Using Fireflies to Measure HIV Replication

Image courtesy of Kara Tao.

Have you ever seen a field of fireflies? If you have, you were probably thinking about how magical the experience was. Or maybe you were wondering how these insects somehow evolved the ability to bioluminescence. But chances are you were not thinking about how firefly luminescence would be a great tool for measuring protein-protein interactions.

A history of co-opting bioluminescence

The phenomenon of bioluminescence, which is the biochemical emission of light by living organisms, has fascinated humans for millennia and has been exploited for just as long. Roman naturalist Pliny the Elder wrote that one could create a torch by rubbing the slime of a luminous jellyfish onto a walking stick. In the 17th century, physician Georg Rumphius documented how indigenous peoples of Indonesia used bioluminescent fungi as flashlights in forests. Then, in 1875, Raphel Dubois reported the first in vitro demonstration of bioluminescence. 

Dubois made two extracts of the bioluminescent clam Pholas, one with hot water and another using cold water. The light in the cold sample eventually disappeared. Furthermore, when he heated the hot sample to near boiling, the glow stopped. But when he mixed those two samples together, he observed light emission once again. Dubois concluded from his observations that a key aspect of bioluminescence comes from a heat-stable organic molecule he named luciferin and an enzyme called luciferase. Today, we understand that luciferase is an enzyme that catalyzes a light-producing reaction in the presence of oxygen and the naturally occurring substrate, luciferin. But scientific interest in the chemistry of the luciferin-luciferase reaction didn’t stop there.

Currently, there are dozens of assays that rely on the activity of luciferase enzymes. For example, the split firefly luciferase complementation assay (SLCA) uses bioluminescence to quantify protein-protein interactions within living cells. The assay uses modified firefly luciferase (FFLUC) split into two pieces, named N-FFLUC and C-FFLUC. On their own, the two pieces of FFLUC are inactive and do not luminesce. When the N-FFLUC and C-FFLUC are brought into close proximity in the presence of oxygen, ATP, and magnesium, the FFLUC will oxidize to produce light. This system can be adapted to measure the interactions between any two small proteins by fusing each of the two interacting proteins to the N or C terminus of luciferase. When the two proteins bind and interact, it brings the N-FFLUC and C-FFLUC close together so that FFLUC will luminesce. The luminescence can then be quantified with a machine called a luminometer, which will provide insight into the level of protein interaction.

Human immunodeficiency virus

Recently, Yale undergraduate Tucker Hansen YC ’22 and his mentor Richard Sutton developed an SLCA to quantify the Human Immunodeficiency Virus type 1 (HIV-1) Rev-Rev interaction. The assay will identify inhibitors that specifically prevent the Rev-Rev interaction of HIV-1 to stop infections.

Human immunodeficiency virus (HIV) is a virus that attacks the body’s immune system. While there are two common subtypes, HIV-1 and HIV-2, most people living with HIV have HIV-1. The virus infects CD4+ T cells, a type of white blood cell also known as helper T cells. These cells help fight infection by triggering the immune system to destroy pathogens in the body. In active CD4+ T cells, infection is caused by the insertion of the viral DNA into the host genome and its subsequent expression into new viral particles. When left untreated, HIV-1 replication causes progressive loss of CD4+ T cells, raising the infected individual’s susceptibility to infectious diseases that would not usually cause illness in a healthy individual. 

There are currently over thirty-eight million people living with HIV-1. Most patients can maintain undetectable viral loads and near-normal life expectancy with the help of antiretroviral medications that inhibit HIV-1 replication. There are currently dozens of FDA-approved medications against HIV-1, including protease, reverse transcriptase, and integrase inhibitors. So why would there be a need for more inhibitors?

A case for new inhibitors of chronic diseases

Viral suppression through existing medications enables immune recovery and the near elimination of the risk of developing AIDS, the more severe and life-threatening stage of HIV infection. However, due to drug resistance, some patients do not respond to the existing medications well. 

HIV-1 drug resistance is caused by changes in the genetic structure of HIV-1 that interfere with the ability of medications to block viral replication. Since RNA viruses such as HIV-1 have an especially high mutation rate that allows for quicker evolution, all retroviral drugs risk becoming ineffective due to the emergence of drug-resistant viral strains. Furthermore, drug resistance can more easily arise if there is poor adherence to prescribed medications. “As with any chronic disease, there is always a need for improvement in current medications, as well as the development of new antivirals,” Hansen said. 

Revving engines

The Rev protein is highly conserved in all subtypes of HIV and is necessary for transporting copies of viral RNA out of the nucleus of the host cell. Without Rev, HIV would not be able to replicate in its host. An essential property of Rev activity is that it must multimerize on the Rev-Response Element (RRE) of HIV RNA to successfully export that RNA from the nucleus to the cytoplasm of the host. This means that multiple Rev proteins must interact with each other to form a multimer. Since the multimerization of Rev is key to its mechanism of action, it could serve as a small molecule drug target.

Sutton, an expert studying HIV for years, knew that the Rev protein was essential to HIV survival. But there are currently no HIV antiviral drugs that target the Rev protein. “[A Rev inhibitor could] be a first-in-class antiviral,” Sutton said. 

Hansen began his project by developing an SLCA that could quantify Rev-Rev interaction in cells. Hansen fused each of the luciferase domains, NLUC and CLUC, to a Rev protein. The fused protein was created by genetically engineering a fusion gene that combined the sequence of the specific luciferase domain with the sequence of the Rev protein. Through a series of experiments, Hansen and Sutton eventually developed a highly sensitive screen for measuring Rev-Rev interaction. When Rev proteins were close enough to multimerize, the NLUC and CLUC would be brought close enough to luminesce. This assay works inside and outside of cells. Thus, even when using just the inner contents of cells, the assay can still accurately quantify Rev-Rev interaction. 

The goal of developing this assay is to find a small molecule inhibitor that can disrupt the Rev-Rev interaction. Hansen demonstrated that this assay could help by testing whether mutant Revs, which would inhibit the wild-type Rev interaction, reduce luminescence levels in the assay. Performing the assay with mutated Rev proteins fused to NLUC and CLUC resulted in much lower luminescence, indicating that this SLCA can be used to screen for an inhibitor that disrupts the Rev-Rev interaction. Similar to how a mutant Rev would not be able to multimerize, a putative inhibitor would be able to disrupt this interaction and result in a much lower luminescence reading. Thus, researchers could use this system to test an unlimited number of small molecules to see whether any can effectively prevent Rev-Rev interactions without being an inhibitor of the luciferase enzyme itself. 

When asked whether he could find such an inhibitor, Sutton admitted that it was unlikely. “Honestly, I don’t think our lab could ever do this,” Sutton said. “It really takes a commercial entity to do it. Can you imagine Tucker screening 200,000 compounds on his own?” A pharmaceutical company, however, has the means and methods to perform large-scale screens to identify potential inhibitors of the rev-rev interaction. 

Sutton is currently applying for a grant from the NIH to fund the next steps of this project. With this funding, he is considering partnering with the local pharmaceutical company, ViiV Healthcare, which focuses on delivering new treatment options for people living with HIV. Sutton and Hansen are hopeful that the potential Rev inhibitors identified through this partnership could serve as a new class of HIV antivirals and present another line of defense for HIV patients with drug-resistance complications.

The work done by Hansen and Sutton is only one of many examples demonstrating the versatility in applications of firefly luciferase. This story highlights the ingenuity of using phenomena in the natural world to create tools and technologies that can facilitate our understanding of biological processes. As we continue to explore and rationalize more of the natural world, Hansen and Sutton’s work reminds us that existing biological processes can be the key to unlocking a whole new world of technology and discovery with innumerable benefits to mankind. 

Further Reading

Jabr, F. (2016, May 10). The Secret History of Bioluminescence. Hakai Magazine.
Rinaldi, A. (2007). Naturally better. Science and technology are looking to nature’s successful designs for inspiration. EMBO Reports, 8(11), 995–999.