Understanding How HIV Infects and Commandeers Host Cells

A team of researchers led by the Rockefeller University and the Aaron Diamond AIDS Research Center (ADARD) have developed a new approach to understanding how viruses attack cells and commandeer their machinery that appears to offer an unprecedented, step-by-step view of how viruses do this. For a study published May 23 in Nature Microbiology, the new approach was applied to the genetically compact HIV-1.

“HIV is truly an expert at living large on a small budget,” said lead author Yang Luo, a postdoc at ADARC and a former graduate student at Rockefeller University. “We asked the question, ‘How does such a compact virus manipulate the host cell to gain entry and replicate itself, all while escaping the immune system?’”

Luo and colleagues focused the study on two viral proteins know to bring about HIV’s infection of human white blood cells: 1) envelope, or Env, which sits on the surface of the virus and helps the membrane that encapsulates the virus fuse with the cell’s outer membrane by binding to receptors on the host cell; and 2) the protein Vif, which destroys an enzyme that host cells produce to defend themselves against the virus.

“Transposon-mediated saturation linker scanning mutagenesis was used to isolate fully replication-competent viruses harboring a potent foreign epitope tag,” wrote the study authors. “Using these viral isolates, we performed differential isotopic labelling and affinity-capture mass spectrometric analyses on samples obtained from cultures of human lymphocytes to classify the vicinal interactomes of the viral Env and Vif proteins as they occur during natural infection.”

These experiments were performed by introducing a genetic sequence into the viral genome—a “tag” that allows one viral protein to be yanked out along with all the other proteins associated with it.

“Inserting a tag sequence into small viruses is a challenge to begin with,” said senior author Mark Muesing, PhD, a principal investigator at ADARC. “If you disrupt their nucleic acid and protein sequences, you can easily compromise the virus's ability to replicate. And HIV represents a particular challenge because it can quickly revert back to its original sequence… We developed a technique to find places in the HIV genome where we can insert stable tags without affecting the virus's capacity to proliferate. In effect, this allowed us to expand cultures of the infected cells along with the tagged viral protein.”

After infecting human cells with viruses carrying the tagged protein sequences, the study investigators were able to pull out and identify many host proteins directly during the infectious process, thereby providing the first evidence that many previously underappreciated host proteins interact with the viral machinery during replication.

“Imagine you have a factory assembly line where only one component of, say, the stamping machine, actually touches the product,” said co-author Michael Rout, PhD, professor and head of Rockefeller’s Laboratory of Cellular and Structural Biology. “Other parts support and power the stamp. Likewise, within an infected cell, we can identify the components of a particular cellular machine, not just the piece that comes in contact with the viral protein.”

Adding to Rout’s thoughts, co-first author Erica Jacobs, PhD, a research associate in the Laboratory of Mass Spectrometry and Gaseous Ion Chemistry at Rockefeller, said, “Every host protein we pull out generates new questions. Does it help the virus invade and coopt the host to replicate itself? Or does it harm it? The answers will not only help us understand the virus, but also shed light on our cells' ability to defend themselves.”

According to co-author Brian Chait, PhD, Camille and Henry Dreyfus Professor and head of the Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, the new approach offers a rare glimpse into the process by which HIV invades and resurrects itself within a cell. “Often, studies of this sort are done with viral proteins in the absence of a true viral infection,” he said. “However, because viral infections are exquisitely orchestrated events, you are likely to miss all kinds of important details if you study the action of these proteins out of their proper context.”

Co-author Ileana Cristea, PhD, an associate professor of molecular biology at Princeton University, added that “deciphering the intricacies of virus-host protein interactions in space and time during the progression of an infection is remarkably. The challenge is to discover which precise interactions are the critical ones.”

Further adding to the conversation, co-first author Todd Greco, an associate research scholar and lecturer in molecular biology in Cristea’s lab, said that “even for host proteins within the same family, their relative stability within HIV-1 protein complexes can be very different. More broadly, by understanding these mechanisms we will better understand the coordinated responses of cells.”