As the cause of chronic hepatitis—a human disease that causes degradation of the liver—the hepatitis C virus (HCV) has long been of interest to researchers and diagnosticians. But HCV is difficult to sustain in a cell culture because liver cells don’t function normally in vitro; this has thwarted much of the research into possible therapeutics.
But this may all be changing, thanks to recent studies by scientists Charles Rice, Sangheeta Bhatia, and colleagues from Rockefeller University’s Center for the Study of Hepatitis C and Laboratory of Virology and Infectious Disease. Their recent work has led to the development of micropatterned cell co-cultures that better sustain HCV for live analysis.
The Rockefeller team has now gone on to develop a new fluorescent reporter system to tag and follow the virus as it infects a host cell. This technique is an upgrade from previous HCV analysis techniques that required either the lysing or fixing of cultured cells. This makes the new technique ideal for precious cell or virus samples, like clinical material, because fixation is not required.
Live analysis, in real time
The new fluorescent reporter system takes advantage of one of HCV’s mechanisms to evade the host immune system. The HCV protease NS3-4A cleaves the protein IPS-1 (also known as MAVS, VISA, or Cardif), which is normally tethered to the cell’s mitochondria and plays a role in innate immune signaling. According to Rockefeller’s Christopher Jones, lead author on the recent Nature Biotechnology paper describing the technique, the researchers used this knowledge to create an artificial, mitochondria-bound substrate that was based on a section of the DNA sequence from IPS-1. The substrate provides control over IPS-1 targeting to the mitochondria as well as NS3-4A recognition of IPS-1. This artificial substrate was then fused to a red fluorescent protein, so that upon HCV infection, NS3-4A completely cleaves the substrate from the mitochondria, releasing the fluorescent protein in an event clearly visible using a fluorescent microscope.
“The real advantage of our reporter system is that the cells can be alive when the imaging is done,” Jones, also of the Center for the Study of Hepatitis C, told BioTechniques. “After cells expressing the reporter are infected, you only need to wait about 14 to 16 hours to see the translocation, which is earlier than most other methods.”
Through their analysis of HCV-infected liver cells in live culture, Jones and his team were able to demonstrate the use of the cell-based fluorescent reporter system to capture live images of drug inhibition of HCV, and the virus spreading by an alternate entry route. The researchers discovered that infected cells undergo a transient stress response, shown by the appearance of stress granules late in infection.
The technique also enabled the imaging of HCV infection in primary human hepatocytes for the first time. “Visualization of primary hepatocyte infection may be an especially valuable advance,” said Jones. “Using this system we may begin to unravel the ways in which HCV causes disease, something that is difficult when studying infection of already cancerous hepatoma cell lines.”
Seeking hepatitis C’s secrets
By enabling the analysis of live cultures, Jones hopes the new system will shed light on some of the mystery surrounding HCV. According to Jones, previous systems were limited to only one viral strain and one cell line. Naturally HCV is far more diverse, with seven distinct genotypes, which the new technique can analyze.
“It would be ideal to study all of these in the lab, but finding new HCV strains that are able to grow in culture requires the ability to detect them,” said Jones. This is exactly what the new analysis technique can do, without destroying the samples, he says.
The new technique is sensitive and able to detect infection earlier than other methods because the fluorescent protein is already highly expressed in the cells and does not need to be made by the virus. Also advantageous is its ability to image individual living cells as they are infected in real time; this allows researchers to correlate infection with other cellular processes.
According to Jones, this technique was specifically designed for hepatitis C, and only works through the NS3-4A and IPS-1 pathway. “We were careful to demonstrate that cleavage of the reporter is specific for HCV infection,” said Jones. “Other viruses—such as yellow fever and Venezuelan equine encephalitis virus, which express their own viral proteases—did not cause cleavage of the reporter. These findings tell us that the reporter was specific for HCV.” However, it may be possible to tailor the reporter to allow detection of other viruses that don’t naturally target IPS-1, which is something the researchers hope to explore.
The Rockefeller researchers will continue to screen for infections using patient isolates in primary hepatocyes to work toward developing treatments for HCV infection. The researchers will also continue to correlate infection and cellular responses to understand the interactions between the virus and host. “Now that we have developed a way of tracking HCV in culture, we will certainly continue to use it to study this important human pathogen,” said Jones.
Funding for this research was provided by grants from the Public Health Service, and the National Institutes of Health Office of the Director Roadmap for Medical Research Initiative. The paper, “Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system,” was published Jan. 31, 2010 in Nature Biotechnology.