Research led by a professor of pathology at Touro University in the United States has identified a novel pathway that the AIDS-causing human immunodeficiency virus (HIV) uses to enter the nucleus of a healthy cell, allowing it to replicate and invade other cells.
The study, published in the peer-reviewed Nature Communications journal in August, was led by Dr. Aurelio Lorico, interim chief research officer at the College of Osteopathic Medicine on Touro’s Nevada campus.
The paper’s findings also identify three proteins that are needed for the virus to invade the nucleus. Lorico and his collaborators have synthesized molecules that can target one of the proteins, possibly leading to new treatments for AIDS. The findings also have implications for better understanding and treatment of other viral infections, cancer, and neurodegenerative diseases such as Alzheimer’s.
The discovery about HIV shared in the paper is an offshoot of research Lorico has done on the role of extracellular vesicles (EVs) in cancer metastasis. EVs are lipid-enclosed spherical particles that are released by cells into the area outside and between cells. They primarily act to communicate and deliver cargo from cell to cell. In the case of cancer’s spread throughout the body, they help cancer cells to grow at the site of metastasis.
“EVs are produced by every cell and then are dumped outside the cell. When you take a drop of blood, you have billions of these EVs in it. You’ll have even more EVs than red blood cells in that drop of blood,” Lorico said.
Lorico was aware of how these EVs enter the cell and its nucleus. When he realized that HIV is very similar to EVs, he began to think that HIV might be using the same pathway.
However, it was initially difficult for him to get scientific journal editors to consider this possibility. Lorico explained that the accepted theory was that HIV makes its way into the cell by fusing with the cell’s plasma membrane. In the fusion process, the external layer of the virus is lost, revealing the naked virus — the capsid that protects the virus’s genetic material. If the capsid does not make it into the cell’s nucleus, then no infection occurs.
“Not all viruses need to enter the nucleus to cause infections, but HIV does. The accepted pathway was that the virus loses its capsid and ‘swims’ into the nucleus through the very tiny nucleopore, which is a series of openings found in the cell’s nuclear membrane serving as channels for selective transport in and out of the nucleus,” Lorico explained.
He contended that it was necessary to consider an alternative idea: that the endosomal pathway into the cell (endocytosis) could also be the means by which HIV enters the nucleus.
Endosomes are “packages” that are formed when there is an invagination (deep indentation) of the cell’s plasma membrane around material to be brought into the cell. The endosomes are then like “cars” or “buses” that transport this material inside the cell — sometimes to be used, sometimes to ultimately be destroyed, and sometimes to be kicked out of the cell.
Lorico brought virologists on board and they started by genetically altering HeLa cancer cells so they could be infected with HIV. Later they switched gears and focused on human T-lymphocyte cells, an important player in our immune system.
Using a super-resolution fluorescent microscope, Lorico was able to observe that HIV was entering the cell’s nucleus using the same invagination process as in endocytosis at the cell membrane.
An endosome transporting HIV pushes the protective nuclear membrane inward to form an indentation, and then moves inside to the invagination’s inner tip and slips into the nucleus.
“When I was hired by Touro, I said to continue the discoveries that I’m doing, I need a super-resolution microscope. We’re talking about a very expensive piece of equipment, but Touro understood the importance and acquired it. I am grateful for their vision,” Lorico said.
The study found that three proteins were critical to this invasion into the nucleus. The first, Rab7, is located on the membrane of the endosome. The second, VAP-A, is on the nuclear membrane where the invagination occurs, and the third, ORP3, connects the first two proteins. An interaction among the three proteins is needed for the invasion to be successful, so targeting any of these proteins could halt the infection.
The researchers decided to target ORP3, as the other two proteins have essential roles in the cell.
“The first thing we did was look in the existing literature to see if there were molecules known to target this bridge protein. There was no ORP3 inhibitor, but there was an inhibitor of other proteins in the family that ORP3 belongs to. It’s an anti-fungal drug called itraconazole, and it is already used clinically,” Lorico said.
The scientists saw that the drug successfully blocked ORP3 and the process by which HIV entered the cell’s nucleus, but there was still the problem that the drug would be toxic if given to patients at the high doses required to tackle infection by the virus.
To surmount this problem, Lorico enlisted synthetic chemists he knew in his native Italy to help create a compound spun off of itraconazole that has much smaller molecules, does not target fungal infections and works against the HIV pathway — and should theoretically be viable, after further testing and clinical trials, for use in human patients.
“We have papers published that say this can work in cancer. And we have unpublished data that shows that in vivo in animal models, it works and blocks metastasis. Now this study shows that it blocks the nuclear transport of HIV. And we’re doing collaborations with other investigators to show that this is blocking other viruses like cytomegalovirus and influenza,” Lorico said.
He is not alone in his enthusiasm about this discovery and his hope that it may prove to be a key insight into the pathogenesis of many types of diseases.
“Many of the recent pandemics have been caused by viral infections. Touro is decidedly excited by the research performed by Dr. Lorico and his colleagues that has uncovered a new pathway necessary for viruses to grow in the body and suggested new ways in which viral infections may be suppressed. The work exemplifies Touro’s strengthed commitment to advancing research,” said Touro University president Dr. Alan Kadish.