >83% proanthocyanidins, against murine norovirus (MNV), a surrogate for human norovirus. GSE showed virucidal activity against MNV in a dose- and time-dependent manner. Atomic force microscopic analysis showed viral particle aggregates after treatment of MNV with GSE. MNV treated with 50 µg/mL of GSE for 10 min resulted in the absence of pathogenicity in an animal model of infection, indicating that GSE has irreversible virucidal activity against MNV particles. Thus, GSE may aid in the development of treatments for norovirus infections.

Characterization of H5N1 Influenza Virus Quasispecies with Adaptive Hemagglutinin Mutations from Single-Virus Infections of Human Airway Cells.

Transmission of avian influenza (AI) viruses to mammals involves phylogenetic bottlenecks that select small numbers of variants for transmission to new host species. However, little is known about the AI virus quasispecies diversity that produces variants for virus adaptation to humans. Here, we analyzed the hemagglutinin (HA) genetic diversity produced during AI H5N1 single-virus infection of primary human airway cells and characterized the phenotypes of these variants. During single-virus infection, HA variants emerged with increased fitness to infect human cells. These variants generally had decreased HA thermostability, an indicator of decreased transmissibility, that appeared to compensate for their increase in α2,6-linked sialic acid (α2,6 Sia) binding specificity and/or in the membrane fusion pH threshold, each of which is an advantageous mutational change for viral infection of human airway epithelia. An HA variant with increased HA thermostability also emerged but could not outcompete variants with less HA thermostability. These results provided data on HA quasispecies diversity in human airway cells.IMPORTANCE The diversity of the influenza virus quasispecies that emerges from a single infection is the starting point for viral adaptation to new hosts. A few studies have investigated AI virus quasispecies diversity during human adaptation using clinical samples. However, those studies could be appreciably affected by individual variability and multifactorial respiratory factors, which complicate identification of quasispecies diversity produced by selective pressure for increased adaptation to infect human airway cells. Here, we found that detectable HA genetic diversity was produced by H5N1 single-virus infection of human airway cells. Most of the HA variants had increased fitness to infect human airway cells but incurred a fitness cost of less HA stability. To our knowledge, this is the first report to characterize the adaptive changes of AI virus quasispecies produced by infection of human airway cells. These results provide a better perspective on AI virus adaptation to infect humans.

Trisaccharide containing α2,3-linked sialic acid is a receptor for mumps virus.

Mumps virus (MuV) remains an important pathogen worldwide, causing epidemic parotitis, orchitis, meningitis, and encephalitis. Here we show that MuV preferentially uses a trisaccharide containing α2,3-linked sialic acid in unbranched sugar chains as a receptor. Crystal structures of the MuV attachment protein hemagglutinin-neuraminidase (MuV-HN) alone and in complex with the α2,3-sialylated trisaccharide revealed that in addition to the interaction between the MuV-HN active site residues and sialic acid, other residues, including an aromatic residue, stabilize the third sugar of the trisaccharide. The importance of the aromatic residue and the third sugar in the MuV-HN-receptor interaction was confirmed by computational energy calculations, isothermal titration calorimetry studies, and glycan-binding assays. Furthermore, MuV-HN was found to bind more efficiently to unbranched α2,3-sialylated sugar chains compared with branched ones. Importantly, the strategically located aromatic residue is conserved among the HN proteins of sialic acid-using paramyxoviruses, and alanine substitution compromised their ability to support cell-cell fusion. These results suggest that not only the terminal sialic acid but also the adjacent sugar moiety contribute to receptor function for mumps and these paramyxoviruses. The distribution of structurally different sialylated glycans in tissues and organs may explain in part MuV's distinct tropism to glandular tissues and the central nervous system. In the crystal structure, the epitopes for neutralizing antibodies are located around the α-helices of MuV-HN that are not well conserved in amino acid sequences among different genotypes of MuV. This may explain the fact that MuV reinfection sometimes occurs.

6SLN-lipo PGA specifically catches (coats) human influenza virus and synergizes neuraminidase-targeting drugs for human influenza therapeutic potential.

OBJECTIVES: The purpose of this study was to develop a new compound to overcome influenza epidemics and pandemics as well as drug resistance. METHODS: We synthesized a new compound carrying: (i) Neu5Acα2-6Galß1-4GlcNAc (6SLN) for targeting immutable haemagglutinins (HAs) unless switched from human-type receptor preference; (ii) an acyl chain (lipo) for locking the compound with the viral HA via hydrophobic interactions; and (iii) a flexible poly-α-L-glutamic acid (PGA) for enhancing the compound solubility and for coating the viral surface, precluding accessibility of the PGA-coated virus to the negatively charged sialic acid on the host cell surface. RESULTS: 6SLN-lipo PGA appears to subvert binding of pandemic H1 and seasonal H3 HAs to receptors, as assessed by using guinea pig erythrocytes, which is critical for virus entry into host cells for multiplication. It shows high potency with IC50 values in the range of 300-500 nM against multiplication of both influenza pandemic H1N1/2009 and seasonal H3N2/2004 viruses in cell culture. It acts in synergism with either of the two FDA-approved neuraminidase inhibitor (NAI) clinical drugs, zanamivir (Relenza(®)) and oseltamivir carboxylate (active form of Tamiflu(®)), and it has the potential to aid NAI drugs to achieve complete clearance of the virus from the culture. CONCLUSIONS: 6SLN-lipo PGA is a new potential candidate drug for influenza control and is an attractive candidate for use in combination with an NAI drug for minimized toxicity, delayed development of resistance, prevention and treatment with the potential for eradication of human influenza.

Antigenic and receptor binding properties of enterovirus 68.

UNLABELLED: Increased detection of enterovirus 68 (EV68) among patients with acute respiratory infections has been reported from different parts of the world in the late 2000s since its first detection in pediatric patients with lower-respiratory-tract infections in 1962. However, the underlying molecular mechanisms for this trend are still unknown. We therefore aimed to study the antigenicity and receptor binding properties of EV68 detected in recent years in comparison to the prototype strain of EV68, the Fermon strain. We first performed neutralization (NT) and hemagglutination inhibition (HI) tests using antisera generated for EV68 strains detected in recent years. We found that the Fermon strain had lower HI and NT titers than recently detected EV68 strains. The HI and NT titers were also significantly different between strains of different genetic lineages among recently detected EV68 strains. We further studied receptor binding specificities of EV68 strains for sialyloligosaccharides using glycan array analysis. In glycan array analysis, all tested EV68 strains showed affinity for α2-6-linked sialic acids (α2-6 SAs) compared to α2-3 SAs. Our study demonstrates that emergence of strains with different antigenicity is the possible reason for the increased detection of EV68 in recent years. Additionally, we found that EV68 preferably binds to α2-6 SAs, which suggests that EV68 might have affinity for the upper respiratory tract. IMPORTANCE: Numbers of cases of enterovirus 68 (EV68) infection in different parts of the world increased significantly in the late 2000s. We studied the antigenicity and receptor binding properties of recently detected EV68 strains in comparison to the prototype strain of EV68, Fermon. The hemagglutination inhibition (HI) and neutralization (NT) titers were significantly different between strains of different genetic lineages among recently detected EV68 strains. We further studied receptor binding specificities of EV68 strains for sialyloligosaccharides using glycan array analysis, which showed affinity for α2-6-linked sialic acids (α2-6 SAs) compared to α2-3 SAs. Our study suggested that the emergence of strains with different antigenicities was the possible reason for the increased detections of EV68 in recent years. Additionally, we revealed that EV68 preferably binds to α2-6 SAs. This is the first report describing the properties of EV68 receptor binding to the specific types of sialic acids.

H6 influenza viruses pose a potential threat to human health.

UNLABELLED: Influenza viruses of the H6 subtype have been isolated from wild and domestic aquatic and terrestrial avian species throughout the world since their first detection in a turkey in Massachusetts in 1965. Since 1997, H6 viruses with different neuraminidase (NA) subtypes have been detected frequently in the live poultry markets of southern China. Although sequence information has been gathered over the last few years, the H6 viruses have not been fully biologically characterized. To investigate the potential risk posed by H6 viruses to humans, here we assessed the receptor-binding preference, replication, and transmissibility in mammals of a series of H6 viruses isolated from live poultry markets in southern China from 2008 to 2011. Among the 257 H6 strains tested, 87 viruses recognized the human type receptor. Genome sequence analysis of 38 representative H6 viruses revealed 30 different genotypes, indicating that these viruses are actively circulating and reassorting in nature. Thirty-seven of 38 viruses tested in mice replicated efficiently in the lungs and some caused mild disease; none, however, were lethal. We also tested the direct contact transmission of 10 H6 viruses in guinea pigs and found that 5 viruses did not transmit to the contact animals, 3 viruses transmitted to one of the three contact animals, and 2 viruses transmitted to all three contact animals. Our study demonstrates that the H6 avian influenza viruses pose a clear threat to human health and emphasizes the need for continued surveillance and evaluation of the H6 influenza viruses circulating in nature. IMPORTANCE: Avian influenza viruses continue to present a challenge to human health. Research and pandemic preparedness have largely focused on the H5 and H7 subtype influenza viruses in recent years. Influenza viruses of the H6 subtype have been isolated from wild and domestic aquatic and terrestrial avian species throughout the world since their first detection in the United States in 1965. Since 1997, H6 viruses have been detected frequently in the live poultry markets of southern China; however, the biological characterization of these viruses is very limited. Here, we assessed the receptor-binding preference, replication, and transmissibility in mammals of a series of H6 viruses isolated from live poultry markets in southern China and found that 34% of the viruses are able to bind human type receptors and that some of them are able to transmit efficiently to contact animals. Our study demonstrates that the H6 viruses pose a clear threat to human health.

Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt.

Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA) from α2,3- to α2,6-linked sialic acid (SA) is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential.

Multiple microRNAs induced by Cdx1 suppress Cdx2 in human colorectal tumour cells.

The mammalian transcriptional factors, Cdx1 and Cdx2 (Cdx is caudal-type homeobox) are paralogues and critical for the cellular differentiation of intestinal or colorectal epithelia. It has been reported previously that in Cdx1 transgenic or knockout mice, endogenous Cdx2 levels are inversely correlated with Cdx1 levels. Recently, we found that exogenous Cdx1 expression can suppress Cdx2 in a human colorectal tumour cell line, SW480, although the underlying molecular mechanisms were unclear. In the present study, we show that several microRNAs induced by exogenous Cdx1 expression directly bind to the CDX2 mRNA 3'UTR (untranslated region) to destabilize these transcripts, finally leading to their degradation. Using microarray analysis, we found that several miRNAs that were computationally predicted to target CDX2 mRNAs are up-regulated by exogenous Cdx1 expression in SW480 cells. Among these molecules, we identified miR-9, miR-16 and miR-22 as having the potential to suppress Cdx2 through the binding of the 3'UTR to its transcript. Importantly, simultaneous mutations of both the miR-9- and miR-16-binding sites in the CDX2 3'UTR were shown to be sufficient to block Cdx2 suppression. The results of the present study suggest a unique feature of miRNAs in which they contribute to homoeostasis by limiting the levels of transcription factors belonging to the same gene family.

Emergence of H5N1 avian influenza viruses with reduced sensitivity to neuraminidase inhibitors and novel reassortants in Lao People's Democratic Republic.

Pandemic influenza viruses can emerge through continuous evolution and the acquisition of specific mutations or through reassortment. This study assessed the pandemic potential of H5N1 viruses isolated from poultry outbreaks occurring from July 2006 to September 2008 in the Lao People's Democratic Republic (PDR). We analyzed 29 viruses isolated from chickens and ducks and two from fatal human cases in 2007. Prior to 2008, all H5N1 isolates in Lao PDR were from clade 2.3.4; however, clade 2.3.2 was introduced in September 2008. Of greatest concern was the circulation of three isolates that showed reduced sensitivity to the neuraminidase (NA) inhibitor oseltamivir in an enzyme inhibition assay, each with different NA mutations - V116A, I222L and K150N, and a previously unreported S246N mutation. In addition, six isolates had an S31N mutation in the M2 protein, which conferred resistance to amantadine not previously reported in clade 2.3.4 viruses. Two H5N1 reassortants were isolated whose polymerase genes, PB1 and PB2, were homologous to those of Eurasian viruses giving rise to a novel H5N1 genotype, genotype P. All H5N1 viruses retained avian-like receptor specificity, but four had altered affinities for alpha2,3-linked sialic acid. This study shows that, in a genetically similar population of H5N1 viruses in Lao PDR, mutants emerged with natural resistance to antivirals and altered affinities for alpha2,3-linked sialic acids, together with reassortants with polymerase genes homologous to Eurasian viruses. These changes may contribute to the emergence of a pandemic influenza strain and are critical in devising surveillance strategies.

Alterations in receptor-binding properties of swine influenza viruses of the H1 subtype after isolation in embryonated chicken eggs.

Alterations of the receptor-binding properties of swine influenza A viruses (SIVs) during their isolation in embryonated chicken eggs have not been well studied. In this study, the receptor-binding properties of classical H1 SIVs isolated solely in eggs or Madin-Darby canine kidney (MDCK) cells were examined. Sequencing analysis revealed substitutions of D190V/N or D225G in the haemagglutinin (HA) proteins in egg isolates, whereas MDCK isolates retained HA genes identical to those of the original viruses present in the clinical samples. Egg isolates with substitution of either D190V/N or D225G had increased haemagglutinating activity for mouse and sheep erythrocytes, but reduced activity for rabbit erythrocytes. Additionally, egg isolates with D225G had increased haemagglutination activity for chicken erythrocytes. A direct binding assay using a sialyl glycopolymer that possessed either a 5-N-acetylneuraminic acid (Neu5Ac) alpha2,6galactose (Gal) or a Neu5Acalpha2,3Gal linkage revealed that the egg isolates used in this study showed higher binding activity to the Neu5Acalpha2,3Gal receptor than MDCK isolates. Increased binding activity of the egg isolates to the Neu5Acalpha2,3Gal receptor was also confirmed by haemagglutination assay with resialylated chicken erythrocytes by Galbeta1,3/4GlcNAcalpha2,3-sialyltransferase. These observations were reinforced by flow-cytometric and N-glycan analyses of the erythrocytes. The alpha2,3-linked sialic acids were expressed predominantly on the surface of mouse and sheep erythrocytes. Chicken erythrocytes expressed Neu5Acalpha2,3Gal more abundantly than Neu5Acalpha2,6Gal, and rabbit erythrocytes expressed both 5-N-glycolylneuraminic acid (Neu5Gc) alpha2,6Gal and Neu5Acalpha2,6Gal. Our results demonstrate clearly that classical H1 SIVs undergo alterations in receptor-binding activity associated with an amino acid substitution in the HA protein during isolation and propagation in embryonated chicken eggs.

Avian and human influenza A virus receptors in trachea and lung of animals.

BACKGROUND: Influenza A viruses are capable of crossing the specific barrier between human beings and animals resulting in interspecies transmission. The important factor of potential infectivity of influenza A viruses is the suitability of the receptor binding site of the host and viruses. The affinities of avian and human influenza virus to bind with the receptors and the distributions of receptors in animals are different. OBJECTIVE: This study aims to investigate the anatomical distribution of avian and human influenza virus receptors using the double staining lectin histochemistry method. METHODS: Double staining of lectin histochemistry was performed to identify both SA alpha2,3 Gal and SA alpha2,6 Gal receptors in trachea and lung tissue of dogs, cats, tigers, ferret, pigs, ducks and chickens. RESULTS: We have demonstrated that avian and human influenza virus receptors were abundantly present in trachea, bronchus and bronchiole, but in alveoli of dogs, cats and tigers showed SA alpha2,6 Gal only. Furthermore, endothelial cells in lung tissues showed presence of SA alpha2,3 Gal. CONCLUSION: The positive sites of both receptors in respiratory tract, especially in the trachea, suggest that all mammalian species studied can be infected with avian influenza virus. These findings suggested that dogs and cats in close contact with humans should be of greater concern as an intermediate host for avian influenza A in which there is the potential for viral adaptation and reassortment.

Analysis of N-glycans in embryonated chicken egg chorioallantoic and amniotic cells responsible for binding and adaptation of human and avian influenza viruses.

The initial step essential in influenza virus infection is specific binding of viral hemagglutinin to host cell-surface glycan receptors. Influenza A virus specificity for the host is mediated by viral envelope hemagglutinin, that binds to receptors containing glycans with terminal sialic acids. Human viruses preferentially bind to alpha2-->6 linked sialic acids on receptors of host cells, whereas avian viruses are specific for the alpha2-->3 linkage on the target cells. Human influenza virus isolates more efficiently infect amniotic membrane (AM) cells than chorioallantoic membrane (CAM) cells. N-glycans were isolated from AM and CAM cells of 10-day-old chicken embryonated eggs and their structures were analyzed by multi-dimensional HPLC mapping and MALDI-TOF-MS techniques. Terminal N-acetylneuraminic acid contents in the two cell types were similar. However, molar percents of alpha2-->3 linkage preferentially bound by avian influenza virus were 27.2 in CAM cells and 15.4 in AM cells, whereas those of alpha2-->6 linkage favored by human influenza virus were 8.3 (CAM) and 14.2 (AM). Molar percents of sulfated glycans, recognized by human influenza virus, in CAM and AM cells were 3.8 and 12.7, respectively. These results have revealed structures and molar percents of N-glycans in CAM and AM cells important in determining human and avian influenza virus infection and viral adaptation.

Mechanisms of the action of povidone-iodine against human and avian influenza A viruses: its effects on hemagglutination and sialidase activities.

BACKGROUND: Influenza virus infection causes significant morbidity and mortality and has marked social and economic impacts throughout the world. The influenza surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), act cooperatively to support efficient influenza A virus replication and provide the most important targets for anti-influenza chemotherapy. In this study, povidone-iodine (PVP-I), which has a broad-spectrum microbicidal property, was examined for its inhibitory effects against influenza virus infection in MDCK cells and the mechanisms of PVP-I action on HA and NA were revealed. RESULTS: Results obtained using a novel fluorescence- and chromogenic-based plaque inhibition assay showed that 1.56 mg/ml PVP-I inhibited infections in MDCK cells of human (8 strains) and avian (5 strains) influenza A viruses, including H1N1, H3N2, H5N3 and H9N2, from 23.0-97.5%. A sialidase inhibition assay revealed that PVP-I inhibited N1, N2 and N3 neuraminidases with IC50 values of 9.5-212.1 microg/ml by a mixed-type inhibition mechanism. Receptor binding inhibition and hemagglutinin inhibition assays indicated that PVP-I affected viral hemagglutinin rather than host-specific sialic acid receptors. CONCLUSION: Mechanisms of reduction of viral growth in MDCK cells by PVP-I involve blockade of viral attachment to cellular receptors and inhibition of viral release and spread from infected cells. Therefore, PVP-I is useful to prevent infection and limit spread of human and avian influenza viruses.

Author Correction: The role of the SWI/SNF chromatin remodeling complex in maintaining the stemness of glioma initiating cells.

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N-glycan structures of human alveoli provide insight into influenza A virus infection and pathogenesis.

The rapidly evolvable influenza A virus has caused pandemics linked to millions of deaths in the past century. Influenza A viruses are categorized by H (hemagglutinin; HA) and N (neuraminidase; NA) proteins expressed on the viral envelope surface. Analyses of past pandemics suggest that the HA gene segment comes from a nonhuman virus, which is then introduced into an immunologically naïve human population with potentially devastating consequences. As a prerequisite for infection, the nonhuman HA molecules of H1-H16 viruses must be able to bind to specific sialyl receptors on the host cell surface along the human respiratory tract. Thus, additional insight into the structures of host cell glycans and how different HAs interact with different glycans might provide new insight into the mechanisms underlying sustained infection and transmission in humans. In this work, we identified the sialyl N-glycans found in normal human alveoli and characterized the influenza viruses that preferentially bound to these different structures. We also determined the amino acid changes in HA that were linked to a switch of receptor-binding preference from nonhuman to pandemic, as well as pandemic to seasonal. Our data provide insight into why seasonal viruses are associated with reduced alveolar infection and damage and suggest new considerations for designing anti-HA vaccines and drugs. The results provide a better understanding of viral tropism and pathogenesis in humans that will be important for prediction and surveillance of zoonotic, pandemic, and epidemic influenza outbreaks. DATABASE: The novel hemagglutinin nucleotide sequences reported here were deposited in GISAID under the accession numbers of EPI685738 for A/Yamaguchi/20/2006(H1N1) and EPI685740 for A/Kitakyushu/10/2006(H1N1).

Tumor suppression via inhibition of SWI/SNF complex-dependent NF-κB activation.

The transcription factor NF-κB is constitutively activated in many epithelial tumors but few NF-κB inhibitors are suitable for cancer therapy because of its broad biological effects. We previously reported that the d4-family proteins (DPF1, DPF2, DPF3a/b) function as adaptor proteins linking NF-κB with the SWI/SNF complex. Here, using epithelial tumor cell lines, A549 and HeLaS3, we demonstrate that exogenous expression of the highly-conserved N-terminal 84-amino acid region (designated "CT1") of either DPF2 or DPF3a/b has stronger inhibitory effects on anchorage-independent growth than the single knockdown of any d4-family protein. This indicates that CT1 can function as an efficient dominant-negative mutant of the entire d4-family proteins. By in situ proximity ligation assay, CT1 was found to retain full adaptor function, indicating that the C-terminal region of d4-family proteins lacking in CT1 would include essential domains for SWI/SNF-dependent NF-κB activation. Microarray analysis revealed that CT1 suppresses only a portion of the NF-κB target genes, including representative SWI/SNF-dependent genes. Among these genes, IL6 was shown to strongly contribute to anchorage-independent growth. Finally, exogenous CT1 expression efficiently suppressed tumor formation in a mouse xenograft model, suggesting that the d4-family proteins are promising cancer therapy targets.

The role of the SWI/SNF chromatin remodeling complex in maintaining the stemness of glioma initiating cells.

Glioma initiating cells (GICs) are thought to contribute to therapeutic resistance and tumor recurrence in glioblastoma, a lethal primary brain tumor in adults. Although the stem-like properties of GICs, such as self-renewal and tumorigenicity, are epigenetically regulated, the role of a major chromatin remodeling complex in human, the SWI/SNF complex, remains unknown in these cells. We here demonstrate that the SWI/SNF core complex, that is associated with a unique corepressor complex through the d4-family proteins, DPF1 or DPF3a, plays essential roles in stemness maintenance in GICs. The serum-induced differentiation of GICs downregulated the endogenous expression of DPF1 and DPF3a, and the shRNA-mediated knockdown of each gene reduced both sphere-forming ability and tumor-forming activity in a mouse xenograft model. Rescue experiments revealed that DPF1 has dominant effects over DPF3a. Notably, whereas we have previously reported that d4-family members can function as adaptor proteins between the SWI/SNF complex and NF-κB dimers, this does not significantly contribute to maintaining the stemness properties of GICs. Instead, these proteins were found to link a corepressor complex containing the nuclear receptor, TLX, and LSD1/RCOR2 with the SWI/SNF core complex. Collectively, our results indicate that DPF1 and DPF3a are potential therapeutic targets for glioblastoma.

MiR-199a Inhibits Secondary Envelopment of Herpes Simplex Virus-1 Through the Downregulation of Cdc42-specific GTPase Activating Protein Localized in Golgi Apparatus.

Because several studies have shown that exogenous miR-199a has antiviral effects against various viruses, including herpesviruses, we examined how miR-199a exerts its antiviral effects using epithelial tumour cell lines infected with herpes simplex virus-1 (HSV-1). We found that both miR-199a-5p and -3p impair the secondary envelopment of HSV-1 by suppressing their common target, ARHGAP21, a Golgi-localized GTPase-activating protein for Cdc42. We further found that the trans-cisternae of the Golgi apparatus are a potential membrane compartment for secondary envelopment. Exogenous expression of either pre-miR-199a or sh-ARHGAP21 exhibited shared phenotypes i.e. alteration of Golgi function in uninfected cells, inhibition of HSV-1 secondary envelopment, and reduction of trans-Golgi proteins upon HSV-1 infection. A constitutively active form of Cdc42 also inhibited HSV-1 secondary envelopment. Endogenous levels of miR-199a in epithelial tumour cell lines were negatively correlated with the efficiency of HSV-1 secondary envelopment within these cells. These results suggest that miR-199a is a crucial regulator of Cdc42 activity on Golgi membranes, which is important for the maintenance of Golgi function and for the secondary envelopment of HSV-1 upon its infection.