Wit, grit and a supercomputer yield chemical structure of HIV capsid

May 29, 2013 — A team led by researchers at the University of Pittsburgh School of Medicine has described for the first time the 4-million-atom structure of the HIV’s capsid, or protein shell. The findings, highlighted on the cover of the May 30 issue of Nature, could lead to new ways of fending off an often-changing virus that has been very hard to conquer.

Scientists have long struggled to decipher how the HIV capsid shell is chemically put together, said senior author Peijun Zhang, Ph.D., associate professor, Department of Structural Biology, University of Pittsburgh School of Medicine.

“The capsid is critically important for HIV replication, so knowing its structure in detail could lead us to new drugs that can treat or prevent the infection,” she said. “This approach has the potential to be a powerful alternative to our current HIV therapies, which work by targeting certain enzymes, but drug resistance is an enormous challenge due to the virus’ high mutation rate.”

Previous research has shown that the cone-shaped shell is composed of identical capsid proteins linked together in a complex lattice of about 200 hexamers and 12 pentamers, Dr. Zhang said. But the shell is non-uniform and asymmetrical; uncertainty remained about the exact number of proteins involved and how the hexagons of six protein subunits and pentagons of five subunits are joined. Standard structural biology methods to decipher the molecular architecture were insufficient because they rely on averaged data, collected on samples of pieces of the highly variable capsid to identify how these pieces tend to go together.

Instead, the team used a hybrid approach, taking data from cryo-electron microscopy at an 8-angstrom resolution (a hydrogen atom measures 0.25 angstrom) to uncover how the hexamers are connected, and cryo-electron tomography of native HIV-1 cores, isolated from virions, to join the pieces of the puzzle. Collaborators at the University of Illinois then used their new Blue Waters supercomputer to run simulations at the petascale, involving 1 quadrillion operations per second, that positioned 1,300 proteins into a whole that reflected the capsid’s known physical and structural characteristics.

The process revealed a three-helix bundle with critical molecular interactions at the seams of the capsid, areas that are necessary for the shell’s assembly and stability, which represent vulnerabilities in the protective coat of the viral genome.

“The capsid is very sensitive to mutation, so if we can disrupt those interfaces, we could interfere with capsid function,” Dr. Zhang said. “The capsid has to remain intact to protect the HIV genome and get it into the human cell, but once inside it has to come apart to release its content so that the virus can replicate. Developing drugs that cause capsid dysfunction by preventing its assembly or disassembly might stop the virus from reproducing.”

The project was funded by National Institutes of Health grants GM082251, GM085043 and GM104601 and the National Science Foundation.

“By using a combination of experimental and computational approaches, this team of investigators has produced a clearer picture of the structure of HIV’s protective covering,” said the National Institutes of Health’s Michael Sakalian, Ph.D., who oversees this and other grants funded through an AIDS-related structural biology program. “The new structural details may reveal vulnerabilities that could be exploited by future therapeutics.”

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Gene therapy gives mice broad protection to pandemic flu strains, including 1918 flu

May 29, 2013 — Researchers at the Perelman School of Medicine, University of Pennsylvania have developed a new gene therapy to thwart a potential influenza pandemic. Specifically, investigators in the Gene Therapy Program, Department of Pathology and Laboratory Medicine, directed by James M. Wilson, MD, PhD, demonstrated that a single dose of an adeno-associated virus (AAV) expressing a broadly neutralizing flu antibody into the nasal passages of mice and ferrets gives them complete protection and substantial reductions in flu replication when exposed to lethal strains of H5N1 and H1N1 flu virus. These strains were isolated from samples associated from historic human pandemics — one from the infamous 1918 flu pandemic and another from 2009.

Wilson, Anna Tretiakova, PhD, Senior Research Scientist, Maria P. Limberis, PhD, Research Assistant Professor, all from the Penn Gene Therapy Program, and colleagues published their findings online this week in Science Translational Medicine ahead of print. In addition to the Penn scientists, the international effort included colleagues from the Public Health Agency of Canada, Winnipeg; the University of Manitoba, Winnipeg; and the University of Pittsburgh. Tretiakova is also the director of translational research, and Limberis is the director of animal models core, both with the Gene Therapy Program.

“The experiments described in our paper provide critical proof-of-concept in animals about a technology platform that can be deployed in the setting of virtually any pandemic or biological attack for which a neutralizing antibody exists or can be easily isolated,” says Wilson. “Further development of this approach for pandemic flu has taken on more urgency in light of the spreading infection in China of the lethal bird strain of H7N9 virus in humans.”

At the Ready Influenza infections are the seventh leading cause of death in the United States and result in almost 500,000 deaths worldwide per year, according to the Centers for Disease Control. The emergence of a new influenza pandemic remains a threat that could result in a much loss of life and worldwide economic disruption.

There is also interest by the military in developing an off-the-shelf prophylactic vaccine should soldiers be exposed to weaponized strains of infectious agents in biologic warfare.

Human antibodies with broad neutralizing activity against various influenza strains exist but their direct use as a prophylactic treatment is impractical. Now, yearly flu vaccines are made by growing the flu virus in eggs. The viral envelope proteins on the exterior, namely hemagglutinin, are cleaved off and used as the vaccine, but vary from year to year, depending on what flu strains are prevalent. However, high mutation rates in the proteins result in the emergence of new viral types each year, which elude neutralization by preexisting antibodies in the body (specifically specific receptor binding sites on the virus that are the targets of neutralizing antibodies).

This approach has led to annual vaccinations against seasonal strains of flu viruses that are predicted to emerge during the upcoming season. Strains that arise outside of the human population, for example in domestic livestock, are distinct from those that normally circulate in humans, and can lead to deadly pandemics.

These strains are also not effectively controlled by vaccines developed to human strains, as with the 2009 H1N1 pandemic. The vaccine development time for that strain, and in general, was not fast enough to support vaccination in response to an emerging pandemic.

Knowing this, the Penn team proposed a novel approach that does not require the elicitation of an immune response, which does not provide sufficient breadth to be useful against any strain of flu other than the one for which it was designed, as with conventional approaches.

The Penn approach is to clone into a vector a gene that encodes an antibody that is effective against many strains of flu and to engineer cells that line the nasal passages to express this broadly neutralizing antibody, effectively establishing broad-based efficacy against a wide range of flu strains.

A Broad Approach The rational for targeting nasal epithelial cells for antibody expression was to focus this expression to the site of the body where the virus usually enters the body and replicates which is the nasal and oral mucosa. Antibodies are normally expressed from B lymphocytes so one challenge was to design vectors that could deliver antibody genes to the non- lymphoid respiratory cells of the nasal and lung passages and could express functional antibody protein.

Targeting the respiratory cells was achieved through the use of a vector based on a primate virus — AAV9 — which was discovered in the Wilson laboratory and evaluated previously by Limberis for possibly treating patients with cystic fibrosis. The team constructed a genetic payload for AAV9 that expressed an antibody that was showed by other investigators to have broad activity against flu.

Efficacy of the treatment was tested in mice that were exposed to lethal quantities of three strains of H5N1 and two strains of H1N1, all of which have been associated with historic human pandemics (including the infamous H1N1 1918).

Flu virus rapidly replicated in untreated animals all of which needed to be euthanized. However, pretreatment with the AAV9 vector virtually shut down virus replication and provided complete protection against all strains of flu in the treated animals. The efficacy of this approach was also demonstrated in ferrets, which provide a more authentic model of human pandemic flu infection.

“The novelty of this approach is that we’re using AAV and we’re delivering the prophylactic vaccine to the nose in a non-invasive manner, not a shot like conventional vaccines that passively transfer antibodies to the general circulation,” says Limberis.

“There’s a long history of using antibodies for cancer and autoimmune disease, but only two have been approved for infectious diseases,” notes Tretikova. “This novel technique has allowed for the development of a prophylactic passive vaccine that is cost effective, easily administered, and quickly manufactured.”

The team is working with various stakeholders to accelerate the development of this product for pandemic flu and to explore the potential of AAV vectors as generic delivery vehicles for countermeasures of biological and chemical weapons.

The research was supported in part by ReGenX, the Public Health Agency of Canada (#531252), the Canadian Institutes of Health Research (#246355) and the National Institutes of Health (GM083602).

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Neuroscientists discover new phase of synaptic development

May 29, 2013 — Students preparing for final exams might want to wait before pulling an all-night cram session — at least as far as their neurons are concerned. Carnegie Mellon University neuroscientists have discovered a new intermediate phase in neuronal development during which repeated exposure to a stimulus shrinks synapses. The findings are published in the May 8 issue of the Journal of Neuroscience.

It’s well known that synapses in the brain, the connections between neurons and other cells that allow for the transmission of information, grow when they’re exposed to a stimulus. New research from the lab of Carnegie Mellon Associate Professor of Biological Sciences Alison L. Barth has shown that in the short term, synapses get even stronger than previously thought, but then quickly go through a transitional phase where they weaken.

“When you think of learning, you think that it’s cumulative. We thought that synapses started small and then got bigger and bigger. This isn’t the case,” said Barth, who also is a member of the joint Carnegie Mellon/University of Pittsburgh Center for the Neural Basis of Cognition. “Based on our data, it seems like synapses that have recently been strengthened are peculiarly vulnerable — more stimulation can actually wipe out the effects of learning.

“Psychologists know that for long-lasting memory, spaced training — like studying for your classes after very lecture, all semester long — is superior to cramming all night before the exam,” Barth said. “This study shows why. Right after plasticity, synapses are almost fragile — more training during this labile phases is actually counterproductive.”

Previous research from Barth’s lab established the biochemical mechanisms responsible for the strengthening of synapses in the neocortex, the part of the brain responsible for thought and language, but only measured the synapses after 24 hours. In the current study, post-doctoral student Jing A. Wen investigated how the synapses developed throughout the first 24 hours of exposure to a stimulus using a specialized transgenic mouse model created by Barth. The model senses its surroundings using only one whisker, which alters its ability to sense its environment and creates a sensory imbalance that increases plasticity in the brain. Since each whisker is linked to a specific area of the cortex, researchers can easily track neuronal changes.

Wen found that during this first day of learning, synapses go through three distinct phases. In the initiation phase, synaptic plasticity is spurred on by NMDA receptors. Over the next 12 hours or so, the synapses get stronger and stronger. As the stimulus is repeated, the NDMA receptors change their function and start to weaken the synapses in what the researchers have called the labile phase. After a few hours of weakening, another receptor, mGluR5, initiates a stabilization phase during which the synapses maintain their residual strength.

Furthermore, the researchers found that they could maintain the super-activated state found at the beginning of the labile phase by stopping the stimulus altogether or by injecting a glutamate receptor antagonist drug at an optimal time point. The findings are analogous to those seen in many psychological studies that use space training to improve memory.

“While synaptic changes can be long lasting, we’ve found that in this initial period there are a number of different things we could play with,” Barth said. “The discovery of this labile phase suggests there are ways to control learning through the manipulation of the biochemical pathways that maintain memory.”

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RA diastolic dysfunction

Nancy Walsh writing in MedPage Today reported, “Patients with rheumatoid arthritis (RA) have an increased incidence of diastolic dysfunction, meaning the heart doesn’t fill with blood properly. This may further raise their already high risk for congestive heart failure, a meta-analysis suggested.” Researchers found that “mean left atrial size was larger in RA patients than in controls. The investigators also found that pulmonary artery pressure was higher. The findings were published in Arthritis Care & Research.Comment: RA patients already are at risk for heart problems. This raises a whole other issue.http://youtu.be/cPP-c_vL8Vc20%

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