How rabies virus moves through nerve cells, and how it might be stopped

Posted on by Catherine Zandonella
Cells in red against black background

To successfully infect its host, the rabies virus must move from the nerve ending to the nerve cell body where it can replicate. In a study published July 20 in the journal PLoS Pathogens, researchers from Princeton University reveal that the rabies virus moves differently compared to other neuron-invading viruses and that its journey can be blocked by a drug commonly used to treat amoebic dysentery.

Most viruses only infect the nervous system accidentally when the immune system is compromised. But some “neurotropic” viruses have evolved to target neurons as part of their normal infectious cycle. The rabies virus, for example, is transmitted when an infected animal bites into a host’s muscle. It then spreads into the end terminals of motor neurons innervating the muscle and travels along the neurons’ long axon fibers to the neuronal cell bodies. From there, the virus can spread throughout the central nervous system and into the salivary glands, where it can be readily transmitted to other hosts. Though rabies infections in humans are rare in the United States, the virus kills nearly 60,000 people annually.

Alpha herpesviruses, such as herpes simplex viruses, also enter peripheral nerve terminals and move along axons to the neuronal cell body, where they can lie dormant for the life of the host.

“Transport to the neuronal cell body is not a passive process, but an active one relying on the neuron’s own motor proteins and microtubule tracks,” said Lynn Enquist, Princeton’s Henry L. Hillman Professor in Molecular Biology, a professor of molecular biology and the Princeton Neuroscience Institute, and the study’s senior author. “Virus particles must engage this machinery for efficient transport in axons, otherwise infection cannot start.”

Viruses moving in a narrow nerve cell
Twenty-four hours after infecting nerve terminals, the rabies virus (red) reaches the cell bodies (green) of control neurons (left) but not neurons treated with emetine (right). Image credit: MacGibeny et al., 2018.

Enquist and colleagues previously found that alpha herpesviruses engage the neuronal transport machinery by stimulating protein synthesis at infected nerve terminals. Viral transport to the cell body can therefore be blocked by drugs that inhibit protein synthesis, as well as by cellular antiviral proteins called interferons.

In the current study, Enquist and colleagues investigated how the rabies virus engages the neuronal transport machinery. The researchers infected neurons with a virulent strain of the virus tagged with a red fluorescent protein, allowing the researchers to observe viral transport in real time by live-cell fluorescence microscopy.

The study was led by Margaret MacGibeny, who earned her Ph.D. in 2018, and associate research scholar Orkide Koyuncu, at Princeton, with contributions from research associate Christoph Wirblich and Matthias Schnell, professor and chair of microbiology and immunology at Thomas Jefferson University.

In contrast to alpha herpesvirus infections, the team found that interferons had no effect on rabies virus transport, perhaps because, until it reaches the neuronal cell body, the rabies virus hides out inside cellular structures called endosomes.

“We also couldn’t detect increased protein synthesis in axons upon rabies virus infection,” MacGibeny said. “But, to our surprise, we saw that a protein synthesis inhibitor called emetine efficiently blocked rabies virus transport to the cell body.”

Emetine had no effect on the transport of endosomes devoid of the rabies virus. But endosomes carrying the virus were either completely immobilized, or were only able to move short distances at slower-than-normal speeds.

Other protein synthesis inhibitors did not block rabies virus transport, however, suggesting that emetine works by inhibiting a different process in infected neurons.

“Emetine has been used to treat amoebic dysentery,” Koyuncu said. “In the laboratory it is widely used to inhibit protein synthesis but there are recent reports indicating that emetine has anti-viral effects that are independent of protein synthesis inhibition. Our study shows that this drug can inhibit rabies virus invasion of the nervous system through a novel mechanism that hasn’t been reported before.”

“The manuscript by MacGibeny et al. both advances and complicates our understanding of how neurotropic viruses make their way from the axon terminus to the cell body,” said Professor Glenn Rall, an expert in neurotropic virus infections at Fox Chase Cancer Center, who was not involved in the study. “Revealing variations in the axonal transport of neurotropic viruses, coupled with intriguing insights into new roles for well-known drugs, has both mechanistic and clinical implications for these life-threatening infections.

“Our next step is to figure out how emetine disrupts rabies virus transport in axons,” Enquist says. “Does it inhibit cell signaling pathways after rabies virus entry, or does it directly block the recruitment of motor proteins to virus-carrying endosomes?”

This study was funded by the US National Institutes of Health (grants P40 OD010996, RO1 NS33506, and F30 NS090640).

The study, Retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons, by Margaret A. MacGibeny, Orkide O. Koyuncu, Christoph Wirblich, Matthias J. Schnell, and Lynn W. Enquist was published in PLoS Pathogens. 2018. DOI: 10.1371/journal.ppat.1007188

Text courtesy of the Department of Molecular Biology

Genetically engineered mice could boost fight against aggressive hepatitis

Posted on by Catherine Zandonella

Article provided by the Department of Molecular Biology

Hepatitis delta virus (HDV) causes the most aggressive form of viral hepatitis in humans, putting at least 20 million people worldwide at risk of developing liver fibrosis, cirrhosis, and liver cancer. Efforts to develop effective treatments against HDV have been hampered by the fact that laboratory mice are not susceptible to the virus. But, in a study published June 27, 2018, in the journal Science Translational Medicine, Alexander Ploss, assistant professor of molecular biology at Princeton University and colleagues describe a genetically engineered mouse that can be persistently infected with HDV.

HDV is a small, RNA-based “satellite” virus that produces just a single protein of its own and therefore requires additional proteins provided by another liver virus, hepatitis B virus (HBV). HDV can infect patients already carrying HBV, or both viruses can infect patients simultaneously. Though infections can be prevented with an anti-HBV vaccine, there are no antiviral therapies available to cure existing HDV infections.

HDV and HBV infect the liver by binding to a protein called NTCP that is present on the surface of liver cells. But the viruses only recognize the version of NTCP present in humans and a few other primates, and therefore can’t infect mice or other small mammals that produce their own versions of NTCP. This has made it difficult to study HBV and HDV infections in the laboratory. Researchers have tried transplanting human liver cells into immunocompromised mice before infecting them with virus, but this approach has produced inconsistent results and is both expensive and time-consuming.

Ploss and colleagues, led by graduate student Benjamin Winer, took a different approach. They generated mice that express the human NTCP protein in their liver cells, allowing these cells to be infected by HBV and HDV.

In these mice, HBV failed to replicate after entering mouse liver cells but HDV was able to establish persistent infection when provided with the HBV proteins it needs to propagate. For example, mice genetically engineered to produce both human NTCP and the entire HBV genome could be infected with HDV for up to 14 days. “To our knowledge, this is the first time the entire HDV life cycle has been recapitulated in a mouse model with inheritable susceptibility to HDV,” Ploss said.

The mice were able to rid themselves of HDV before they developed any liver damage, apparently by mounting an immune response involving antiviral interferon proteins and various white blood cell types, including Natural Killer (NK) cells and T cells. Accordingly, mice expressing human NTCP and the HBV genome, but lacking functional B, T, and NK cells could be infected with HDV for two months or more.

These immunocompromised animals allowed Ploss and colleagues to test the effectiveness of two drugs that are currently being developed as treatments for HDV infection. Both drugs—either alone or in combination—suppressed the levels of HDV in immunocompromised mice after viral infection. But the drugs were not able to completely clear the mice of HDV; viral levels rose again within weeks of stopping treatment.

“This is largely in line with recently reported data from clinical trials, showing the utility of our model for preclinical antiviral drug testing,” Winer said.

“Our model is amenable to genetic manipulations, robust, and can be adopted as a method to rapidly screen for potential treatments,” Ploss added.

Timothy M. Block, president of the Hepatitis B Foundation and its Baruch S. Blumberg Institute who was not involved in the study, said “These systems should be able to provide practical, and presumably economical  tools. Their work is urgently needed, and a desperate community welcomes it. I emphasize that it is often the new methods in science that revolutionize a field such as drug discovery, almost as much as the new drugs themselves.”

The research team included collaborators from Princeton University; Weill Medical College of Cornell University; The Jackson Laboratory; University Medical Center Hamburg-Eppendorf, Hamburg; New York University Medical Center; and North Carolina State University College of Veterinary Medicine.

This study was supported by grants from the National Institutes of Health (R01 AI079031, R01 AI107301, R21AI117213 to Alexander Ploss), a Research Scholar Award from the American Cancer Society (RSG-15-048-01-MPC to Alexander Ploss), a Burroughs Wellcome Fund Award for Investigators in Pathogenesis (to Alexander Ploss) and a Graduate fellowship from the Health Grand Challenge from the Global Health Fund of Princeton University (to Benjamin Y. Winer). The NYU Experimental Pathology Immunohistochemistry Core Laboratory is supported in part by the Laura and Isaac Perlmutter Cancer Center Support Grant; NIH/NCI P30CA016087 and the National Institutes of Health S10 Grants NIH/ORIP S10OD01058 and S10OD018338. Benjamin Y. Winer is a recipient of F31 NIH/NRSA Ruth L. Kirschstein Predoctoral awarded from the NIAID. Julie Sellau and Elham Shirvani-Dastgerdi are both recipients of postdoctoral fellowships from the German Research Foundation. Michael V. Wiles was funded by The Jackson Laboratory.

Benjamin Y. Winer, Elham Shirvani-Dastgerdi, Yaron Bram, Julie Sellau, Benjamin E. Low, Heath Johnson, Tiffany Huang, Gabriela Hrebikova, Brigitte Heller, Yael Sharon, Katja Giersch, Sherif Gerges, Kathleen Seneca, Mihai-Alexandru Pais, Angela S. Frankel, Luis Chiriboga, John Cullen, Ronald G. Nahass, Marc Lutgehetmann, Jared Toettcher, Michael V. Wiles, Robert E. Schwartz, and Alexander Ploss. Preclinical assessment of antiviral combination therapy in a genetically humanized mouse model for persistent hepatitis delta virus infection. Science Translational Medicine. 2018. DOI: 10.1126/scitranslmed.aap9328

Researchers find an alternative mode of bacterial quorum sensing

Posted on by Catherine Zandonella

By the Department of Molecular Biology

Whether growing in a puddle of dirty water or inside the human body, large groups of bacteria must coordinate their behavior to perform essential tasks that they would not be able to carry out individually. Bacteria achieve this coordination through a process called quorum sensing in which the microorganisms produce and secrete small molecules called autoinducers that can be detected by neighboring bacterial cells. Only when a large number of bacteria are present can the levels of secreted autoinducer build up to the point where the community can detect them and respond as a coordinated group.

In a paper published last month in PLoS Pathogens, a team of researchers led by postdoctoral research associate Sampriti Mukherjee and professor Bonnie Bassler of the Department of Molecular Biology at Princeton University revealed the existence of a new quorum-sensing molecule that increases the virulence of the pathogenic bacterium Pseudomonas aeruginosa. The finding could help researchers develop new antimicrobial drugs to treat the serious infections caused by this bacterium.

P. aeruginosa is an incredibly adaptable organism that can grow in environments ranging from soil and freshwater to the tissues of plants and animals. It thrives on the surfaces of medical equipment and is therefore a major cause of hospital-acquired infections, causing life-threatening conditions such as pneumonia and sepsis in vulnerable patients. The bacterium has become resistant to commonly used antibiotics, making the development of new antimicrobials a priority for both the Centers for Disease Control and Prevention and the World Health Organization.

Quorum sensing is crucial for P. aeruginosa‘s adaptability. The process regulates the development of biofilms, the three-dimensional structures formed by large bacterial communities that promote their ability to establish and maintain infections. “P. aeruginosa strains harboring mutations in the quorum-sensing machinery are attenuated for virulence, and thus, interfering with quorum sensing holds promise for the development of novel anti-microbial therapies,” said Bassler, the Squibb Professor in Molecular Biology at Princeton University and a Howard Hughes Medical Institute Investigator.

P. aeruginosa possesses similar quorum-sensing machinery to other species of bacteria. For example, it produces an enzyme called RhlI that synthesizes an autoinducer molecule known as N-butanoyl-L-homoserine lactone, or C4-HSL. This molecule can then bind and activate a protein called RhlR that regulates the expression of multiple genes that P. aeruginosa needs to form a biofilm and/or infect a host.

Biofilms
Caption: Compared to a typical, or wild-type (WT) colony (left), P. aeruginosa cells lacking RhlR form a much more wrinkled biofilm (middle), while cells lacking RhlI form a biofilm that is abnormally smooth (right). Credit: Mukherjee et al., 2017.

In theory, removing RhlI or RhlR should have the same effect on P. aeruginosa cells, since the latter protein shouldn’t be able to work without the autoinducer produced by the former. But the researchers, led by postdoctoral fellow Sampriti Mukherjee, noticed that bacterial colonies lacking RhlI formed unusually smooth biofilms, whereas strains lacking RhlR formed biofilms that were much more wrinkled than normal.

The researchers went on to show that in biofilms, many genes only depended on RhlR, not on RhlI. “That suggested that RhlR can be activated by an alternative molecule, in addition to C4-HSL,” Bassler said.

The researchers found that bacteria lacking RhlI, which are therefore unable to synthesize the C4-HSL autoinducer, still secrete a molecule capable of activating RhlR. Bassler and colleagues don’t yet know what this molecule is, but it seems to be quite different from C4-HSL. “We are currently working to purify and identify this molecule,” Bassler said.

Researchers at Princeton discovered that a molecule called RhlR, which is important for the ability of P. aeruginosa to infect animals, can be activated by an alternative molecule, in addition to C4-HSL. Image credit: Mukherjee et al., 2017.

Crucially, the activation of RhlR by this unknown molecule may be important for P. aeruginosa‘s ability to infect animals. Mukherjee and the team found that bacteria lacking RhlI were just as effective as wild-type bacteria in infecting both roundworms and mice. But bacteria lacking RhlR were much less virulent and far less able to grow inside these animals. “Targeting RhlR with small-molecule inhibitors could provide an exciting path forward for the development of novel antimicrobial drugs,” Bassler said.

The ability of RhlR to be activated by distinct molecules might also help explain P. aeruginosa‘s adaptability. Bassler and colleagues speculate that different environments could stimulate discrete levels of production of the different autoinducer molecules, each of which could activate RhlR, or a related protein called LasR, to induce expression of the specific genes the bacteria need to thrive in that particular location.

The work was supported by the Howard Hughes Medical Institute, the National Institutes of Health (grant 2R37GM065859), the National Science Foundation (grant MCB-0948112), and a Life Science Research Foundation Postdoctoral Fellowship through the Gordon and Betty Moore Foundation (grant GBMF2550.06).

In addition to Mukherjee and Bassler, the team consisted of postdoctoral fellow Dina Moustafa and professor Joanna Goldberg in the Department of Pediatrics at Emory University School of Medicine, and Chari Smith, a research consultant at Princeton University.

The study, “The RhlR quorum-sensing receptor controls Pseudomonas aeruginosa pathogenesis and biofilm development independently of its canonical homoserine lactone autoinducer,” by Sampriti Mukherjee, Dina Moustafa, Chari D. Smith, Joanna B. Goldberg, and Bonnie L. Bassler, was published in the journal PLoS Pathogens on July 17, 2017. DOI:10.1371/journal.ppat.1006504.

An immune signaling pathway for control of Yellow Fever Virus infection

Posted on by Catherine Zandonella

By the Department of Molecular Biology

Princeton University researchers have uncovered a critical role for a new immune signaling pathway in controlling infection by the flavivirus Yellow Fever Virus (YFV).  The paper describing this discovery was published today in the journal mBio.

Infection with YFV causes a devastating illness with a mortality rate of up to 50%.  Like other members of its viral family—which includes West Nile Virus, Dengue Virus and Zika Virus—YFV is transmitted to humans by mosquitos that are expanding into new areas across the globe, exposing more people to these dangerous viruses. Fortunately, there is an effective vaccine for YFV: a live-attenuated strain of the virus, called YFV-17D, which differs by only a few amino acids from the virulent viral strain YFV-Asibi, but nonetheless provokes a potent and durable protective immune response in humans.

“An improved understanding of the complex mechanisms regulating YFV-17D attenuation will provide insights into key viral-host interactions that regulate host immune responses and infection outcomes, [and] open novel avenues for the development of innovative vaccine strategies,” said Alexander Ploss, assistant professor at Princeton’s Department of Molecular Biology, who led the study with first author Florian Douam, a postdoctoral research associate. However, research efforts have been hampered due to the fact that mice, which are used in the study of viral infections, are resistant to YFV infection. Nonetheless, recent mouse experiments have pointed to an important role for cytokines called interferons (IFN) in controlling the virus.

Mice, like humans, possess three types of interferons, molecules produced by the immune system during infection: type I interferons, which signal through the widely distributed IFN-α/β receptor; type II interferons that act on IFN-γ receptors present in most tissues; and type III interferons, which activate signaling by IFN-λ receptors found on epithelial cells. Mice lacking type I receptors die after infection by YFV-Asibi, but survive YFV-17D infection despite extensive viral replication at early stage of infection.Type II IFN signaling has also been shown to be important for clearing up late stage YFV-Asibi and YFV-17D infection when type I IFN signaling is defective. By contrast, the contribution of type III IFN signaling to control of YFV infection was unknown.

To address this question, Douam and colleagues studied YFV-17D infection in mice lacking the type III receptor. Initial experiments showed that these mice were able to control viral replication and rapidly cleared YFV-17D, indicating that type III signaling alone wasn’t necessary for resistance to YFV-17D. However, mice lacking both type I and type III receptors succumbed after YFV-17D infection, suggesting type III signaling does contribute to the antiviral immune response.

To find out more, the authors examined YFV-17D levels in various tissues. Early in the infection, the virus was present in every tissue of each mouse model examined. However, although viral loads were low in wild-type mice and type III receptor-deficient mice, they were much higher in type I and type I/III receptor-deficient mice. Surprisingly, the viral loads in brains of type I/III receptor-deficient mice increased over time in comparison to type I receptor-deficient mice, showing that loss of type III IFN signaling enhances the susceptibility of type I-receptor deficient animals to brain infection. This is significant because the presence of viruses in the brain can cause brain damage such as spongiosis or encephalitis. The low level of YFV-17FD brain invasion in wild-type mice caused mild spongiosis, whereas type I/III receptor-deficient mice had severe spongiosis—potentially explaining YFV-17D lethality in those animals.  However, this raised the question of why YFV-17 was present at such high levels in the animals’ brains.

Brain tissues
Left panel: Loss of Type I IFN signaling leads to active replication of the attenuated YFV strain (YFV-17D), which is accompanied by viral invasion of the brain and damage to brain tissues (spongiosis). Right panel: The additional loss of Type III IFN signaling in Type I IFN-deficient mice impairs the integrity of the blood-brain-barrier and alters immune cell function, which aggravates spongiosis and is ultimately lethal. Image Credit: Florian Douam and Alexander Ploss

Another study recently showed that type III IFN signaling affects the epithelial cells that make up the blood brain barrier (BBB), and modulates BBB integrity during infection by another flavivirus, West Nile Virus. Consistent with this, Ploss’s group observed that the BBB of type I/III receptor-deficient mice was especially leaky to a blue dye. But this wasn’t the only way that loss of type III IFN signaling impaired the body’s response to YFV; the researchers also found evidence that type III receptor deficiency provokes strong imbalances in several different kinds of immune cells during YFV-17D infection. In particular, type I/III receptor-deficient mice were defective in the activation of T cells, critical immune cells that control YFV-17D infection.

“We uncovered a critical role of type III IFN-mediated signaling in preserving the integrity of the blood brain barrier and preventing viral brain invasion,” Ploss said. More work is needed to explore how type III IFN signaling affects YFV infection in primates, but this study already provides important new insights about a poorly understood immune signaling pathway.

The study was supported by grants from National Institutes of Health (R01 AI107301, R21AI117213 to Alexander Ploss and R01 AI104669 to Sergei Kotenko). Additional funding included a Research Scholar Award from the American Cancer Society (RSG-15-048-01-MPC), the Princeton Environmental Institute‘s Grand Health Challenge program from Princeton University, and an Investigator in Pathogenesis Award by the Burroughs Wellcome Fund (all to Alexander Ploss).

The study, “Type III Interferon-mediated signaling is critical for controlling live attenuated Yellow Fever Virus infection in vivo.,” by Florian Douam, Yentil E. Soto-Albrecht, Gabriela Hrebikova, Evita Sadimin, Christian Davidson, Sergei V. Kotenko and Alexander Ploss was published in the journal mBio.  (2017). doi:10.1128/mBio.00819-17