What Happens to a Society When It Turns Against Many of Its Children and Imprisons Them?
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Darren was kneeling, but he was not praying. Baptized Catholic and brought up in the church, Darren had turned to his faith to get him through the early years of a decade-long confinement, including months spent on isolation units.
Now he found himself forced to his knees, hands cuffed behind his back, stripped to his boxers, one in a long row of boys in the same supplicant pose. They would be required to hold this position for two full weeks, their only respite a daily three-hour block allotted for sleep and a quick meal—a cheese sandwich, milk, and an egg.
So it was that all the boys in Darren's unit were brought to the gymnasium, stripped to their underwear, and ordered to their knees. They would remain in that posture, bound together in a long row, until the guards chose to release them. Silence was strictly enforced.
Darren was a college student when he spoke with me about his long sojourn through the California Youth Authority (CYA). Earnest and deliberate, he chose his words as carefully as he did his attire—a zip-top blue sweater and matching wool knit cap, a carved silver stud anointing each ear, and the hint of a goatee framing a handsome brown face. There was no apparent anger in his tone, even when he described the worst of the abuses. His voice simply dropped, infused with a quiet, contemplative sorrow.
It was difficult to imagine this poised young man in the supplicant, dehumanized position he had described to me. But formal investigations into conditions at the CYA (now known as the Division of Juvenile Justice) confirmed Darren's account and worse.
Prostrate before grown men to whom he might as well have been invisible, listening to the guards laugh and joke among themselves as their charges knelt before them, visibly suffering, Darren silently sought answers. How can the people here do the the things they do to children? he asked the heavens. No answer was forthcoming.
Abuse of the young inside state-run juvenile prisons is both rampant and extreme. A federal survey published in 2010 found widespread abuse and maltreatment in America's youth corrections facilities. More than a third of youth in secure corrections facilities or camp programs reported that staff used force unnecessarily, and 30 percent said that staff placed youth into solitary confinement or locked them up alone as discipline. About half had experienced some kind of group punishment, as Darren described, and half said staff applied punishment unfairly. Fear of abuse was equally pervasive: 38 percent of youth said they feared being physically attacked by staff or other youth. Twenty-five percent feared attack from another youth, and 22 percent were afraid of attack from a staff member. Many lived in fear of both.
In the context of the juvenile prison, the term "abuse" takes on multiple meanings. The following are just a sample of incidents that have come to light in recent years.
In Florida, a three-hundred-pound guard crushed a twelve-year old boy with his body, suffocating the sixty-five-pound child to death.
In California, groups of correctional officers slammed handcuffed boys face-first into walls or set attack dogs on them, often in full view of security cameras.
In Louisiana, guards assaulted sleeping children, or entertained themselves by pitting boys in fights against each other, leaving them with broken jaws, fractured eye sockets, and an array of other injuries.
In Georgia, guards assaulted children who were anemic, injured, mentally disabled, and as young as nine. When one boy collapsed during a punishing exercise regime, a supervisor broke his arm. A boy who talked in line was punched in the ear so hard that his eardrum was punctured.
In Mississippi, guards ripped the clothing from suicidal girls, then hog-tied them, naked, and tossed them into solitary. They also shackled girls to poles and forced them to run in hot weather carrying logs. Those who threw up from the heat and the strain were forced to eat their own vomit.
In Arkansas, young people were left naked in solitary with the air-conditioning turned on high, or hog-tied and set outside at night in freezing weather.
In the absence of a consistent reporting mechanism, the best source of information about abuse behind bars often comes from litigation initiated by family members, advocacy groups, or the U.S. Department of Justice. The federal entity charged with enforcing the Eighth Amendment (which forbids cruel and unusual punishment), the Department of Justice is responsible for investigating allegations of abuse behind bars and can sue those jurisdictions in which abuse is egregious enough to test constitutional limits.
A 2011 report from the Annie E. Casey Foundation includes an overview of successful civil rights suits across the country. Abuse is pervasive throughout the nation's juvenile facilities, author Richard Mendel concludes, with documentation of "systemic violence, abuse, and/or excessive use of isolation or restraints" in thirty-nine states plus Washington, D.C., and Puerto Rico, going back to 1970.
In New York, a supervisor with that state 's Office of Children and Family Services, which oversees both the delinquency and dependency systems, said young people sometimes returned from upstate juvenile facilities with bruises, contusions, or even broken bones. Each time, she would pick up the phone and report these instances of alleged child abuse to a branch of the same agency suspected of inflicting them. Only a handful of states, it turns out, have not been determined to have systematically brutalized the youth in their care. A review of all fifty states found only eight where there was not conclusive evidence of system-wide mistreatment.
Reading the results of Department of Justice investigations or class action lawsuits—catalogues of brutality running on for pages—is a painful experience. But there is also something bracing about the bald exposure of abuses behind bars. Surely, one imagines, once these horrors are exposed both in the press and at the highest levels of our legal system, something will be done about them. Reforms will be instituted, the children made safe.
Those who author reports on abuse in juvenile prisons tend to foster this reformist faith, ending the most chilling investigation with an optimistic list of suggested reforms. The language ranges from academic to outraged to crisply legalistic, depending on the authors, but the recommendations themselves are generally quite similar: conduct further inquiries, form new committees, consult experts, draft protocols, hold trainings, and the like. Beneath these procedural suggestions lies a single mandate: quit beating up the children.
Federal law and a number of court decisions have made it clear that incarcerated youth do not forfeit their human rights along with their liberty. Yet as lawsuit follows lawsuit and consent decrees pile up, abuse remains endemic to our nation's juvenile prisons. It has persisted for decades, despite investigations, expos.s, admonitions, and court orders; despite public displays of outrage and official commitments to change.
No matter how vehemently we profess ourselves "scandalized" when another investigation brings new abuses to light, the seemingly gratuitous cruelty of captor toward captive is so widespread, and so long-standing despite multiple reform efforts, that it raises a troubling question: is it even possible to eradicate the abuses that occur with such regularity when large numbers of vulnerable young people are held captive far from the public eye? Or is there something inherent to this particular structure that makes such abuses inevitable?
From "chemical restraints" to shield-bearing "extraction teams" to age-old tools like the fist and the boot, today's incarcerated youth face a range of mechanisms of assault that taxes the imagination. The restraint chair, for example, may sound like an artifact from a medieval dungeon, but it is used in juvenile facilities to this day.
"They strap you down," he repeated, his voice rising with a fury the passage of time seemed not to have diminished.
Copyright 2014 by Nell Bernstein. The excerpt originally appeared in Burning Down the House: The End of Juvenile Prison published by The New Press here with permission.
Nell Bernstein is the Author of Burning Down the House, from The New Press, and All Alone in the World (The New Press), a Newsweek "Book of the Week." She is a former Soros Justice Media Fellow and a winner of a White House Champion of Change award. Her articles have appeared in Newsday, Salon, Mother Jones, and the Washington Post, and TruthOut, among other publications. She lives in Albany, California.
Richard Ross is a photographer, researcher and professor of art based in Santa Barbara, California. Ross has been the recipient of grants from the National Endowment for the Arts, the Annie E. Casey and MacArthur Foundations. Ross was awarded both Fulbright and Guggenheim Fellowships. His most recent work, the — In Justice series, turns a lens on the placement and treatment of American juveniles housed by law in facilities that treat, confine, punish, assist and, occasionally, harm them. Two books and traveling exhibitions of the work continue to see great success while Ross collaborates with juvenile justice stakeholders, using the images as a catalyst for change.
This piece was reprinted by EmpathyEducates with permission or license. We thank the Author, Nell Bernstein for her kindness, awareness, research and for the lesson. We are extremely grateful for Photographer Ricahrd Ross' strong voice and visual presence. We cannot begin to express our abundant appreciation for each of the two and the many vital conversations each inspires.
How Could Such a Rich Country as Ours Produce So Many Poor People?


The ET tech I'm referring to is the stuff we've all read about and heard testimony on for years like the chair, and the rocks that look like rock until the right person in the right frame of mind touches them, (so maybe a rock isn't just a rock, sometimes…) ET tech like craft and orbs; the part we see being the membrane or difference in the frequency of the two states of matter between dimensions (thank you Jason Quitt for such a succinct definition!!! ) that the eye can perceive, the ET tech THAT IS CONSCIOUSNESS - built from consciousness fields - frequency boundaries ( there is a CARAT dot here, somewhere).
Maybe it all boils down to: ET tech IS Consciousness. ET's being the key, being used as an analog/analogy, a DOORWAY into other....places.... in this story ??? And maybe the Whack-a-mole game they are playing right now desperately trying to spin the narrative is all directed at covering this up?
The video makes a pretty good case for this. Worth the watch. Really.


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Don't let a chemical industry insider weaken health protections
You may have already heard that the Trump administration is taking actions behind the scenes to weaken an Environmental Protection Agency (EPA) proposal to protect us from PFAS chemicals. You may be outraged (but probably not surprised) to learn that these actions have been driven by a former chemical industry lobbyist—who's now nominated to run our top consumer protection agency.
That's right, this is the kind of plot thickening that movies are made of! But unlike a good movie, this story gets a little wonky and detailed right off the mark, so it's important for us to break it all down so we can understand what we need to do to get our happily ever after.
What's going on? And what's a SNUR?
The EPA proposal that the Trump administration is trying to weaken is called a "SNUR," which seems like the sound of a muppet snoring but stands for Significant New Use Rule. A SNUR is a rule that EPA issues in order to get advance notice about the new use of a chemical that could harm human health or the environment. ("New use" can also mean the resurrection of an old one.) Under the SNUR, EPA either approves or disallows the specific use.
This particular "SNUR" is for per- and poly-fluoroalkyl substances (PFAS), which have contaminated the tap water of millions of Americans and are present in everyday products like nonstick pans and waterproof jackets. This is problematic because exposure to these toxic chemicals is linked to cancer, immune diseases, and other serious illnesses. Chemical companies unleashed this class of chemicals with little or no safety testing. Today we find ourselves dealing with this toxic mess as a result.
What would EPA's proposed SNUR do?
In 2006, EPA and eight major companies in the PFAS chemical industry entered into a voluntary agreement to phase out U.S. production of PFOA and PFOS, two of the oldest PFAS chemicals, by 2015. In 2015, EPA proposed a rule (the SNUR) that amounted to a de facto ban on certain PFAS chemicals (including but not limited to PFOA and PFOS) for which there were no ongoing uses. It also included an important de facto ban on resuming abandoned uses of these types of PFAS chemicals that had previously been allowed. In addition, the rule proposed to remove exemptions in the Toxic Substances Control Act (TSCA) that allowed anyone to import these chemicals as part of a consumer product.
That 2015 SNUR was never finalized. However, when Congress passed the 2020 National Defense Authorization Act (NDAA), one of the provisions we won required EPA to take final action on it by June 20, 2020. But then, a plot twist.
The proposed PFAS SNUR is now a much weaker version of the original proposal
Unfortunately, the version of the proposed SNUR that EPA released for public comment two months ago significantly watered down the earlier proposal. It limited which new PFAS-containing products would require SNUR notification and review before they can be imported to the US. This opened up a loophole through which imports containing hazardous PFAS could be imported and sold in the U.S., exposing families and continuing the cycle of harm.
In the comments we submitted along with 30 other environmental health groups, Safer Chemicals Healthy Families argued that this defeats EPA's own goal of achieving maximum reduction of the presence of PFOA and PFOS—a subset of the larger class of PFAS chemicals—in people and the environment.
So, what happened?! What caused EPA to change course?
Answer: Nancy Beck. She is a former lobbyist for the chemical industry who was hired by the Trump administration for a senior position in the EPA's chemical safety office. She is the White House staffer who has been front and center in this latest erosion of our public health protections.
In her current role at the White House National Economic Council, Dr. Beck has taken a lead role on PFAS chemicals. Senate Environment and Public Works Ranking Member Tom Carper sent a letter to EPA Administrator Andrew Wheeler outlining his objections to the proposal. With that letter, Senator Carper included a very good timeline of the White House's involvement in watering down the proposal, which perfectly aligns with Beck's time in this role.
You may also remember when Dr. Beck used her proverbial red pen to weaken the Toxic Substances Control Act (TSCA) "framework rules" for evaluating chemicals like asbestos and methylene chloride in 2017. This was the issue behind Safer Chemicals Healthy Families, et al v. US. EPA, a lawsuit that we won last fall.
Nancy Beck is now the Trump Administration's nominee to chair the Consumer Product Safety Commission, but she must be stopped. She is the absolute wrong choice to protect Americans from dangerous products.
How can we change the story?
Let's not allow the chemical industry to write the script for our real lives. The EPA is supposed to protect our health and therefore must strengthen its final rule on these dangerous chemicals.
Let's stop this from happening. The Senate should reject her nomination.
In the movies, just when it looks like the bad guys could win, heroes like you step in and become a powerful force. Grab your cape and get in the fight with us.
TAKE ACTION: Tell your senators that we need a consumer advocate leading the CPSC, not a chemical industry insider!
Liz Hitchcock
Director of Safer Chemicals Healthy Families
Liz Hitchcock is director of Toxic-Free Future's federal policy program. She leads advocacy efforts with Congress, EPA and other federal agencies.
Consumer Product Safety Commission, Environmental Protection Agency, Nancy Beck, PFAS, TSCA reform

redpajama_set_name: RedPajamaCommonCrawl


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Genome Replication and Regulation of Viral Gene Expression
Basic Residues within the Ebolavirus VP35 Protein Are Required for Its Viral Polymerase Cofactor Function
Kathleen C. Prins, Jennifer M. Binning, Reed S. Shabman, Daisy W. Leung, Gaya K. Amarasinghe, Christopher F. Basler
Kathleen C. Prins
Jennifer M. Binning
Reed S. Shabman
Daisy W. Leung
Gaya K. Amarasinghe
Christopher F. Basler
DOI: 10.1128/JVI.00925-10
The ebolavirus (EBOV) VP35 protein binds to double-stranded RNA (dsRNA), inhibits host alpha/beta interferon (IFN-α/β) production, and is an essential component of the viral polymerase complex. Structural studies of the VP35 C-terminal IFN inhibitory domain (IID) identified specific structural features, including a central basic patch and a hydrophobic pocket, that are important for dsRNA binding and IFN inhibition. Several other conserved basic residues bordering the central basic patch and a separate cluster of basic residues, called the first basic patch, were also identified. Functional analysis of alanine substitution mutants indicates that basic residues outside the central basic patch are not required for dsRNA binding or for IFN inhibition. However, minigenome assays, which assess viral RNA polymerase complex function, identified these other basic residues to be critical for viral RNA synthesis. Of these, a subset located within the first basic patch is important for VP35-nucleoprotein (NP) interaction, as evidenced by the inability of alanine substitution mutants to coimmunoprecipitate with NP. Therefore, first basic patch residues are likely critical for replication complex formation through interactions with NP. Coimmunoprecipitation studies further demonstrate that the VP35 IID is sufficient to interact with NP and that dsRNA can modulate VP35 IID interactions with NP. Other basic residue mutations that disrupt the VP35 polymerase cofactor function do not affect interaction with NP or with the amino terminus of the viral polymerase. Collectively, these results highlight the importance of conserved basic residues from the EBOV VP35 C-terminal IID and validate the VP35 IID as a potential therapeutic target.
Ebolaviruses (EBOVs) are enveloped, nonsegmented, negative-strand RNA viruses belonging to the family Filoviridae (34). Because filoviruses cause outbreaks of severe, often lethal hemorrhagic fever, they are of concern as potential bioweapons and as an emerging public health threat. Currently, the determinants of EBOV virulence are incompletely defined, but enhanced understanding of the biochemical and structural properties of EBOV proteins will facilitate development of prophylactic or therapeutic measures toward these viruses. One EBOV protein that functions as a virulence determinant is VP35. VP35 binds to double-stranded RNA (dsRNA), inhibits host innate immune responses, is a viral structural protein, and serves as a component of the viral RNA polymerase complex (1).
VP35 inhibits alpha/beta interferon (IFN-α/β) production, activation of the IFN-inducible protein kinase R (PKR) antiviral protein, and RNA silencing (3, 10, 12, 35). Among these functions, inhibition of IFN-α/β production clearly contributes to efficient virus replication in cell culture and in vivo (15-17, 33). Inhibition of IFN-α/β production primarily occurs through inhibition of retinoic acid-inducible gene I (RIG-I)-dependent signaling, which activates interferon regulatory factor 3 (IRF-3) and IRF-7, transcription factors that regulate IFN-α/β gene expression (2, 6, 7, 18, 32, 33). Recent studies demonstrated that VP35 dsRNA binding activity strongly correlates with IFN inhibition (6, 25, 33). However, VP35 likely interacts with and inhibits additional signaling molecules downstream of RIG-I (7, 25, 32). Altogether, these combined inhibitory activities likely contribute to the IFN suppression observed in cells expressing VP35 or infected with EBOV (11, 14, 17, 33). In addition to immune suppression, VP35 is an essential cofactor in the filoviral polymerase complex (28-30). The functional viral polymerase complex includes four EBOV proteins: nucleoprotein (NP), the VP35 and VP30 proteins, and the large protein (L), which is the RNA-dependent RNA polymerase (29, 30). In this complex, VP35 interacts with NP and L, and it is thought that VP35 bridges the catalytic subunit of the polymerase complex, L, to the NP-associated viral RNA (4, 8). Both VP35-NP and VP35-L interactions are therefore expected to be essential for viral RNA synthesis (4).
Recent structural studies of the carboxy-terminal dsRNA binding domain of VP35, referred to as the interferon inhibitory domain (IID), identified several structural features that are important for VP35 interaction with dsRNA and for inhibition of IFN-α/β production (21, 23, 25). Specifically, a central basic patch within the VP35 IID was demonstrated to make contacts with the phosphodiester backbone of dsRNA, and a hydrophobic pocket was found to form an end cap that recognizes the blunt ends of dsRNA (25). Mutation of either central basic patch residues or end-cap residues disrupted VP35-dsRNA interaction and impaired its ability to block signaling by RIG-I, a cellular protein which is likely to be the primary sensor of EBOV in most cell types (13). However, mutations impairing dsRNA binding and IFN-antagonist functions did not affect the VP35 polymerase cofactor function (25).
In addition to the central basic patch, structural studies identified a separate cluster of conserved basic residues, called the first basic patch, as well as additional conserved basic residues that border the central basic patch (23, 25). From these studies, it was not clear if these additional basic residues within the structurally defined VP35 IID region are important for VP35-mediated functions. In the present study, we demonstrate that basic residues outside the central basic patch are not critical for IFN inhibition but are important for viral RNA synthesis, as mutation of these basic residues abrogates viral RNA synthesis by the polymerase complex. Interestingly, residues located within the first basic patch are also important for VP35 interaction with NP. However, not all basic residues important for replication are required for NP binding, as several basic residue mutants that show diminished replication can bind to NP. These results support a model where VP35 bridges viral NP and L. Together, these results further support the role of EBOV VP35 as a critical, multifunctional virulence factor and a key component of the viral RNA replication complex.
Antibodies.Monoclonal antibodies against Zaire EBOV VP35 and against Zaire EBOV NP were generated in collaboration with the Mount Sinai Hybridoma Center and have been described previously (6, 27). A monoclonal anti-hemagglutinin (anti-HA) antibody was purchased from Sigma (St. Louis, MO). A monoclonal anti-maltose binding protein (anti-MBP) antibody was purchased from New England Biolabs.
Cell lines and viruses.HEK293T cells and Vero cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Sendai virus (SeV) strain Cantell was grown in 10-day-old embryonated chicken eggs for 2 days at 37°C.
Plasmids.The Zaire EBOV VP35 expression plasmid pCAGGS-VP35 and an IFN-β promoter reporter plasmid were described previously (2, 3). VP35 point mutations were generated by standard PCR-based methods and cloned into the mammalian expression plasmid pCAGGS (31). Firefly luciferase was expressed from the pCAGGS plasmid in the minigenome assays (see below). The pRL-TK Renilla luciferase expression plasmid was purchased from Promega (Madison, WI). Sequences encoding Zaire EBOV L amino acids 1 to 505 (HA-L amino acids 1 to 505) were amplified from plasmid pTM1-L by PCR (33), cloned with an amino-terminal HA tag, and cloned into pCAGGS. The expression plasmid for Zaire EBOV NP, pcDNA3 EBOV NP, has been described previously (27).
MBP-fusion VP35 IID and VP35 IID protein expression and purification.MBP-fusion VP35 IID proteins were expressed and purified as described previously (23, 24). Briefly, BL21(DE3) cells expressing MBP-fusion VP35 IID proteins were lysed using an EmulsiFlex-C5 homogenizer (Avestin) and clarified by centrifugation at 30,000 × g at 4°C for 30 min. The supernatant was purified by affinity and ion-exchange chromatography, prior to final purification by size-exclusion chromatography. For VP35 IID proteins, the fusion tags were removed prior to final purification by cleavage with tobacco etch virus (TEV) protease followed by size-exclusion chromatography. The purity of the protein samples was assessed by SDS-PAGE.
Isothermal titration calorimetry assays.Quantitative analysis of VP35 IID protein binding interactions with dsRNA was performed on a microcalorimeter (VP-ITC; Microcal, North Hampton, MA) by isothermal titration calorimetry (ITC), using protein samples dialyzed against 500 ml of dialysis buffer [10 mM HEPES (pH 7.0), 150 mM NaCl, 1 mM MgCl2, 2 mM Tris(2-carboxyethyl)phosphine (TCEP)] for 12 h. The resulting raw microcalorimeter data were processed and analyzed to determine n (number of binding sites) and KD (binding constant) using ORIGIN software.
IFN-β-luciferase reporter assay.HEK293T cells were transfected by using Lipofectamine 2000 (Invitrogen) with the indicated amounts of expression plasmid, an IFN-β-firefly luciferase reporter plasmid (400 ng), and a constitutively expressed Renilla luciferase reporter plasmid (pRLTK, 200 ng). To induce reporter gene expression, cells were infected with SeV at a multiplicity of infection of 10. Twenty-four hours posttransfection, the cell lysates were assayed with the dual luciferase reporter assay (Promega), and firefly luciferase activity was normalized to Renilla luciferase activity. The results are presented as percent induction of the positive control (SeV infected, empty vector transfected [no VP35]), the value for which was set equal to 100%.
EBOV transcription/replication assay.The EBOV transcription/replication assay was based on a previously described system (30). HEK293T cells were cotransfected by the calcium phosphate precipitation method with phage T7-driven expression plasmids encoding the Zaire EBOV NP, L, and VP30 and VP35 proteins. Also transfected were a T7 RNA polymerase expression plasmid, a plasmid that expresses from a T7 promoter a Zaire EBOV minigenome which encodes a fused green fluorescent protein (GFP)-chloramphenicol acetyltransferase (CAT) reporter gene. This is flanked by the cis-acting sequences necessary for replication and transcription of the RNA by a reconstituted EBOV polymerase complex. Also transfected was a constitutively expressing luciferase expression plasmid that served as a transfection control. At 36 h posttransfection, cells were lysed with reporter lysis buffer (Promega) and both CAT and luciferase reporter activities were determined. CAT activity was normalized to luciferase activity. Minigenome reporter activation is presented as percent activity relative to that of the positive-control reaction (250 ng of wild-type [WT] VP35 plasmid), which was set equal to 100%. Error bars represent the standard deviation (SD) from at least three experiments.
Immunoprecipitations (IPs).To immunoprecipitate full-length VP35 proteins, lysates from transfected cells were incubated with 1 μg of anti-VP35 monoclonal antibody overnight at 4°C, followed by 1 h incubation with protein G-Sepharose beads (Roche). The beads were washed five times with lysis buffer. After the beads were washed, they were resuspended in SDS-PAGE sample loading buffer, separated by 10% SDS-PAGE, and analyzed by Western blotting, as indicated.
MBP-VP35 IID fusion protein interactions with NP.Forty-five micrograms of pcDNA3 EBOV NP was transfected into 3 × 107 HEK293T cells. At 24 h posttransfection, the cells were lysed in 4 ml NP-40 lysis buffer (50 mM Tris-HCl [pH 8], 280 mM NaCl, 0.5% NP-40, 0.2 mM EDTA, 2 mM EGTA, and 10% glycerol with protease inhibitors). Three hundred fifty microliters of clarified lysate was incubated with the VP35 IID domain fused with MBP or equivalent amounts of the MBP-mutant IID fusion proteins at 4°C. Twenty-four hours later, MBP fusion proteins were bound with 40 μl of amylose resin for 30 min. The resin was washed three times, and MBP fusion proteins were eluted with 100 μl of 10 mM maltose. A fraction of the elution was analyzed by Western blotting. MBP was detected with an anti-MBP antibody (New England Biolabs), and NP was detected with the previously described (see above) anti-NP mouse monoclonal antibody.
To determine if the MBP-VP35 IID fusion proteins could interact with NP in the presence of dsRNA, lysates expressing NP were generated as described above. Before addition of the MBP-VP35 IID protein to the cellular lysate, increasing amounts of poly(I:C) (Invivogen) were first added to the lysate. Poly(I:C) was added at a concentration of either 16 or 98 nM, while either WT or mutant VP35 IID was used at a concentration of 2.4 μM. Following a 24-h incubation period, samples were processed as described above.
First basic patch residues retain dsRNA binding activity.The locations of the first basic patch, central basic patch, and basic residues that border the central basic patch are illustrated in Fig. 1. We previously determined that mutation to alanine of VP35 IID central basic patch residues or of end-cap residues impairs IID dsRNA binding activity and impairs the IFN-antagonist function of full-length VP35 (25). The same mutations, however, had little impact on VP35 polymerase cofactor function (25). In contrast, mutation to alanine of first basic patch residue K222, R225, K248, or K251 did not impair IFN-antagonist function (25). To determine whether the first basic patch mutants affect either the global structure of the VP35 IID or its dsRNA binding activity, we performed a series of nuclear magnetic resonance (data not shown) and ITC experiments. First, 1H/15N heteronuclear single quantum coherence (HSQC) spectra were collected for each mutant from the first basic patch and compared to the corresponding WT spectrum. The results indicated that while some local changes result from the mutation, no global changes occur upon introduction of the alanine substitution. Consistent with these observations, our dsRNA binding studies, using ITC, show that all WT and mutant proteins bind to dsRNA with a similar high affinity, further demonstrating that residues within the first basic patch are not important for dsRNA binding (Fig. 2). In contrast, mutation of residues in the central basic patch, such as R305, K309, R312, K319, R322, and K339, was previously shown to diminish dsRNA binding (25). Altogether, these observations are consistent with previous data indicating that the extent of dsRNA binding by VP35 strongly correlated with its ability to inhibit IFN-α/β production, but it is unlikely that the first basic patch contributes to the IFN antagonism (25).
Highly conserved basic residues are located in three regions within VP35 IID. Ribbon representation of VP35 IID highlighting residues from the first basic patch (A) and central basic patch (B) and border basic residues located outside the central basic patch (C). Electrostatic surface representation of the first basic patch (D) and central basic patch and bordering basic residues (E) (scale, −10 kT/e to +10 kT/e).
ITC binding isotherms and corresponding raw data for 8-bp dsRNA binding to WT (A) and mutant R225A (B), K248A (C), and K251A (D) VP35 IIDs. The corresponding average KD values for WT, R225A, K248A, and K251A are 0.7, 1.0, 2.4, and 0.8 μM, respectively.
First basic patch residues are critical for VP35 polymerase cofactor function.To determine whether the first basic patch mutants are critical for VP35 to function in the viral polymerase complex, a minigenome assay was employed (25, 29, 30, 33). This assay measures expression of a fused GFP-CAT reporter gene expressed from a noninfectious, model viral RNA. Expression of GFP-CAT is entirely dependent upon the presence of a functional EBOV RNA polymerase complex, which is reconstituted by coexpression of the viral NP, VP35, VP30, and L. In the absence of VP35, no reporter gene expression is detected, while in the presence of optimal concentrations of VP35, maximal reporter gene activation is detected (25, 33). These plasmids are cotransfected with a constitutively expressed Renilla luciferase plasmid which serves as a control for transfection efficiency (25, 33). The mutation of R225, K248, or K251 to alanine completely abrogated GFP-CAT reporter gene expression, while the K222A mutant retained activity similar to that of WT VP35, with optimal reporter activation occurring at 125 to 250 ng of VP35 plasmid DNA (Fig. 3). These data demonstrate that three of four first basic patch residues in VP35 are important for viral polymerase cofactor function.
Viral polymerase cofactor function of basic residue-to-alanine first basic patch mutants. Viral polymerase activity was assessed in the minigenome assay. Empty vector (−), WT VP35 (VP35), or the indicated VP35 mutants were assessed. Cells were transfected with empty vector or with increasing amounts of wild-type or mutant VP35 plasmid and expression plasmids for ZEBOV NP, VP30, L, T7 RNA polymerase, and luciferase. A luciferase expression plasmid was cotransfected as a control for transfection efficiency. A plasmid expressing a negative-sense minigenome RNA and reporter construct was also transfected. Minigenome activity was quantified by measuring CAT activity, and this was normalized to the level of expression of luciferase. Error bars represent 1 standard deviation for three experiments. (Lower panel) Western blots (WB) for VP35 proteins. The concentrations of VP35 plasmids used were 63 ng, 125 ng, and 250 ng. WT VP35 at 250 ng was set as 100% minigenome activity.
We also individually substituted glutamic acid residues for the first basic patch residues R225, K248, and K251. For this analysis, we also generated an H240E mutant, since H240 is located near the first basic patch. Previously, an H240A mutant was found to exhibit marginal IFN-antagonist function, but because H240A was poorly expressed in the minigenome assay, the ability of this mutant to function as a polymerase cofactor was uncertain (25, 33). Each of the mutants with the glutamic acid substitution expressed VP35 comparably to WT VP35 by both the minigenome assay and the IFN-antagonist assay (Fig. 4). As was seen with mutation to alanine, mutation to glutamic acid in each of the first basic patch residues also abrogated VP35 polymerase cofactor function, as evidenced by loss of expression from the minigenome (Fig. 4A). Although H240A was poorly expressed in assays for polymerase function (25), the H240E mutation was readily expressed but the mutant exhibited no detectable activity (Fig. 4A). Despite a lack of polymerase cofactor activity, the R225E, K248E, and K251E mutants did retain the ability to inhibit IFN-β promoter activation (Fig. 4B). Consistent with data obtained with the H240A mutant, the H240E mutant exhibited, at best, a minimal ability to inhibit IFN-β promoter activation (IFN-β promoter activity was approximately 90% of that of the no-VP35 control; Fig. 4B).
Viral polymerase cofactor function and IFN-antagonist function of basic residue-to-glutamine first basic patch mutants. (A) Empty vector (EV), wild-type VP35 (VP35), or the indicated first basic patch VP35 mutants were assessed in the minigenome assay, as described in the legend to Fig. 3. (B) Inhibition of IFN-β (IFNb) promoter activation by wild-type and mutant VP35 proteins. HEK293T cells were transfected with empty vector (−) or increasing amounts (1, 2, or 4 μg) of WT VP35 (VP35) or mutant VP35 expression plasmid, an IFN-β promoter-firefly luciferase reporter plasmid, and a constitutively expressed Renilla luciferase reporter plasmid. Firefly luciferase activity was normalized to the level of Renilla luciferase activity. Data are presented such that the activity of the empty vector, SeV-infected samples (+), is set equal to 100%. Error bars represent 1 SD, and corresponding Western blots (WB) of cell lysates are shown below the graphs.
Mutation of basic residues bordering the central basic patch lose polymerase cofactor function.To determine whether the first basic patch residues are unique in their importance for viral polymerase function, the basic residues K282, R283, R298, and R300 were individually mutated to alanine. These residues are located on the periphery of the central basic patch (Fig. 1C). The resulting minigenome data, shown in Fig. 5 A, revealed diminished polymerase cofactor function (Fig. 5A). In order to determine whether the border residue mutants are globally defective for VP35 functions, they were assayed for IFN-antagonist function (Fig. 5B). VP35 with the K282A mutation displayed a modest loss of activity relative to that of WT VP35, which is consistent with the electrostatic interactions between the K282 ε amino group and the phosphodiester backbone of the dsRNA in the VP35 IID/8-bp dsRNA complex structure (25). The remaining border residue mutants exhibited IFN-inhibitory activity comparable to that of WT VP35 (Fig. 5B).
Viral polymerase cofactor function and IFN-antagonist function of border residue mutants. (A) Empty vector (first bar), WT VP35 (VP35), or the indicated border basic residue mutants were assessed for function in the minigenome assay, as described in the legend to Fig. 3. (B) Inhibition of IFN-β promoter activation by WT and mutant VP35 proteins. HEK293T cells were transfected with empty vector (first bar) or increasing amounts (1, 2, or 4 μg) of WT VP35 or mutant VP35 expression plasmid, an IFN-β promoter-firefly luciferase reporter plasmid, and a constitutively expressed Renilla luciferase reporter plasmid. Firefly luciferase activity was normalized to the level of Renilla luciferase activity. Data are presented such that the activity of empty vector, SeV-infected samples, is set equal to 100%. Error bars represent one SD, and corresponding Western blots (WB) of cell lysates are shown below the graphs.
The basic nature of VP35 IID residues, not their identity, is the critical determinant for polymerase cofactor function.To assess whether the basic charge or the identity of the residue is the critical determinant for VP35-mediated polymerase cofactor function, several arginine residues were mutated to lysine, while lysine residues were mutated to arginine in the first basic patch (R225K, K248R, K251R), in a representative border residue (R283K), and at two representative central basic patch positions (R312K and R322K). These mutations were assessed in the minigenome assay, revealing that all VP35 proteins with these mutations functioned similarly to wild-type VP35. Although VP35 proteins with the R283K, R312K, and R322K mutations displayed a moderate decrease in function, this is most likely due to differences in expression of the various plasmids (Fig. 6), as opposed to direct differences in function. These data suggest that having a basic charge at these particular sites is important for VP35 copolymerase function and that arginine and lysine residues can be used interchangeably at these positions with limited perturbation of the VP35 polymerase cofactor function.
Viral polymerase cofactor function of arginine-to-lysine and lysine-to-arginine mutations for representative residues from the first basic patch, border residues, and the central basic patch. Empty vector (−), WT VP35 (VP35), or the indicated mutants were assessed for function in the minigenome assay, as described in the legend to Fig. 3. Error bars represent SDs, and corresponding Western blots (WB) of cell lysates are shown below the graphs. The concentrations of VP35 plasmids used were 125 ng, 250 ng, and 500 ng. Data were expressed as the fold induction over that by the negative control (no VP35).
First basic patch mutants are impaired for interaction with the viral nucleoprotein.VP35 interacts with NP, and this interaction is presumed to be essential for viral polymerase activity (4). To determine whether loss of polymerase activity could be explained by loss of VP35-NP interaction, co-IPs were performed with WT and mutant full-length VP35 proteins which were expressed with NP in HEK293T cells (Fig. 7). First basic patch mutants R225A and K248A displayed reduced levels of VP35 expression in this experiment relative to the level of WT VP35 expression and an unusual migration pattern in which a significant amount of each protein, as detected in the Western blot, ran at a lower rate than was expected. These mutants did not display any detectable interaction with NP. The K251A mutant displayed a greatly reduced interaction with NP. The H240A mutant was also assessed. When it was expressed from the plasmid used in the co-IP, the H240A mutant was detectably expressed, but a fraction of the protein exhibited aberrant migration, similar to that seen with the R225A and K248A mutants. This mutant also lacked the ability to efficiently interact with NP (Fig. 7). The VP35 mutants with a first basic patch mutation to glutamic acid were also tested for their interaction with NP. These VP35 mutants showed a significant reduction in NP binding compared to WT VP35 (data not shown). This reduced binding to NP most likely explains the lack of copolymerase activity demonstrated by these mutants in Fig. 4A. In contrast to the first basic patch mutants, the border residue mutants retained the capacity to interact with NP, despite their loss of polymerase cofactor function. The loss of polymerase cofactor function without the loss of NP interaction is not unique to mutants at these border basic residues. For example, we also assessed interaction with NP by mutants with mutations at either of two phenylalanines important for VP35 end capping of dsRNAs. Mutant F235A was previously demonstrated to lose function as a polymerase cofactor, while mutant F239A retained this function (25). However, both mutants retain the ability to interact with NP (Fig. 7).
Full-length WT and mutant VP35 interactions with EBOV NP. Cells were transfected with WT or mutant VP35 and EBOV NP. The left-most lane (−) corresponds to a sample in which NP was expressed in the absence of VP35. Lysates were subjected to IP with anti-VP35 antibody. Proteins were detected by Western blotting with anti-NP and anti-VP35 monoclonal antibodies.
VP35 IID is sufficient to interact with NP.We next asked whether the VP35 IID is sufficient to interact with NP. Pull-downs were performed by mixing purified MBP-VP35 IID fusions with lysates of HEK293T cells transfected with the NP expression plasmid. MBP alone was used as a negative control. NP was not precipitated in the absence of added protein or in the presence of MBP alone. In contrast, WT MBP-VP35 IID precipitated NP, demonstrating that the VP35 IID is sufficient for interaction with NP (Fig. 8 A). When the first basic patch mutants were assessed for NP binding, only the MBP-VP35 IID K222A mutant retained binding to NP, consistent with the fact that this mutant retains its function in the polymerase complex. In contrast, the other first basic patch mutants, which lost function as a polymerase cofactor, also lost their interaction with NP. Specifically, MBP-VP35 IID R225A and K248A mutants lacked any detectable binding to NP, whereas the MBP-VP35 IID K251A mutant showed significantly reduced coprecipitation with NP (Fig. 8B). Also consistent with the results of the co-IP assays, MBP-VP35 IID F235A and F239A mutants retained the ability to bind to NP, while MBP-VP35 IID H240A did not pull down NP (Fig. 8B).
The VP35 IID is sufficient to interact with NP, and this interaction is impaired by either specific point mutations or the presence of dsRNA. (A) MBP or MBP-VP35 IID was added to NP-transfected cell lysates. MBP was immobilized by amylose resin, and proteins were detected with anti-MBP (α-MBP) or anti-NP (α-NP) antibodies. (B) MBP alone, MBP-VP35 IID WT, or mutants with point mutations were added to lysates containing EBOV NP. MBP was precipitated using amylose resin and was detected with NP and MBP antibodies. (C) MBP, WT MPB-VP35 IID, or MBP-VP35 IID mutants (R312A, K322A, or F239A) were added to NP-transfected lysates in the absence or presence of poly(I:C). The final concentration of each MBP was 2.4 μM, and poly(I:C) was present at either 16 or 98 nM. MBPs were pulled down, and both MBP and NP were detected in the same way as described for panels A and B. The input whole-cell extract lane (WCE) expressing NP represents 4% of the total lysate used for each group, while the MBP pull-down lane represents 15% of the total pull down.
Recent structural studies suggest that dsRNA binding can lead to high-order complex formation between dsRNA and VP35 (D. W. Leung, G. K. Amarasinghe, and C. F. Basler, unpublished observations). In order to assess if VP35-NP interactions are sensitive to the dsRNA binding functionality of VP35, we analyzed VP35-NP interactions in the presence of dsRNA. To this end, increasing amounts of poly(I:C) were added to a MBP-VP35 IID-NP coprecipitation experiment. The left panel of Fig. 8C represents the input amount of NP before the addition of each MBP. As the poly(I:C) concentration increased, the WT VP35 IID-NP interaction was impaired (Fig. 8C, right panel). In contrast, the ability of the central basic patch dsRNA binding mutants R312A and R322A to interact with NP was unaffected by the dsRNA (Fig. 8C, right panel). F239A is an end-cap residue mutant that also displays reduced dsRNA binding (25). As demonstrated above (Fig. 7), this mutant exhibits reduced binding to NP. Consistent with the results obtained with dsRNA binding mutants R312A and R322A, interaction of this mutant with NP was unaffected by addition of poly(I:C) (Fig. 8C). Therefore, these results suggest that dsRNA binding by the VP35 central basic patch can modulate the VP35-NP interaction.
Impact of basic residue mutations on VP35 interaction with L.VP35 interaction with L is also required for filoviral RNA synthesis (28). Characterizing the interactions of filovirus L with other components of the viral RNA polymerase complex is complicated by difficulties in detecting the expression of full-length L. However, a previous study demonstrated that the marburgvirus (MARV) VP35 can interact with the amino-terminal 503 amino acids of the MARV L (28). This interaction required the presence of a functional coiled-coil oligomerization motif within the amino-terminal half of VP35 (28). We therefore screened WT and mutant full-length ZEBOV VP35 proteins for interaction with a similar construct, HA-tagged ZEBOV L (encompassing amino acids 1 to 505). Full-length VP35, as well as all mutants tested, retained interaction with L (Fig. 9). Therefore, loss of interaction between VP35 and L does not appear to explain why mutation of the basic residues surrounding the central basic patch causes loss of polymerase cofactor function.
Full-length WT and mutant VP35 interactions with EBOV L. Cells were transfected with WT or mutant VP35 and EBOV HA-L amino acids 1 to 505 (L1-505). Lysates were immunoprecipitated with anti-VP35 antibody and protein G beads. Proteins were detected with HA or VP35 monoclonal antibodies. WT and mutant full-length VP35 can coimmunoprecipitate with HA-L amino acids 1 to 505. WC, whole-cell extract.
The molecular basis for the high virulence of EBOVs is incompletely understood. Recent studies have, however, provided insight into the structure and function of VP35, a protein that functions as a virulence factor, that suppresses innate immunity, and that acts as an essential component of the viral polymerase (21, 23, 25, 26). To date, VP35 virulence factor function has been correlated with its ability to bind to dsRNA and to suppress IFN-α/β responses. Its function as part of the viral polymerase complex appears to require its interaction with the viral NP and L (4, 29, 30). Notable structural features of VP35 relevant to its IFN-antagonist function are a central basic patch and a set of hydrophobic residues that end cap dsRNAs within the carboxy-terminal IID (25). Mutations to either of these sets of residues can disrupt VP35-dsRNA binding and impair its ability to inhibit IFN-α/β production. Central basic patch mutations are also sufficient to severely attenuate virus replication (15, 33). The present studies have identified additional features within the VP35 IID that are also of functional importance. In contrast to the central basic patch and end-cap residues, the current study demonstrates that the residues of the first basic patch as well as other basic residues surrounding the central basic patch are not required for VP35 interaction with dsRNA. These residues are, however, essential for VP35 polymerase cofactor function.
VP35 is an essential component of the filoviral polymerase complex. This has been demonstrated with systems analogous to the minigenome system described above (28-30). The minimal set of filoviral proteins required for transcription and replication for MARV are NP, VP35, and L, although recovery of recombinant MARVs from cDNA also required expression of VP30 (9). Transcription of EBOV subgenomic replicon RNAs requires NP, VP35, VP30, and L; however, the replication reaction can proceed in the absence of VP30 (5, 30). Omission of VP35 from either system abolishes reporter gene expression, demonstrating an essential role for VP35 as a viral polymerase cofactor. The importance of VP35 for viral polymerase function is also supported by the observation that Zaire EBOV VP35 could not functionally substitute for Reston EBOV VP35 when the remaining nucleocapsid proteins, NP, VP30, and L, were Reston EBOV derived (5).
VP35 interactions with NP and with L are expected to be essential for viral polymerase function. Similar to rhabdovirus and paramyxovirus systems, where viral phosphoprotein is thought to be the functional equivalent of VP35, VP35 can complex with L, the enzymatic subunit of viral polymerase (4). Because viral genomes and antigenomes are NP associated, VP35 presumably brings L to its template RNAs. On the basis of similarity to rhabdoviruses and paramyxoviruses, VP35 may also chaperone NP to prevent its association with cellular RNAs. Consistent with these models, VP35 from either EBOV or MARV has been demonstrated to interact with NP and L by colocalization and coimmunoprecipitation studies (4, 28). Our current work demonstrates that the VP35 C-terminal IID is sufficient for interaction with NP and that the first basic patch residues are critical for both viral polymerase function and VP35-NP interaction. These studies also provide the first residue-specific interaction sites on VP35 that are important for VP35-NP binding. The behavior of these mutants in the minigenome system further demonstrates that the interactions between VP35 and NP are critical for viral polymerase function. Our data presented above indicate that the C-terminal VP35 IID is sufficient to interact with NP, suggesting that VP35 may possess a single NP-interacting interface, although this remains to be formally tested, as the VP35 N-terminal oligomerization domain may contain additional interaction elements (28). Additionally, our results from VP35-NP binding in the presence of dsRNA suggests that although residues in the central basic patch are not important for VP35-NP binding, this interaction may be sensitive to the oligomerization state(s) that results from VP35 binding to dsRNA. Several lines of evidence, including our own unpublished data, suggest that VP35 IID can form multimeric complexes upon dsRNA binding. Therefore, the observed loss of VP35-NP binding for WT VP35 but not for VP35 mutants that lack dsRNA binding suggests that the NP binding site is likely to include elements outside the first basic patch.
The basis for loss of polymerase function upon mutation of the basic residues bordering the central basic patch remains undefined. MARV VP35 was previously demonstrated to interact with amino acids 1 to 503 of MARV L by coprecipitation, and the amino-terminal 879 amino acids of Reston EBOV L were sufficient to interact with the Zaire or Reston EBOV NP-VP35 complex, as assessed by colocalization studies (5, 28). We therefore tested ZEBOV WT and mutant VP35 proteins for interaction with ZEBOV L amino acids 1 to 505. Because MARV VP35 requires an intact oligomerization function and because this oligomerization function is mediated by an amino-terminal coiled-coil motif, we tested full-length VP35 rather than monomeric VP35 IID for interaction with the L amino terminus. Our data demonstrate that the N-terminal region of L is sufficient to interact with full-length VP35 and that all of the mutants tested retain the ability to interact with this region of L. Therefore, the loss of polymerase cofactor function upon mutation of the basic residues that border the central basic patch is unlikely to be due to the loss of VP35-L interactions.
EBOV VP35 is reported to interact with a variety of other viral and host proteins (7, 20, 22, 32). For example, VP35 is reported to interact with NP to form filamentous nucleocapsids when these proteins are coexpressed with the viral VP24 protein (19, 36). EBOV VP35 interaction with the EBOV matrix protein VP40 has been demonstrated by a mammalian two-hybrid assay and by immunofluorescence studies, and this interaction could mediate packaging of a model viral genomic RNA into virus-like particles (20). VP35 interactions with cellular kinases IκB kinase ε and TANK binding kinase 1 (TBK-1) and with interferon regulatory factor 7 (IRF7), ubiquitin-conjugating enzyme 9 (Ubc9), and protein inhibitor of activated signal transducer and activators of transcription 1 (PIAS1) are reported to influence its capacity to inhibit IFN-α/β production (7, 32). VP35 also inhibits the IFN-induced, dsRNA-activated kinase PKR, potentially countering the antiviral effects of PKR (10, 35), and can also inhibit RNA silencing (12). Whether the basic residues studied here affect these interactions and functions remains to be determined.
Currently, there are no approved vaccines or treatment for the EBOVs, in large part due to the paucity of knowledge regarding the specific molecular features critical for the function of viral components. Therefore, the identification of structural features, such as those described here, can potentially facilitate development of antivirals. To this end, we recently demonstrated that mutation to alanine of two basic residues within the central basic patch, K319 and R322, abrogated VP35 dsRNA binding activity and greatly reduced its ability to inhibit IFN-α/β production. This mutation did not significantly impair VP35 function as a polymerase cofactor (33). However, when they were introduced into a recombinant EBOV, the same mutations in VP35 greatly attenuated virus replication in cells that produce IFN-α/β and rendered the virus avirulent in guinea pigs (33). Mutation of another central basic patch residue, R312, also impaired the ability of a recombinant EBOV to suppress IFN responses and greatly attenuated replication in mice (15-17). Together, these data validate the central basic patch as a potential target for antivirals. Similarly, the hydrophobic residues that mediate VP35 interaction with the ends of dsRNA are required for full IFN-antagonist function and are therefore candidate targets as well (25). The present study defines additional elements within the VP35 IID, including the first basic patch and residues that border the central basic patch, as specific targets for antiviral development. Strategies to target these residues through mutations or with inhibitors that interact with these residues would be predicted to impair virus polymerase function, thereby limiting virus replication.
This work is supported by NIH grants (grant 1R56AI089547 to C.F.B. and G.K.A., grant 1F32AI084324 to D.W.L., grants R01AI059536 and AI057158 [Northeast Biodefense Center-Lipkin] to C.F.B., grant 1F32AI084453 to R.S.S., and grant R01AI081914 to G.K.A.), an MRCE developmental grant (grant U54AI057160-Virgin to G.K.A.), and the Roy J. Carver Charitable Trust (grant 09-3271 to G.K.A.).
Received 29 April 2010.
Accepted 27 July 2010.
↵*Corresponding author. Mailing address: Department of Microbiology, Mount Sinai School of Medicine, Box 1124, 1 Gustave L. Levy Place, New York, NY 10029. Phone: (212) 241-4847. Fax: (212) 534-1684. E-mail: chris.basler{at}mssm.edu
↵▿ Published ahead of print on 4 August 2010.
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Journal of Virology Sep 2010, 84 (20) 10581-10591; DOI: 10.1128/JVI.00925-10
You are going to email the following Basic Residues within the Ebolavirus VP35 Protein Are Required for Its Viral Polymerase Cofactor Function


A Dimensionally Interchangeable with THK HSR A, B, CA & CB carriage has hardened steel load bearing plates. The rail HSR R and TR and Thomson 511 E 35. 23. 23.5. 36. 29.5. 22. 88. 57. 45. M6. M8. M6. 7. 10.7. 12. 8. 10. 60. 3000.
HKR / miniHK series are mounted on floating bearings and so no lateral forces . PMK 35-7. Example: ordering our MK series. The tables on the overview pages . HSR..A, HSR..B. MK/MKS 1501 A. 24. 1. HSR..R. MK/MKS 1501 A. PMK 15-4 LB, HSR..CA, HSR..HA,. HSR..CB, HSR..HB. MK/MKS 3501 A. PMK 35-3. 48.
mounted on floating bearings, which means that the lifting movements of the contact . HSR. 15. A, B. MK/MKS 1501 A. 24. 1. R. MK/MKS 1501 A. PMK 15-4. 28. 20 PMK 35-7. 55. 45. A, LA, B, LB, CA, HA, CB, HB. MK/MKS 4501 A. 60. 46.
BMC 35*. HGH 35HA. LGH 35HA. HSR 35LR SHS 35LR. LH 35BN. 1624-3XX. LWHDG BMA 35*. HGW 35CC. LGW 35CB. HSR 35B. HSR 35CB*. LH 35FL. LWH 35..B . Replace with rail/bearing combination from Hiwin's HG & EG products.
and LM block used, type HSR is in use in a wide range of heavy-cutting . HSR 35 L. HSR 45. HSR 45 L. HSR 55. HSR 55 L. HSR 65. HSR 65 L. HSR 85.
HK 3501 A. PMK 35-4. 48. 31. 45. SR..W, SR..TB. HK 4501 A. 60. 43. 55. SR..W, SR..TB. HK 5501 S. 68. 48. HSR. 15. HSR..A, HSR..AM, HSR..B, HSR..BM, HSR.
M 5 x 6. LAH25 AN/ANZ. 79. 58. 35. 11.5. LAH25 BN/BNZ. 40. 7. 12.5. 48. 35. 107. 86. 50. 18 .. Using the plastic provisional rail supplied, slide linear bearing on to the linear rail. 2. Linear Guide Series. NSK. THK. Thomson. LH. HSR. CG. Linear Guide Sizes. NSK .. Mexico: NSK Rodamientos Mexicana, S.A. de C.V..
35. CAD DRAWING DATA. For LE/LH/LS/LU & LW-Series go to NSK technical website www.npa.nsk.com. For 3D NSK has a plating for linear bearings and rails to cover conditions requiring wet, corrosive and clean room RAIL PART NUMBER. ASSEMBLY PART NUMBER. LAH 20. AN. Z. HSR. 20. R HSR##TB/CB/B.
With cage-less, full-ball types of ball bearings, balls make metallic contact with one Model SHS has the same dimensions as model HSR, which is the de facto .. H1. Shoulder height for the LM block. H2. E. Unit: mm. 15. 20. 25. 30. 35. 45.
21 Dec 2008 Translation of a reporter transcript bearing the HSR was inhibited by 24-h, . acid mixture and translation-grade [35S]methionine (Perkin Elmer) and cytosolic . Liu L, Cash TP, Jones RG, Keith B, Thompson CB, Simon MC.


text:
Q: QTcpServer retrieve more than one address I use QTcpServer from the Qt-Framework with C++.
I started it with
ret = tcpServer->listen(QHostAddress::Any, 9871)

With
QHostAddress serverAddress () const

I retrieve the Address from it. Which is 0.0.0.0. In my opinion it should listen on more than one address (like 127.0.0.1 and the LAN Address).
Is there a way to retrieve more than one address from this class?

A: I'm not sure what your question is exactly, but...
Usually, a 0.0.0.0 means that the socket listen to all interfaces that are both up and configured with a valid IPv4 address.
If you want to get all the available IP addresses on the system, you should enumerate the network interface then query their respective IPv4 address (Probably with an ioctl, specifying SIOCGIFSWADDR).

A: I agree with ereOn's answer above.
If you want to list your network interface adresses, take a look at QNetworkInterface.

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Graphics display system having a moving cursor, such as a cursor, for selecting a displayed entity with the capability of determining which one of several lower level entities in the vicinity of the cursor is to be selected. The correlation of a cursor with a entity intended to be selected is performed by calculating an index that does not represent the actual distance, which is time consuming, but rather represents a value, more quickly calculated, such that the size order among the indices is the same as the size order of the distances they represent.
U.S. Pat. No. 3,997,891 is hereby incorporated by reference to illustrate one of the input methods and its practice with which this invention would be useful.
The subject matter of this patent application is related to the subject matter of copending patent application Ser. No. 926,928 filed Nov. 3, 1986, U.S. Pat. No. 4,731,609.
This invention relates to computer-aided design systems, i.e., computer controlled graphics and particularly to computer-aided design applications where one of many drawings, views, patterns, lines, or points can be selected for modification by the user. For purposes of explanation, computer-aided drawings comprise a hierarchy, the top being a drawing, a drawing being comprised of one or more views, a view being comprised of one or more patterns, a pattern being comprised of one or more lines, and lines being defined by points.
An entity is a member of any level in the hierarchy.
The subject matter includes cursor mark position control having a manual means which determines the position and controls particular energization of a display element to which the control by the input means is directed.
In computer controlled graphics, a cursor is an on-screen symbol used to supply feedback to the user by visually indicating the position and set membership of selected displayed entities. A moveable cursor can be used to select entities for purposes of altering the displayed graphics.
Where many entities appear on the screen, e.g., boxes, circles, nuts and bolts, and polygons, selecting the desired entity by moving a selection device to the proximity of the entity to be identified is not an easy process. The mathematical computations require more time because of the increase in the number of points and lines to be taken into account.
The selection devices for computer-aided design (CAD) applications include styli on digitized tablets, mouse, light pens, or joy sticks. Such devices function to return a set of x,y coordinates that indicate where the selection device specifying the cursor has been positioned with respect to the system of coordinates used in the display system. When trying to select a given point or line with a selection device, it is difficult to place the selection device exactly at the x,y coordinates of the entity to be selected.
Closely spaced entities increase the difficulties of selecting the desired entity. Correlation, as used herein, is the ability to associate a selected x,y coordinate to a screen entity without ambiguity. Existing techniques use mathematical algorithms for calculating whether each individual vector displayed on a graphic screen is correlated to the x,y position returned by an input device. As the number of vectors displayed on the screen increases, the time required to correlate increases proportionately, since more calculations have to be made on more vectors until the correct vector is identified.
When CAD workstations were connected to large main frame processing units, which were very fast, the time required for the calculations was short. Using such CAD applications on smaller processors, such as personal computers, requires longer periods of time for correlation.
A substantial amount of time is required to correlate graphic data on a CRT screen, and when a large amount of geometry is displayed, the correlation calculations use a large amount of time. Complex geometry in many systems cannot be conveniently edited because of the amount of time required. In graphic programs run on personal computers, correlation usually degrades performance of the system.
The invention to be described has a correlation time, independent from the amount of data and provides almost instantaneous correlation of the selected entity, even when used with slow processors. The invention to be described is device independent and can be adapted for CADAM, e.g., trapping.
Low cost raster devices have graphic hardware that are not capable of differentiating between a single line or multiple lines occupying the same end points. Therefore, in the case of multiple lines having the same x,y coordinates of end points, if one line is to be erased, the system cannot determine whether there are lines beneath it, leaving a space where the line should have been. The effect is that the screen does not display the actual information relating to the drawing. To restore the lines beneath a deleted line, the system has to redraw the display, which is a slow process and requires and operator to constantly work with inaccurate information, constantly selecting the redraw operation. The invention eliminates the above described problems.
U.S. Pat. No. 3,997,891 describes a light pen detection system for detecting the character display are on a cathode ray tube (CRT) display. The character near the pen is marked and, if not the desired one, the detection process continues until an indication is provided indicating that the correct character has been detected. The system according to this patent responds to light on the detecting surface of the pen which means that the selected character is the one having a portion thereof at the point on the display where the pen is positioned.
In accordance with the invention a graphics system for displaying entities from a bit map storing the pels of said entities, including a correlation array for storing tags, each tag associated a separate entity and stored in locations in the array corresponding to the bit map locations occupied by the associated entity, and also having a moveable cursor for selecting a displayed entity by converting the x,y coordinates of the cursor to a location in said correlation array to extract the tag of the selected entity, correlates a part of said selected entity with said cursor selecting whether said part of said selected entity is a line or a point, representing each displayed entity by a command vector stored in a buffer memory, the command vector including the end points of lines comprising the displayed entity, and storing in a file buffer a list of tags identifying the displayed entities cross referencing the buffer memory address of the command vector corresponding to the displayed entity for each tag. The cross referenced address is accessed in said file buffer corresponding to the selected entity and, from the buffer memory, the command vector corresponding to the selected entity is retrieved. The vector includes the x,y coordinates of the end points of the lines comprising the selected entity which are used to calculate an index value corresponding to the distance between the cursor and each selected part of the entity. The selected part thereof having the lowest index value is selected as the desired part of the selected entity.
The invention is described in detail by referring to the various figures which illustrate specific embodiments of the invention, and wherein like numerals refer to like elements.
FIG. 1 is a block diagram of a system in which the invention is useful.
FIG. 2 is an illustration of a buffer command entry or vector.
FIG. 3 is an illustration showing the relationship between a cursor and the points defining a polygon.
FIG. 4 is an illustration showing the relationship between two cursors and two line.
FIG. 1 demonstrates a system in which the invention can be utilized and shows the details of interconnections among the various devices and of memory entries.
The input to the system is shown as supplied by a digitizing tablet 101, e.g., commercially available as an IBM Model 5083 II, which supplies signals identifying the x- and y-coordinates of the stylus (or pointer) 102 on the tablet and a select signal which is activated by the operator, e.g., by pushing down on the stylus to activate a switch therein, to signify that a selected point is to be used.
A signal from control unit 110, usually a programmable processor, enables the system to specify a desired sensitivity, i.e., to specify the size of the area around the pointer which should react to the positioning of the pointer.
The display is stored as bits in a screen map 17 which has a bit cell for storing each dot as presented on the screen of a CRT display 16. Normally, if a bit in the screen map 17 is set, i.e., is true, turned on, has a binary value of one, the corresponding point on the screen of the display 16 will be lighted. If a bit in the screen map 17 is reset, i.e., is false, turned off, has a binary value of zero, then the corresponding point on the screen of the display 16 is dark.
When a point is indicated by a moveable cursor, the x,y coordinates of the cursor are to be associated with one of the displayed entities. In this explanation, it will be assumed that the entity selected to be displayed is at the pattern level and that some information as to the selected line or point is also to be determined.
The usual method for associating a cursor point with an entity is to compare the x,y coordinates of the cursor with all the x,y coordinates of the displayed entities. Such a comparison can be expedited by determining whether the cursor's x,y coordinates fall on a line between two points that define a line instead of comparing the coordinates with all the points of the same line. Such calculation is well known in analytic geometry and involves computing the equation of the line associated with its two end points followed by a substitution of x,y values to determine whether the cursor is on the line. Other comparisons are required to ascertain that the cursor is between the two end points since the cursor could be beyond the end points and still fulfill the mathematical requirements for being part of the line.
where m is the number of cells per row.
This method provides a fast correlation between a cursor's position and the entity at that position. In such a display system, it is desirable to have high resolution which requires that the screen map 17 have a large capacity. As the number of dots on the display screen, sometimes called pels (picture elements), the ability to place the cursor exactly on an entity becomes more difficult. It also requires that the correlation array 10 be large as well.
An entity to be drawn may first be selected from a menu on the screen of display 16 using the digitized tablet 101 and stylus 102 to position the pointer on the display screen 15. When the desired figure is selected, a description of the procedure to draw the selected figure is displayed on the screen. For example, the instructions for drawing a line may comprise selecting each end point defining the line.
where x1,y1 and x2,y2 are the selected end points of the line. (Similar algorithms are available for circles, text, and other forms.) The formula may be used by substituting for x all the intermediate values from x1 to x2 to find the corresponding y intermediate values. These points are converted to memory addresses in the the screen map 17 and the corresponding bits set. At the same time, the corresponding cells in the correlation map can be calculated as explained above and the tag assigned to the entity stored therein. That is, the correlation array cells (or elements) corresponding to the intermediate points are computed and each computed element in the array corresponding to all the points of the line will contain the tag number representing the line stored therein.
Assuming that the-tag of the line in the illustration is assigned the value of 1, then the array elements 4, m+3, m+2, 2m+1, and 2m will store the tag value of 1.
A buffer memory 19 stores command entries in the form illustrated in FIG. 2. Each such entry or command vector has a field for specifying the color of the figure to be displayed. One field contains a command illustrated in FIG. 2 as DRAW contains the x- and y-coordinates of the end points of the vectors making up the entity corresponding to the command entry. The example in FIG. 2 comprises a typical display list structure. An actual structure may be more complicated, but the important point is that, because x,y coordinates are stored in a display list in a simple, low precision format (typically sixteen bits) and because the list is stored in memory, the computations necessary to perform the correlation to a particular point or line can be accomplished extremely fast. After selection, the tag can be used to extract high precision x,y coordinates from a disk-based data base.
In FIG. 1, execution unit 18 converts the vector information to a raster scan format, storing the desired display information in a screen map 17. The display information is bit-mapped from the screen map 17 to the raster scan display unit 16, shown with a line 15 displayed as the vector stored in the correlation map 10.
The control unit 110 also stores an element list 11. For each entity corresponding to a tag, the beginning and ending addresses of the command vector corresponding entity in the buffer memory 19.
A data base is also maintained in the control unit 110 to permit manipulation of the entities at several levels in a hierarchy as described above.
Color fill-ins and cross-hatching of entities can be stored and moved about as separate entities.
The point xt,yt in FIG. 3 represents the position of a cursor defined by the position of a stylus on a digitized tablet, of a light pen on a CRT display, of a mouse on a surface, and the like.
The cursor is first correlated to an entity. In the illustrated example, it is assumed that the cursor is within n (where n continues to represent the group size of pels) pels of the line from x1,y1 to x2,y2 or of the line from x2,y2 to x3,y3. The entity, i.e., the rectangle 30, will be highlighted on the display screen to indicate that it has been selected.
The computations being described are being continually carried on because the cursor may be in motion. Also, the wrong entity is sometimes selected so the user will move the cursor closer to the desired entity until the desired entity has been selected. Highlighting, i.e., brightening the lines of the entity, is one of the methods of indicating which entity has been selected. Other indications may include linking the lines comprising the selected entity or changing their color.
Besides selecting a desired entity, it may also be desirable to select a given line or a given point within that entity. Therefore, in addition to selecting the desired entity and highlighting it, it is also necessary to select the nearest line or point, whichever has been previously designated by the user. In this illustration, a point will be marked by blinking a marker at the point and a line, by blinking a marker at the center point of the line.
To correlate the cursor to the nearest point, an index value related to the distance of the cursor from each point in the entity being stored and displayed--the rectangle 30 in this illustrative example shown in FIG. B--is calculated and the stored point corresponding to the smallest index value is determined to be the selected point.
In the prior art, the actual distance between the cursor and all other points is calculated using the square root of the quantity (xt-xi)2 +(yt-yi)2.
When the entity has been selected, the element list 11 is accessed and the beginning and ending address of the command vector representing the rectangle 30 in the buffer memory 19 are used to extract the command vector. The four points of representing the entities corners are available from the command vector so retrieved.
where the square root is not necessary because the actual distance is not required, merely a value related to the distance and permits ordering the points according to distance. This reduces the time required to calculate each index.
As illustrated in FIG. 3, the point x2,y2 is closest to the cursor and is, therefore, selected. It is usually emphasized on the display as a brighter portion or highlighted cross on the point. Alternatively, the selected point may be identified by a blinking marker such as a small cross centered on the selected point.
where xk,yk and xj,yj are the point pairs that identify each line tested in the display.
When testing for proximity to a line, an additional test is required to verify that the cursor is not correlated to a phantom line such as the extension of a line. In FIG. 4, a dotted line 46 shows an extension of a line defined by the end points x7,y7 and x8,y8. A cursor at the location xt2,yt2 would be incorrectly correlated to the extension line because it is closer thereto than it is to the line with the end points x5,y5 and x6,y6. It is, however, desired to have the cursor xt2,yt2 correlated to the latter line.
where M is actually the slope of the line designated by end points x1,y1 and x2,y2. Division by two is easily accomplished in microprocessors by shifting the quantity to be divided one bit position to the right. Error accumulation if y2 is close to y1 or if x2 is close to x1 can be reduced by standard techniques well known in the computer art.
After calculating the values of X and Y, a test is made to insure that X falls between x1 and x2 and that Y falls between y1 and y2. If they do not, then the line is not considered as a candidate and the end point pairs are not stored even though the index value is less than the index value of the previous line.
While the invention has been particularly shown and described with reference to a preferred embodiment thereof, it will be understood by those skilled in the art that various changes and modifications in form and details may be made therein without departing from the spirit and scope of the invention according to the following claims.
selecting as the desired part of the selected entity the selected part thereof having the lowest index value.
discarding as the selected line any line which would not be intersected by a normal from the cursor.
DE19873789856 DE3789856D1 (en) 1986-11-03 1987-09-04 Fast point / line correlations in graphical structures.
DE19873789856 DE3789856T2 (en) 1986-11-03 1987-09-04 Fast point / line correlations in graphical structures.
Bantz and Hatfield, "Correlation of Coded Graphic Representations with Cursor Position", IBM Technical Disclosure Bulletin, vol. 20, No. 2, 1977, pp. 806-807.