Bottleneck breakthrough

Four decades after the discovery of HIV-1, the retrovirus that cause AIDS, a vaccine still eludes researchers because of the virus' complex and challenging biology.

HIV-1 evolves about 1 million times faster than mammalian DNA, and once it invades an individual, it generates thousands of genetic variants, requiring any potential vaccine to play an endless game of whack-a-mole.

But if we better understood how HIV-1 infects the first cell when it invades a new body, we could prevent it from taking hold and spinning off new variants.

Researchers at Fred Hutch Cancer Center help explain why some of those variants are better at infecting new individuals than others in a paper published today in the journal Nature Microbiology.

“If we could understand why they are selected, maybe we can stop them from being transmitted,” said Hannah Itell, PhD, the lead author of the study conducted in the lab of Julie Overbaugh, PhD, in the Human Biology Division at Fred Hutch.

Itell, now a postdoctoral researcher at Vanderbilt University, and her colleagues discovered that there’s something about the human host that favors some HIV-1 variants while blocking others, a consistent, widespread finding that is rare for the field.

“We are quite excited about this finding," said Overbaugh, who holds the Endowed Chair for Graduate Education. "This is a question we have been trying to answer for some time.”

Something about the host cell’s biology favors some variants

Although HIV-1 generates thousands of variants in people with HIV who have not received treatment, only a few of those variants manage to squeeze through a tight bottleneck to infect a new person.

The first cells that HIV-1 productively infects in a new human host are a type of white blood cell involved in the immune system. To enter those cells, viruses seek two kinds of doors called coreceptors, known as R5 and X4 for short.

Though both doors are readily available on the surface of the cell, HIV-1 variants that favor the R5 door get in much more often than variants that favor the X4 door.

Why R5 variants but not X4 ones squeeze through the bottleneck and infect a new host has puzzled the researchers in the field for a long time.

Itell and her colleagues hypothesized that something about the biology of those white blood cells might give advantage to the variants favoring the R5 door, but they didn’t know what that something was.

They figured that it was most likely a protein that somehow blocks variants favoring the X4 door on the white blood cell. To find that protein, they would need to systematically turn off genes until they knocked one out that made a difference.

Fred Hutch HIV researcher Michael Emerman, PhD, and his team had already developed a CRISPR-Cas9 gene editing technique to screen for protein-coding genes that restrict HIV in some way.

Researchers typically use cancer cells in such screens because they grow rapidly and relentlessly, a feature that makes them deadly in humans yet useful in labs.

But Itell and her colleagues wanted to see what was going on in healthy white blood cells from donors who had no history of HIV-1.

They adapted the Emerman Lab’s screen for use on healthy donor cells, and the screens turned up a conspicuous protein with a long name, SLC35A2 or SLC for short.

“That’s how we homed in on that one gene,” Itell said.

A rare finding

Because HIV biology is so complex, it’s difficult to generalize findings that happen with just one or two variants.

So, they put SLC to the test, exposing healthy white blood cells with and without the SLC gene to a wide range of HIV-1 variants.

“We intentionally selected viruses that are quite different from one another,” Itell said.

They exposed healthy white blood cells from two donors to 10 different HIV-1 variants, five that preferred the R5 door and five that preferred the X4 door.

They had to work quickly, finishing each round in about 21 days, because the shelf life of healthy white blood cells is much shorter than the cancer cells typically used in research.

Across each combination of donor and variant, SLC stood out.

“Time and time again, we saw how consistent this effect was,” Itell said. “That was when we knew we had something very interesting because it is so rare in the HIV field to see such consistent results across virus strains.”

The results were unambiguous, providing a clear dividing line for SLC’s influence between R5 and X4 variants.

“It’s not only blocking X4 viruses, but it’s helping the R5 viruses to make this a very clean selection process,” Itell said.

That gene carries the instructions for making the SLC protein, which helps add sugars to molecules, a process called glycosylation that guides many important interactions within and between cells.

It’s unclear why sugaring of the white blood cell makes a difference to HIV-1 viruses. But it’s unlikely Itell and her colleagues would have discovered the effect using cancer cells because those cells often have compromised sugaring that would have muddied the waters and obscured SLC’s blocking role.

“It was very exciting to find this because there’s actually not another protein that we’re aware of that has this type of an effect,” Itell said.

And that gives researchers a potential target for a drug or a prophylactic that could stop HIV-1 before it enters the first cell.

This work was supported by grants from the National Institutes of Health.