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Rockefeller University Team Profiles HIV-Host Interactome


NEW YORK (GenomeWeb) – A team led by scientists at The Rockefeller University has generated a profile of the HIV-host interactome in actual infected cells.

In the study, detailed in a paper published this week in Nature Microbiology, the researchers used a combination of epitope tagging and affinity-capture mass spec to investigate interaction partners of two HIV proteins as they exist in replication-competent viruses.

The method allowed the team to circumvent issues that have limited previous such efforts and identify a variety of new interactors, Mark Muesing, a Rockefeller researcher and senior author on the paper, told GenomeWeb. The approach should also be broadly applicable to virus-host interactome studies involving other pathogens, he noted.

The HIV proteome is quite compact, featuring just nine open reading frames. However, high-quality antibodies to these proteins are lacking, which has hindered interactome studies using affinity-capture mass spec.

Researchers have done interactome studies by transfecting cells with single HIV genes, Muesing said, but such models "don't truly reflect what is going on during replication," he noted. "So we wanted to find a system where we could look at the interactome directly during infection."

Muesing and his colleagues tackled this problem by using transposon-mediated saturation linker scanning mutagenesis to insert a 3xFLAG epitope tag into HIV proteins of interest and then isolate viruses that remained fully functional after insertion of the tag. This insertion allowed the researchers to later pull down viral proteins and their interactors using antibodies to the 3xFLAG tag and then analyze them via mass spec.

Getting the virus to accept the tag was not without its challenges, though. "This tag is 35 amino acids in a very compact virus," Muesing said, "so it's not an easy task" to insert it.

By using saturation mutagenesis, the researchers essentially let the virus determine where in the proteins the tag should go, said Yang Luo, a postdoc at Rockefeller and first author on the paper.

"For compact viruses like HIV, it is really hard to find a place in the genome for the tag," she said. "The way that we did the saturation mutagenesis was to let nature select for where the tag should be, [which] is very exciting and elegant and applicable to many other viruses where people have had difficulty inserting [epitope] tags."

Ultimately, they selected three proteins for study, two of which they detailed in the Nature Microbiology paper. These featured proteins were Env, the HIV envelope protein, which, Muesing said, is a target of much HIV vaccine research, and Vif, which is involved in countering host defense mechanisms.

Comparing the interaction partners they identified for these two proteins with previously identified interactors, the researchers found "significant overlap," but also identified a number of new interactors, Muesing said.

"Previous work didn't look at when the virus was replicating, and so you missed out on a lot of things," he said.

For instance, he noted, "because it was a live infection, we were able to identify molecules from the cell surface that had not been picked up before, and we showed that the virus has impacts on these surface molecules, some of which were totally unexpected.

In particular, the study shed new light on the virological synapse, a molecular complex that allows viruses to transmit themselves between cells.

Due to this structure, "viruses pass really efficiently [between] two cells," Muesing said. "And we think that we have recovered some of that apparatus."

Traditionally, it has been "a nebulous thing," he said. "People know they can see it by, for instance, immunofluorescence, but they don't know what the components are."

In their work, the researchers identified some adhesion molecules that had previously been considered likely components of the virological synapse, Muesing said, but they also identified as putative components signaling molecules that had been "totally unappreciated."

Many viruses use the virological synapse for transmission between cells but it is particularly important in the case of HIV in that "it has been shown that the broadly neutralizing antibodies that people have recovered in some cases cannot inhibit that type of transmission," Muesing said. Additionally, he noted, other researchers have shown that restriction factors don't work as well at countering that type of transmission.

"It is a really protected environment that the virus constructs to protect itself from host mechanisms that could interfere with that transmission," he said. "So this has clinical implications."

To help ensure the identified interactions were, in fact, true interactions as opposed to non-specific binding events, the researchers developed an isotopic labeling-based method called I-DIRT (for isotopic differentiation of interactions as random or targeted), in which they used heavy and light isotopes to label cultures infected with, respectively, untagged and tagged strains. This allowed them to assess to what extent peptides were being pulled down due to their association with the tagged proteins as opposed to non-specific binding to the anti-3xFLAG antibody used for the pulldown, and to account for such non-specific binding in their findings.

Muesing said he and his colleagues plan in future studies to expand their tagging to include a number of other HIV proteins. He noted that thus far they have been successful in the majority of their attempts to tag these molecules, including with highly conserved proteins like integrase.

In addition, they hope to insert the tag into different parts of the proteins, which could allow them to better observe interactions at different stages in the infection cycle, he said. For instance, in the case of the Env protein, the researchers found that they were only able to observe it at a specific point in its life cycle, right when it was initially translated in the endoplasmic reticulum.

"The location of the tag was such that either it was buried in the secondary structure [of the Env] at different times or buried by further modifications of sugar" making it impossible to pulldown at those times, Muesing said.

Inserting the tag in different locations should make it accessible at different points in its existence and at different locations within the cell, he noted. "So it becomes more immunoreactive at different times and we can look at the different [interactions] along its pathway."