Discovery of HIV-1 Protein Structure Could Lead to New Therapies

BALTIMORE--Following the battlefield tactic of divide and conquer(in this case, dividing a molecule into two fragments), Universityof Maryland Baltimore County (UMBC) researchers have determinedthe three-dimensional structure of a key part of the HIV-1 p24capsid protein (see illustration on page 1).

This protein forms a shell within the AIDS virus that encapsulatesthe virus' RNA and some enzymes vital to its ability to infectcells and replicate.

"This new information suggests potential new targets fordrug development and provides new insights into the process ofHIV replication," Jack Killen, MD, director of the divisionof AIDS at the National Institute of Allergy and Infectious Diseases,said in a statement.

The HIV-1 capsid (CA) fragment contains within it the bindingsite for cyclophilin A, a protein that is found in most bloodcells and is the target for the immunosuppressant cyclosporin.Earlier work by Columbia University researchers demonstrated thatHIV's capsid protein must bind cyclophilin A for HIV-1 to infectcells.

"We now have a detailed picture of the cyclophilin A bindingsite, and that information should be crucial to scientists whoare designing antiviral agents based on inhibiting cyclophilinA's interaction with HIV-1," Michael F. Summers, PhD, professorof chemistry at UMBC and associate investigator at the HowardHughes Medical Institute, said at a media briefing.

Surprisingly, the HIV-1 CA fragment has a structure unlike anyother viral coat protain listed in the Brookhaven National Laboratorydatabase.

The new findings, detailed in Science (273:231-235, 1996), capa 6-year effort by Dr. Summers to unravel the structure of theHIV-1 capsid protein. HIV contains eight proteins and scientistshave now determined the 3D structure of six. He and his coworkershad previously resolved the features of two other HIV-1 structuralproteins besides p24--p7 nucleocapsid, a protein inside the viralcore, and the p17 matrix protein that lies between the core andthe viral membrane.

A Puzzle With 2,000 Pieces?

The CA protein links together inside the virus to form a cone-shapedprotective shell surrounding the genetic material at HIV-1's core.For a long time, the UMBC team focused on the entire capsid protein,using nuclear magnetic resonance (NMR) spectroscopy in an effortto reveal its structure.

"Picture the core of HIV as an eggshell composed of 2,000identical copies of the p24 capsid protein," Dr. Summersexplained. "Each piece of the eggshell is designed to aggregatewith the others, and when we put these proteins in solution, theystuck together, becoming too large for us to study by NMR."

Dr. Summers and his coworkers--postdoctoral researcher RossitzaK. Gitti, PhD, doctoral student Brian M. Lee, and undergraduateJill Walker--finally made a breakthrough about a year ago. SaidDr. Summers: "We figured out how to use enzymes to cut offthe third of the molecule responsible for aggregation, which leftus with a section of the molecule, called the amino-terminal coredomain, that we could study."

Wesley I. Sundquist, PhD, and Sanghee Yoo of the University ofUtah, using a genetic engineering technique developed by Dr. Sundquist,provided the UMBC team with large quantities of the bigger proteinfragment. This piece, about two thirds of the whole molecule,contains the cyclophilin A binding site. Dr. Summers' team successfullyexplored this fragment with NMR.

Alpha Helices Predominate

When they finally obtained their visualization of the CA fragment,however, its shape stunned them. "The structure is completelydifferent from what we expected," Dr. Summers said. "Foryears, people had hypothesized that it would be composed predominantlyof the beta-sheet motif common to other viruses. We found thatthe CA fragment is almost entirely composed of alpha helices andis unlike any previously discovered viral coat protein."

Its complex structure consists of seven corkscrew-shaped alphahelices, two regions called beta hairpins, and a single exposedloop. Dr. Summers envisions the loop as particularly importantbecause it contains Pro90, an amino acid that binds cyclophilinA. HIV carries the bound protein with it when it buds from aninfected cell.

The UMBC team also discovered that Pro90 comes in two differentconformations. One may be necessary for the CA protein to assembleinto the core shell and the other for the core to disassemble."Pro90 may serve as a molecular switch for capsid assemblyor disassembly, with cyclophilin A either flipping the switchor locking it into a particular position," Dr. Summers said.

"We have felt that if we knew the structure of this proteinin HIV-1, it would make it easier to design drugs that targetthe CA protein and possibly keep HIV from infecting cells,"he added. Dr. Summers said that he expected several drug companiesto quickly incorporate the new findings into their searches fornew AIDS drugs.

The UMBC research received support from the Howard Hughes MedicalInstitution, the National Institute of Allergy and InfectiousDiseases (NIAID), and the National Institute of General MedicalSciences.