Researchers study mechanism of HIV infection

Carnegie Mellon biophysicists Stephanie Tristram-Nagle and John Nagle have uncovered details as to what allows HIV to infect immune T-cells.

According to an article published in Biophysical Journal, “HIV-1 Fusion Peptide Decreases Bending Energy, Promotes Curved Fusion Intermediates,” data from this research will aid in computer simulations of HIV infection and will help further drug discovery.

Enveloped viruses, which develop outer layers from their host cells, infect cells by fusing with the cells’ membranes and inserting their genetic contents into the cells, essentially turning them into virus-producing factories. In order for HIV to infect immune T-cells and deliver its RNA contents, it must fuse its viral envelope with the T-cell membrane.

More specifically, HIV lowers the energy barriers of T-cell membranes to curve the membranes and allow a pore to form. The protein gp41, which is located on the viral envelope, enables HIV to penetrate the membrane.

According to a Carnegie Mellon press release, the Nagle lab discovered that a short stretch of the gp41 protein, known as fusion peptide 23 (FP-23), decreases the energy needed to bend a cell membrane, making it easier for the HIV to infect the cell.

A cell’s lipid membrane is normally highly resistant to bending. Researchers used diffuse X-ray scattering data from Cornell University’s CHESS synchrotron to measure the bending modulus of a lipid bilayer and found that FP-23 causes the bending modulus to decrease.

According to the article in Biophysical Journal, a smaller bending modulus lessens the free energy barriers that must be overcome to pass through a cell membrane. The HIV particle can then undergo fusion with the membrane and infect it.

The Nagle lab was able to study the intermediate stage of viral infection of a hydrated lipid bilayer by using novel research techniques and data analysis.

“Another method to study viruses and how they infect cells is to look at the protein-membrane interaction via protein crystallography,” said Tristram-Nagle. “This, however, focuses on pre-fusion and post-fusion states of viral infection. Our lab was able to use a novel technique to probe the intermediate fusion state.”

Researchers developed a method to validate and perform data analysis on the structure and properties of fully hydrated lipids. This type of analysis was critical in their discovery of how FP-23 interacts with the membrane.

“It is important to determine the structure and interactions of fully hydrated, fluid-phase lipid bilayers, because they are the underlying component of all plant and animal cell membranes,” Tristram-Nagle said. “Different lipids have different areas and thicknesses as well as different material properties, and these differences turn out to be essential for membrane protein function. ”

Researchers pioneered a method of data analysis that involves applying theory from liquid crystal literature to the lipid membrane. The shape of a cellular membrane, represented by the fluid mosaic membrane model, is very similar to that of a liquid crystal.

“Our lab pioneered the use of liquid crystal theory to correct the X-ray scattering intensity collected from fully hydrated lipids and determine a structure from this kind of data,” said Tristram-Nagle. “Without the correction, the X-ray data are weakened due to thermal undulations in fully hydrated lipid membranes and
the structures are not able to be determined.” X-ray scattering data was used to calculate the bending modulus of the membrane and validate its significance.

The preparation of the hydrated lipid samples, known as the rock-and-roll procedure, was also pioneered by Tristram-Nagle. It involves putting hydrated lipids — which are similar to those found in a cell membrane — on silicon wafers, using them, and then hydrating them in a humidity chamber.

Researchers from the Nagle lab said that they will continue to work on how HIV proteins affect the cell membrane. Currently, they are studying the pretransmembrane section of the gp41 protein, an important cholesterol recognition site. The HIV membrane is 80 percent cholesterol, which Tristram-Nagle hypothesizes could be important if the cholesterol is involved in the change of gp41 during HIV infection of the T-cell.

“I have been working with a CMU undergrad for about one year, Alex Greenwood — now a graduate student at Cornell — to characterize interaction of the CRAC motif peptide with lipid/cholesterol mixtures, and this work is nearing completion,” she said.

The fusion peptide research was carried out over the course of five years. John Nagle and Stephanie Tristram-Nagle have worked together since 1982. The Nagles won the Avanti Award at the Biophysical Society in 2003 for their novel approach to analyzing X-ray data of hydrated lipids.