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Stem Cell-Derived Human Intestinal Organoids as an Infection Model for Rotaviruses

ABSTRACT Directed differentiation of stem cell lines into intestine-like tissue called induced human intestinal organoids (iHIOs) is now possible (J. R. Spence, C. N. Mayhew, S. A. Rankin, M. F. Kuhar, J. E. Vallance, K. Tolle, E. E. Hoskins, V. V. Kalinichenko, S. I. Wells, A. M. Zorn, N. F. Shroyer, and J. M. Wells, Nature 470:105-109, 2011). We tested iHIOs as a new model to cultivate and study fecal viruses. Protocols for infection of iHIOs with a laboratory strain of rotavirus, simian SA11, were developed. Proof-of-principle analyses showed that iHIOs support replication of a gastrointestinal virus, rotavirus, on the basis of detection of nonstructural viral proteins (nonstructural protein 4 [NSP4] and NSP2) by immunofluorescence, increased levels of viral RNA by quantitative reverse transcription-PCR (qRT-PCR), and production of infectious progeny virus. iHIOs were also shown to support replication of 12/13 clinical rotavirus isolates directly from stool samples. An unexpected finding was the detection of rotavirus infection not only in the epithelial cells but also in the mesenchymal cell population of the iHIOs. This work demonstrates that iHIOs offer a promising new model to study rotaviruses and other gastrointestinal viruses. IMPORTANCE Gastrointestinal viral infections are a major cause of illness and death in children and adults. The ability to fully understand how viruses interact with human intestinal cells in order to cause disease has been hampered by insufficient methods for growing many gastrointestinal viruses in the laboratory. Induced human intestinal organoids (iHIOs) are a promising new model for generating intestine-like tissue. This is the first report of a study using iHIOs to cultivate any microorganism, in this case, an enteric virus. The evidence that both laboratory and clinical rotavirus isolates can replicate in iHIOs suggests that this model would be useful not only for studies of rotaviruses but also potentially of other infectious agents. Furthermore, detection of rotavirus proteins in unexpected cell types highlights the promise of this system to reveal new questions about pathogenesis that have not been previously recognized or investigated in other intestinal cell culture models. Gastrointestinal viral infections are a major cause of illness and death in children and adults. The ability to fully understand how viruses interact with human intestinal cells in order to cause disease has been hampered by insufficient methods for growing many gastrointestinal viruses in the laboratory. Induced human intestinal organoids (iHIOs) are a promising new model for generating intestine-like tissue. This is the first report of a study using iHIOs to cultivate any microorganism, in this case, an enteric virus. The evidence that both laboratory and clinical rotavirus isolates can replicate in iHIOs suggests that this model would be useful not only for studies of rotaviruses but also potentially of other infectious agents. Furthermore, detection of rotavirus proteins in unexpected cell types highlights the promise of this system to reveal new questions about pathogenesis that have not been previously recognized or investigated in other intestinal cell culture models.

Role of Histone Deacetylation in Cell-specific Expression of Endothelial Nitric-oxide Synthase

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in cell-specific expression of endothelial nitric-oxide synthase (eNOS) are not fully understood. In this study we investigated whether histone deacetylation was involved in repression of eNOS expression in non-endothelial cells. Induction of eNOS expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate was observed in all four different types of non-endothelial cells examined. Chromatin immunoprecipitation assays showed that the induction of eNOS expression by TSA was accompanied by a remarkable increase of acetylation of histone H3 associated with the eNOS 5′-flanking region in the non-endothelial cells. Moreover, DNA methylation-mediated repression of eNOS promoter activity was partially reversed by TSA treatment, and combined treatment of TSA and 5-aza-2′-deoxycytidine (AzadC) synergistically induced eNOS expression in non-endothelial cells. The proximal Sp1 site is critical for basal activity of eNOS promoter. The induction of eNOS by inhibition of HDACs in non-endothelial cells, however, appeared not mediated by the changes in Sp1 DNA binding activity. We further showed that Sp1 bound to the endogenous eNOS promoter and associated with HDAC1 in non-endothelial HeLa cells. Combined TSA and AzadC treatment increased Sp1 binding to the endogenous eNOS promoter but decreased the association between HDAC1 and Sp1 in HeLa cells. Our data suggest that HDAC1 plays a critical role in eNOS repression, and the proximal Sp1 site may serve a key target for HDCA1-mediated eNOS repression in non-endothelial cells.

Autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-β signaling is required for rotavirus replication

Significance This study describes a unique mechanism of virus-initiated autophagy and exploitation of autophagy membranes for virus replication. Autophagy is a highly regulated cellular process in which cells destroy and recycle their own components in lysosomes. The mechanism most viruses use to induce autophagy is unknown. We show a rotavirus pore-forming protein activates a calcium-dependent signaling pathway to initiate autophagy. Rotavirus hijacks autophagy membranes to transport viral proteins to sites of virus replication for assembly of infectious particles and interferes with autophagy maturation. Inhibition of the signaling pathway blocks virus production, suggesting a therapeutic target to fight infection. Autophagy is a cellular degradation process involving an intracellular membrane trafficking pathway that recycles cellular components or eliminates intracellular microbes in lysosomes. Many pathogens subvert autophagy to enhance their replication, but the mechanisms these pathogens use to initiate the autophagy process have not been elucidated. This study identifies rotavirus as a pathogen that encodes a viroporin, nonstructural protein 4, which releases endoplasmic reticulum calcium into the cytoplasm, thereby activating a calcium/calmodulin-dependent kinase kinase-β and 5′ adenosine monophosphate-activated protein kinase-dependent signaling pathway to initiate autophagy. Rotavirus hijacks this membrane trafficking pathway to transport viral proteins from the endoplasmic reticulum to sites of viral replication to produce infectious virus. This process requires PI3K activity and autophagy-initiation proteins Atg3 and Atg5, and it is abrogated by chelating cytoplasmic calcium or inhibiting calcium/calmodulin-dependent kinase kinase-β. Although the early stages of autophagy are initiated, rotavirus infection also blocks autophagy maturation. These studies identify a unique mechanism of virus-mediated, calcium-activated signaling that initiates autophagy and hijacks this membrane trafficking pathway to transport viral proteins to sites of viral assembly.

Rotavirus Disrupts Calcium Homeostasis by NSP4 Viroporin Activity

ABSTRACT Many viruses alter intracellular calcium homeostasis. The rotavirus nonstructural protein 4 (NSP4), an endoplasmic reticulum (ER) transmembrane glycoprotein, increases intracellular levels of cytoplasmic Ca2+ ([Ca2+]cyto) through a phospholipase C-independent pathway, which is required for virus replication and morphogenesis. However, the NSP4 domain and mechanism that increases [Ca2+]cyto are unknown. We identified an NSP4 domain (amino acids [aa] 47 to 90) that inserts into membranes and has structural characteristics of viroporins, a class of small hydrophobic viral proteins that disrupt membrane integrity and ion homeostasis to facilitate virus entry, assembly, or release. Mutational analysis showed that NSP4 viroporin activity was mediated by an amphipathic α-helical domain downstream of a conserved lysine cluster. The lysine cluster directed integral membrane insertion of the viroporin domain and was critical for viroporin activity. In epithelial cells, expression of wild-type NSP4 increased the levels of free cytoplasmic Ca2+ by 3.7-fold, but NSP4 viroporin mutants maintained low levels of [Ca2+]cyto, were retained in the ER, and failed to form cytoplasmic vesicular structures, called puncta, which surround viral replication and assembly sites in rotavirus-infected cells. When [Ca2+]cyto was increased pharmacologically with thapsigargin, viroporin mutants formed puncta, showing that elevation of calcium levels and puncta formation are distinct functions of NSP4 and indicating that NSP4 directly or indirectly responds to elevated cytoplasmic calcium levels. NSP4 viroporin activity establishes the mechanism for NSP4-mediated elevation of [Ca2+]cyto, a critical event that regulates rotavirus replication and virion assembly. IMPORTANCE Rotavirus is the leading cause of viral gastroenteritis in children and young animals. Rotavirus infection and expression of nonstructural protein 4 (NSP4) alone dramatically increase cytosolic calcium, which is essential for replication and assembly of infectious virions. This work identifies the intracellular mechanism by which NSP4 disrupts calcium homeostasis by showing that NSP4 is a viroporin, a class of virus-encoded transmembrane pores. Mutational analyses identified residues critical for viroporin activity. Viroporin mutants did not elevate the levels of cytoplasmic calcium in mammalian cells and were maintained in the endoplasmic reticulum rather than forming punctate vesicular structures that are critical for virus replication and morphogenesis. Pharmacological elevation of cytoplasmic calcium levels rescued puncta formation in viroporin mutants, demonstrating that elevation of calcium levels and puncta formation are distinct NSP4 functions. While viroporins typically function in virus entry or release, elevation of calcium levels by NSP4 viroporin activity may serve as a regulatory function to facilitate virus replication and assembly. Rotavirus is the leading cause of viral gastroenteritis in children and young animals. Rotavirus infection and expression of nonstructural protein 4 (NSP4) alone dramatically increase cytosolic calcium, which is essential for replication and assembly of infectious virions. This work identifies the intracellular mechanism by which NSP4 disrupts calcium homeostasis by showing that NSP4 is a viroporin, a class of virus-encoded transmembrane pores. Mutational analyses identified residues critical for viroporin activity. Viroporin mutants did not elevate the levels of cytoplasmic calcium in mammalian cells and were maintained in the endoplasmic reticulum rather than forming punctate vesicular structures that are critical for virus replication and morphogenesis. Pharmacological elevation of cytoplasmic calcium levels rescued puncta formation in viroporin mutants, demonstrating that elevation of calcium levels and puncta formation are distinct NSP4 functions. While viroporins typically function in virus entry or release, elevation of calcium levels by NSP4 viroporin activity may serve as a regulatory function to facilitate virus replication and assembly.

Increased Collagen Deposition and Elevated Expression of Connective Tissue Growth Factor in Human Thoracic Aortic Dissection

Background— Thoracic aortic dissection (TAD) is characterized by dysregulated extracellular matrix. Little is known about the alterations of collagen and stimulators of collagen synthesis, eg, connective tissue growth factor (CTGF), in patients with TAD. In this study, we examined their roles in TAD. Methods and Results— Surgical specimens of the aortic wall of TAD patients (n=10) and controls (n=10) were tested for collagen types I and III and CTGF expression. When compared with controls, protein levels of type I and III collagen and CTGF were significantly increased by 3.2-, 3.7-, and 5.3-fold, respectively (P<0.05 for all). Similar patterns were shown in mRNA levels of type I&agr; and I&agr;2 collagen and CTGF. Using immunohistochemistry and trichrome staining, we also observed elevated levels of collagen in the aortic media and adventitia. Treatment with recombinant human CTGF increased collagen synthesis in cultured aortic smooth muscle cells in a dose- and time-dependent fashion, in which expression of collagens increased from 506±108 counts per minute to 2764±240 cpm by 50 ng/mL CTGF, and from 30±43 cpm to 429±102 cpm at 48 hours. Conclusions— TAD patients exhibited significantly increased expression of aortic collagen types I and III as well as CTGF, which is likely to be responsible for the compromised aortic distensibility and systemic compliance. Because CTGF can increase collagen expression, CTGF may be a new target molecule in the pathogenesis and progression of TAD.

Golgi localization of ERManI defines spatial separation of the mammalian glycoprotein quality control system

The current study provides mechanistic insight into the overlapping dynamics by which glycoprotein folding and quality control use distinct intracellular compartments as part of the proteostasis network in mammalian cells.

Human Cytomegalovirus Causes Endothelial Injury Through the Ataxia Telangiectasia Mutant and p53 DNA Damage Signaling Pathways

Atherosclerosis is the leading cause of death in the United States, and human cytomegalovirus (HCMV), a member of the herpes virus family, may play a role in the development of the disease. We previously showed that HCMV regulated endothelial apoptosis. In this study, we investigated the induction of apoptosis and signal transduction pathways regulating this process in HCMV-infected endothelial cells. As observed previously, HCMV induced a typical cytopathic effect in human aortic endothelial cells (HAECs), ie, the formation of single nucleated or multinucleated giant cells. Although infected HAECs were resistant to apoptosis at earlier stages of infection, they became apoptotic with prolonged infection as demonstrated by positive staining using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). This apoptotic process was mediated by the caspase-dependent mitochondrial apoptotic pathway as indicated by increased expression and cleavage of caspases 3 and 9 as well as increased expressions of pro-apoptotic molecules Bax and Bak. Blocking caspases 3 or 9 significantly inhibited the HCMV-induced apoptosis. Further exploration of the upstream pathway demonstrated upregulation of the tumor suppressor p53 gene and activation of the ataxia telangiectasia mutant (ATM) pathway in the infected cells. Blocking p53 inhibited HCMV-stimulated Bax and Bak expression as well as caspase-3 activation and blocking the ATM pathway inhibited HCMV-stimulated p53 activation. Although early infection may render cells antiapoptotic, prolonged infection, however, induced endothelial apoptosis through ATM and p53-dependent activation of the mitochondrial death pathway. This proapoptotic effect may be relevant to endothelial dysfunction and HCMV-associated vascular diseases.

Evaluations for In Vitro Correlates of Immunogenicity of Inactivated Influenza A H5, H7 and H9 Vaccines in Humans

Background Serum antibody responses in humans to inactivated influenza A (H5N1), (H9N2) and A (H7) vaccines have been varied but frequently low, particularly for subunit vaccines without adjuvant despite hemagglutinin (HA) concentrations expected to induce good responses. Design To help understand the low responses to subunit vaccines, we evaluated influenza A (H5N1), (H9N2), (H7N7) vaccines and 2009 pandemic (H1N1) vaccines for antigen uptake, processing and presentation by dendritic cells to T cells, conformation of vaccine HA in antibody binding assays and gel analyses, HA titers with different red blood cells, and vaccine morphology in electron micrographs (EM). Results Antigen uptake, processing and presentation of H5, H7, H9 and H1 vaccine preparations evaluated in humans appeared normal. No differences were detected in antibody interactions with vaccine and matched virus; although H7 trimer was not detected in western blots, no abnormalities in the conformation of the HA antigens were identified. The lowest HA titers for the vaccines were <1∶4 for the H7 vaccine and 1∶661 for an H9 vaccine; these vaccines induced the fewest antibody responses. A (H1N1) vaccines were the most immunogenic in humans; intact virus and virus pieces were prominent in EM. A good immunogenic A (H9N2) vaccine contained primarily particles of viral membrane with external HA and NA. A (H5N1) vaccines intermediate in immunogenicity were mostly indistinct structural units with stellates; the least immunogenic A (H7N7) vaccine contained mostly small 5 to 20 nm structures. Summary Antigen uptake, processing and presentation to human T cells and conformation of the HA appeared normal for each inactivated influenza A vaccine. Low HA titer was associated with low immunogenicity and presence of particles or split virus pieces was associated with higher immunogenicity.

Activation of the Endoplasmic Reticulum Calcium Sensor STIM1 and Store-Operated Calcium Entry by Rotavirus Requires NSP4 Viroporin Activity

ABSTRACT Rotavirus nonstructural protein 4 (NSP4) induces dramatic changes in cellular calcium homeostasis. These include increased endoplasmic reticulum (ER) permeability, resulting in decreased ER calcium stores and activation of plasma membrane (PM) calcium influx channels, ultimately causing a 2- to 4-fold elevation in cytoplasmic calcium. Elevated cytoplasmic calcium is absolutely required for virus replication, but the underlying mechanisms responsible for calcium influx remain poorly understood. NSP4 is an ER-localized viroporin, whose activity depletes ER calcium, which ultimately leads to calcium influx. We hypothesized that NSP4-mediated depletion of ER calcium activates store-operated calcium entry (SOCE) through activation of the ER calcium sensor stromal interaction molecule 1 (STIM1). We established and used a stable yellow fluorescent protein-expressing STIM1 cell line (YFP-STIM1) as a biosensor to assess STIM1 activation (puncta formation) by rotavirus infection and NSP4 expression. We found that STIM1 is constitutively active in rotavirus-infected cells and that STIM1 puncta colocalize with the PM-localized Orai1 SOCE calcium channel. Expression of wild-type NSP4 activated STIM1, resulting in PM calcium influx, but an NSP4 viroporin mutant failed to induce STIM1 activation and did not activate the PM calcium entry pathway. Finally, knockdown of STIM1 significantly reduced rotavirus yield, indicating STIM1 plays a critical role in virus replication. These data demonstrate that while rotavirus may ultimately activate multiple calcium channels in the PM, calcium influx is predicated on NSP4 viroporin-mediated activation of STIM1 in the ER. This is the first report of viroporin-mediated activation of SOCE, reinforcing NSP4 as a robust model to understand dysregulation of calcium homeostasis during virus infections.

Effect of Nuclear Actin on Endothelial Nitric Oxide Synthase Expression

Background—Previously, we showed that the 27nt repeat polymorphism in endothelial nitric oxide synthase (eNOS) intron 4 was associated with altered eNOS mRNA and protein levels, nitric oxide (NO) production and vascular disease risk; the 27-nt repeats had a cis-acting role in eNOS promoter function. In the present study, we investigated nuclear protein that binds the 27nt repeat and mediates eNOS expression. Methods and Results—Using 5′-biotin-labeled 27nt DNA duplex and streptavidin–agarose beads pull-down assay and mass spectrometry, we identified that nuclear β-actin was one of the major 27nt binding proteins. Using the pGL3 reporter vectors containing the 5×27nt repeats as an enhancer in an in vitro transcription assay, we found that exogenous β-actin significantly increased reporter gene transcription efficiency. The β-actins upregulating effect was compromised when exogenous 27nt RNA duplex was added. Furthermore, the eNOS expression was reduced when β-actin gene was silenced by specific siRNA, and actin overexpression upregulated eNOS expression >3-fold. Conclusion—Our data demonstrate that β-actin as a transcription factor stimulates eNOS expression; and the transcriptional effect appears to be 27nt-dependent. Our findings represent a novel molecular mechanism regulating eNOS expression, which could potentially lead to discoveries of eNOS specific pharmaceutical agents, eg, active peptides, with clinical applications.