Several minute intermediate stage in virus-cell fusion discovered; possible window of opportunity for drug development
La Jolla, CA – To ignite a life-threatening infection in the body, a virus such as HIV invades body cells by first merging, or fusing, with the cell's outer membrane. Once inside the cell, the invading microbe's genetic material takes over, turning the 'host' cell into a factory to produce more copies of the virus, which then spill out to invade other cells in the body.
Scientists had assumed that once a virus begins fusing with a cell's membrane, infection of the host cell was inevitable. Thus, antiviral drug development has largely focused on preventing events that happen either before or after this step.
However, a multi-institutional team of researchers is reporting that it has detected an intermediate stage between the virus' merger with the cell membrane and the microbe's delivery of its genetic contents into the cell, when the fate of the host cell still hangs in the balance.
This intermediate stage, which can last several minutes, may represent a window of opportunity for drug development. The remarkable findings, captured on video, were published by the Proceedings of the National Academy of Sciences online on June 3.
Biophysicist Gregory Melikyan of Rush University Medical Center in Chicago and microbiologist John Young of the Salk Institute for Biological Studies filmed individual viruses fusing with a host cell membrane. Avian sarcoma and leukosis virus (ASLV), a virus that is in the same class as HIV, was used in the study.
The researchers discovered that once the virus fuses with the host cell membrane the hole, or pore, through which the virus unloads its deadly genetic cargo into the host cell does not open up right away; instead, a small pore can persist for several minutes before adopting its final size, or (in rare cases) closing permanently.
This 'intermediate stage,' as the scientists describe this time interval, was not known to exist for virus-cell fusion events. The net effect is that the invading virus is held up for a significant amount of time and, in the rare cases in which the pore does not open, it fails to infect the cell at all.
"It's like a space craft docking on a space station," said Young. "If you try to open the inner door before the pressures have equalized, you can tease it open a little bit but it keeps closing on you until there's enough pressure in the airlock to allow it to open all the way."
Melikyan explained, "The pore is an unstable structure at that moment: some pores will open and some won't. It's a crucial point in viral entry because it's critical for the pore to enlarge sufficiently for the genetic material to pass into the host cell."
The researchers are excited by this study because even a brief pause during the process by which a virus invades a cell provides a possible new drug target in the fight against HIV and other similar microbes.
"Our experimental system does pause frequently for hundreds of seconds," said Melikyan. "How this happens in real life is hard to say.
"But our model," he added, "is likely to apply to any virus such as HIV that fuses with the cell membrane and shares the same fusion proteins, so it certainly provides a new target. Also, existing drugs can be re-evaluated to pinpoint at what stage they actually work, to fine-tune their activity."
The experimental set-up for this study was devised by Young and Melikyan last year to investigate viral infection in a manner as close to 'real life' as possible. The researchers filmed the microscopic viruses, which are only 100 nanometers across, by labeling them with fluorescent dyes and recording at one frame every 7 seconds. (For comparison purposes, a human hair is 80,000 nanometers thick.) ASLV is a useful model for several viruses, including Ebola, the 'flu, and measles, as well as HIV.
"This system is especially well suited for these types of studies because the fusion mechanism of this virus can be finely manipulated and monitored in the lab, allowing for an exquisite level of control of the whole process," said Young. "This allows us to do things that are not possible with other systems."
The next stage is for researchers to confirm that other viruses such as HIV also have an intermediate step. Preliminary data from the Melikyan laboratory indicate that this may be the case for HIV. "We already know that the entire HIV fusion sequence is very slow, sometimes taking several hours," said Melikyan. "It is not inconceivable that it, too, has an intermediate step that lasts several minutes, giving us an adequate time window for drug action."
An independent, non-profit research organization, the Salk Institute for Biological Studies was founded by Dr. Jonas Salk in 1960 just five years after he discovered the first safe and effective polio vaccine. Five years later, the institute's main research campus, featuring two mirror image buildings designed by the famed architect Louis Kahn, was completed. Today Salk scientists conduct basic research in the biological sciences to identify the molecular origins of health and disease.