HCV is a significant human pathogen, infecting more than three percent of the world’s population. The incidence of infection in the United States has been estimated to be as high as 4 million cases.
In the March issue of the journal PLoS Pathogens, Timothy Tellinghuisen, an assistant professor in the Department of Infectology at Scripps Florida, and his colleagues describe how they used mutations of the viral NS5A phosphoprotein to disrupt virus particle production at an early stage of assembly. NS5A has long been proposed as a regulator of events in the HCV life cycle, but exactly how it orchestrates these events has been unclear.
“The interesting thing about this mutant is that while it triggers totally normal RNA replication, it causes severe defects in the output of infectious virus—in fact, it releases no infectious virus that we can detect,” says Tellinghuisen. “And though this discovery isn’t a cure for HCV, it is an important research tool that stops the assembly pathway.” Total disruption of the replication process would be a cure for the disease, he adds, and that’s the team’s long-term goal.
HCV infection is roughly five to seven times more prevalent than HIV, underscoring the pandemic nature of HCV infection. HCV occurs when blood from an infected person enters the body of someone who is not infected. Most new HCV infections are due to illegal drug injections and sharing needles. However, those who had blood transfusions prior to blood donor screening in 1991, healthcare workers who had needle stick accidents, and hemodialysis patients are also at risk for developing HCV infection. The virus predominantly infects the liver, and following many decades of virus reproduction serious disease such as hepatitis (liver inflammation), cirrhosis (liver scarring), and carcinoma (liver cancer) develop. Ultimately, HCV infection destroys the liver, resulting in death. Attempts at curing HCV infections with drug therapy have been only marginally successful.
Before more effective therapies can be developed, scientists need to understand, at the molecular level, the detailed mechanisms HCV uses to infect cells, replicate itself, assemble progeny virus, and exit the cell. Each of these processes could potentially be a target for a new drug to eliminate HCV infection. HCV, like all viruses, requires the normal cellular machinery for its replication and has developed strategies to utilize normal cell physiology for its own benefit (often to the detriment of the host).
The Tellinghuisen team, which includes Research Assistants Katie L. Foss and Jason Treadaway, has focused recent efforts on NS5A to understand the regulation of events used by the virus to assemble infectious copies of itself and exit the cell. NS5A is a three-domain protein, which means it is comprised of three compactly folded regions roughly 50 to 300 amino acids in length. The requirement of domains I and II for RNA replication is well documented. NS5A domain III, however, is not required for RNA replication, and the function of this region in the HCV life cycle is unknown.
Using standard molecular biology, the researchers removed from domain III of NS5A a coding sequence corresponding to roughly 15 amino acids. Then they generated a clone of the virus, transcribed the RNA from that clone, and purified the RNA. This RNA, which is directly infectious, was then transfected into a liver cell line where it produced all the HCV proteins that are encoded by that RNA genome.
“Those proteins assemble in the cell to make a structure called a replicase that then copies the viral RNA,” Tellinghuisen explains. “We measured that RNA accumulation and observed no defect in RNA replication, but found, surprisingly, that no infectious viral particles were released from the cells.” The team also found that no viral RNA nor nucleocapsid protein are released from cells, indicating that an early event in virus assembly had been affected.
Using genetic mapping and biochemical analyses, the authors were able to show that their deletion altered a phosphorylation signal controlling the switch from RNA replication to virus particle assembly. This signal was attributed to the activity of a cellular kinase that when inhibited by genetic or chemical means led to a reduction in infectious virus production without altering HCV RNA replication.
“These data provide the first evidence for a function of domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV,” Tellinghuisen says. “The ability to uncouple virus production from RNA replication may be useful in understanding HCV assembly and may become therapeutically important.”
Charles M. Rice, head of the Center for the Study of Hepatitis C at Rockefeller University, comments, “This is a spectacular advance linking a specific phosphorylation event by a cellular kinase to hepatitis C virus assembly. Remarkably, the target is a viral nonstructural protein, NS5A, and the data point to a pivotal role for this protein in regulating RNA amplification and infectious virus production. These new data make this multifunctional protein an even more attractive target for developing new anti-virals for treating hepatitis C.”
This project was funded by a Career Development Award from the National Institutes of Allergy and Infectious Diseases of the National Institutes of Health, and by the State of Florida.
About The Scripps Research Institute
The Scripps Research Institute is one of the world's largest independent, non-profit biomedical research organizations, at the forefront of basic biomedical science that seeks to comprehend the most fundamental processes of life. Scripps Research is internationally recognized for its discoveries in immunology, molecular and cellular biology, chemistry, neurosciences, autoimmune, cardiovascular, and infectious diseases, and synthetic vaccine development. Established in its current configuration in 1961, it employs approximately 3,000 scientists, postdoctoral fellows, scientific and other technicians, doctoral degree graduate students, and administrative and technical support personnel. Scripps Research is headquartered in La Jolla, California. It also includes Scripps Florida, whose researchers focus on basic biomedical science, drug discovery, and technology development. Currently operating from temporary facilities in Jupiter, Scripps Florida will move to its permanent campus in 2009.
For information:Keith McKeown
Keith McKeown | EurekAlert!
Exciting Plant Vacuoles
14.06.2019 | Julius-Maximilians-Universität Würzburg
A microscopic topographic map of cellular function
13.06.2019 | University of Missouri-Columbia
Light can be used not only to measure materials’ properties, but also to change them. Especially interesting are those cases in which the function of a material can be modified, such as its ability to conduct electricity or to store information in its magnetic state. A team led by Andrea Cavalleri from the Max Planck Institute for the Structure and Dynamics of Matter in Hamburg used terahertz frequency light pulses to transform a non-ferroelectric material into a ferroelectric one.
Ferroelectricity is a state in which the constituent lattice “looks” in one specific direction, forming a macroscopic electrical polarisation. The ability to...
Researchers at TU Graz calculate the most accurate gravity field determination of the Earth using 1.16 billion satellite measurements. This yields valuable knowledge for climate research.
The Earth’s gravity fluctuates from place to place. Geodesists use this phenomenon to observe geodynamic and climatological processes. Using...
Discovery by Brazilian and US researchers could change the classification of two species, which appear more akin to jellyfish than was thought.
The tube anemone Isarachnanthus nocturnus is only 15 cm long but has the largest mitochondrial genome of any animal sequenced to date, with 80,923 base pairs....
Researchers at Chalmers University of Technology, Sweden, have discovered a completely new way of capturing, amplifying and linking light to matter at the nanolevel. Using a tiny box, built from stacked atomically thin material, they have succeeded in creating a type of feedback loop in which light and matter become one. The discovery, which was recently published in Nature Nanotechnology, opens up new possibilities in the world of nanophotonics.
Photonics is concerned with various means of using light. Fibre-optic communication is an example of photonics, as is the technology behind photodetectors and...
Fraunhofer IZM is joining the EUROPRACTICE IC Service platform. Together, the partners are making fan-out wafer level packaging (FOWLP) for electronic devices available and affordable even in small batches – and thus of interest to research institutes, universities, and SMEs. Costs can be significantly reduced by up to ten customers implementing individual fan-out wafer level packaging for their ICs or other components on a multi-project wafer. The target group includes any organization that does not produce in large quantities, but requires prototypes.
Research always means trying things out and daring to do new things. Research institutes, universities, and SMEs do not produce in large batches, but rather...
29.04.2019 | Event News
17.04.2019 | Event News
15.04.2019 | Event News
14.06.2019 | Information Technology
14.06.2019 | Materials Sciences
14.06.2019 | Medical Engineering