Restricting HIV

This year’s Retrovirus Meeting at Cold Spring Harbor Laboratory featured research on all aspects of HIV biology, but host cell restriction factors took center stage.

Regular attendees of the annual Cold Spring Harbor Laboratory meeting on retroviruses know that the science served up at the meeting can be a lot to digest. With 120 talks and almost 200 posters all crammed into a mere five days, this year’s meeting—which took place from May 21 to 26—was no exception. “It’s an intense experience,” admitted Vineet KewalRamani of the National Cancer Institute in Frederick, Maryland, one of the organizers this year.

While the meeting covers just about every aspect of retroviral biology, most of the talks dealt with HIV. Host cell restriction factors, which inhibit viral replication or infection, were a major topic of discussion. Research into these factors has exploded over the past decade, says Jonathan Stoye, a virologist from the MRC National Institute for Medical Research in London. “There has been a remarkable gold rush in interest in restriction factors,” he said in his Keynote address. In 2000, he observed, the Cold Spring Harbor meeting on retroviruses featured just two talks on such factors. This year, more than a quarter of the talks were devoted to them.

One full session this year focused on the recently identified restriction factor SAMHD1, including reports that it might prevent productive HIV infection in resting CD4+ T cells. Other notable talks covered the host cell restriction factor tetherin, its possible role as a pattern recognition receptor (PRR) that induces an innate immune response to HIV, and what it takes for the virus to successfully switch to a new host that expresses different restriction factors. Reports on other areas of retroviral biology described the identification of new types of antiviral host factors, the first in vivo evidence of virological synapses (structures involved in direct cell-to-cell spread of retroviruses) and new insights into where specifically HIV integrates in the nucleus of the host cell.

It was at this meeting last year that researchers first announced that SAMHD1 is the cellular restriction factor targeted by HIV Vpx (see Research Briefs, IAVI Report, May-June 2011). Studies suggested that SAMHD1 expression in dendritic cells (DCs) and macrophages accounts for their resistance to infection by HIV-1. At this year’s session on SAMHD1, two groups reported that this may also explain why resting CD4+ T cells are resistant to HIV-1 infection.

One report came from Hanna-Mari Baldauf of Oliver Keppler’s group at the University of Heidelberg in Germany, who found SAMHD1 to be expressed in resting primary CD4+ T cells taken from the blood of healthy volunteers. The study, done in collaboration with Oliver Fackler’s lab at the University of Heidelberg, also showed that the resistance of these cells to HIV-1 infection can be overcome by inhibiting SAMHD1, suggesting that the restriction factor confers HIV-1 resistance to the early phase of infection in resting CD4+ T cells.

The resistance to infection could also be overcome by adding deoxynucleotides, the products of which—dNTPs—HIV-1 reverse transcriptase needs to synthesize DNA from viral RNA. This is consistent with SAMHD1 inhibiting HIV-1 replication by degrading dNTPs. Yet Baldauf and colleagues also found SAMHD1 expression in activated CD4+ T cells, which are primary targets of HIV-1. Why such cells can be productively infected by the virus with relative ease, even though they express SAMHD1, remains unclear. But previous studies have found that dNTP levels tend to be higher in activated than in resting CD4+ T cells. This suggests that higher dNTP levels could make it more difficult for SAMHD1 to inhibit HIV-1 replication by degrading dNTPs in activated CD4+ T cells. 

The role of virological synapses in HIV infection
SAMHD1 expression in DCs is one reason why these cells resist productive HIV-1 infection: If the virus enters DCs the traditional way (binding the CD4 or CCR5 coreceptors), it either can’t replicate or gets degraded.

But HIV-1 sometimes escapes this fate: Every once in a while, DCs take up the virus and transmit it directly to CD4+ T cells they are in contact with through structures known as virological synapses. These are sites of cell-cell contact through which HIV and other retroviruses can be directly transmitted from an infected cell to an uninfected cell. “It’s known that DCs are helping in virus dissemination,” said Rahm Gummuluru, an associate professor of microbiology at Boston University School of Medicine. “But how do they do it?”

At the meeting, Gummuluru offered one possible explanation: to use DCs to spread to CD4+ T cells, HIV must enter DCs in a manner that does not depend on Env binding the CD4- or CCR5 coreceptors. Instead, he proposed, HIV-1 must enter the DCs through the binding of a lipid called GM3 in its outer membrane to a receptor on the DCs.

Part of the evidence for this mechanism of entry comes from the observation by Gummuluru’s team that only HIV particles with GM3 in their membrane can spread to CD4+ T cells from DCs in contact with them (1). At the meeting, Gummuluru reported that a receptor on the DCs is also important for this process. This suggests, said Gummuluru, that if it is to spread by “transinfection” to CD4+ T cells via virological synapses HIV must enter DCs using a pathway dependent on GM3 interaction with this receptor. “We would argue that it’s this receptor-ligand interaction is what is targeting viruses to transinfection,” he said.

Currently, Gummuluru is trying to see where in the body DCs expressing high levels of the receptor are located. “Those dendritic cells which express this [receptor] protein in vivo would aid in virus dissemination as opposed to virus destruction,” he said, adding that he expects to see them in tissues known for their high levels of virus replication in CD4+ T cells, such as the gut.

Keeping HIV from entering DCs via binding to that receptor could also help prevent mucosal HIV transmission, Gummuluru said. That’s because DCs are among the first cells HIV encounters at the mucosa (even before it encounters CD4+ T cells). HIV might therefore first enter DCs and then spread from there to CD4+ T cells, instead of first infecting CD4+ T cells directly. 

Gummuluru is planning animal experiments with a microbicide that contains agents that keep HIV from binding to the receptor. He already has in vitro evidence that one such agent, GM3-containing liposomes, can prevent viral entry into DCs, he said. One advantage of this approach is that because the newly identified receptor and GM3 are both host molecules, the virus should not be able to easily develop resistance mutations against agents that inhibit their interaction.

While Gummuluru and others believe that virological synapses are an important pathway of HIV transmission, the existence of these synapses still hasn’t been shown in vivo. “There is only in vitro stuff,” said Walther Mothes, an associate professor at Yale University School of Medicine. “Nobody knows what happens in vivo.” 

At the meeting, Xaver Sewald from Mothes’ lab reported the first in vivo evidence for these synapses, at least for a retrovirus that infects mice. Sewald took primary lymphocytes from the blood of mice that express red fluorescent protein (RFP) in all of their cells, and infected the cells ex vivo with Murine Leukemia Virus (MLV) particles that carried a green fluorescent protein (GFP) fused to their Gag protein. He then injected different types of MLV-infected RFP-cells, including B cells and CD4+ T cells, into the lymph nodes of unlabeled, living mice. Whenever MLV-infected B cells in the lymph nodes were in contact with other, unlabeled cells, the viral Gag-GFP was localized to the side where the cells were in contact with the other cells. This was only the case for infected B cells expressing the viral Env protein.

Because virological synapses are induced by migration of Gag toward the area where the infected cell contacts an uninfected cell, and because Env is required for their formation, this suggested that the infected B cells had formed virological synapses with the uninfected cells in the lymph nodes of living mice. “This is the first proof of principle that in vivo you see these structures,” Sewald said.

Using cell-type specific antibodies, Sewald and colleagues then found that the MLV-infected B cells typically formed virological synapses with uninfected B cells or CD4+ T cells.

Mothes plans to use a similar approach to look for in vivo evidence of virological synapses in HIV transmission.

A second role for tetherin
The discovery that the restriction factor tetherin might have a secondary role as a virus-specific PRR that can activate an innate immune response to HIV also caused considerable excitement this year.

If true, this would make tetherin the second host cell restriction factor found to have such a dual role. Just last year, Jeremy Luban’s group at the University of Geneva reported that Trim5 functions as a PRR, in that it activates an innate immune response when it encounters HIV (2). At the time, Luban told IAVI Report that other host cell restriction factors such as tetherin might also act as PRRs (see A Flurry of Updates from Keystone, IAVI Report, March-April 2011).

It now appears Luban was right, at least about tetherin. Speakers from three groups reported evidence at Cold Spring Harbor that tetherin can indeed activate NFκB, a factor that in turn activates the innate immune response. One of them, Paul Bates, a professor of microbiology at the Perelman School of Medicine at the University of Pennsylvania, said he got the idea that this might be the case when one of his graduate students showed him a paper from almost ten years ago where researchers expressed a large number of cDNAs in cultured cells to see if the proteins they encode could activate NFκB (3). Tetherin, known as BST-2 at the time, was one of the top hits, although its involvement in HIV-1 restriction wasn’t known yet.

That role is, of course, well known today: Tetherin, as its name implies, ties HIV-1 particles to the surface of infected cells so they can be endocytosed and degraded instead of infecting other cells. HIV-1, in turn, uses a protein called Vpu to counteract tetherin by promoting its degradation inside the host cell.

Bates reported that he found two forms of tetherin in human cells: A shorter one that’s resistant to Vpu-mediated degradation, and a longer one that isn’t. As for tetherin potentially inducing NFκB, Bates said he was initially skeptical, but when his graduate student consistently found that the longer, but not the shorter, form of tetherin could activate NFκB at least 20-fold in a cultured human cell line, he was convinced. “That absolutely sold it for me that it was a real activity,” Bates said. “I think it makes sense. We never liked the idea that all that tetherin did was hold [HIV] particles on the surface and allow them to be endocytosed. That makes no sense for a restriction factor. You wanna be able to signal.”

Bates said he has so far only been able to demonstrate NFκB activation by tetherin in cultured human cell lines, but he is currently trying to show this in primary CD4+ T cells as well.

But Stuart Neil from King’s College London has already obtained indirect evidence that this might be the case. He found that primary CD4+ T cells express tetherin. When these cells are infected with tetherin-sensitive HIV-1 (which doesn’t have Vpu to induce tetherin degradation, allowing tetherin to stay around and activate an innate immune response), they produce more proinflammatory cytokines of the type known to be activated by NFκB. This implies—though it doesn’t directly show—that tetherin might indeed activate NFκB in primary CD4+ T cells. 

Still, whether tetherin can actually sense HIV hasn’t yet been established, Bates said. “It’s suggested, but it’s clearly not proven,” he said, adding that he believes tetherin likely recognizes the shape change the host cell membrane undergoes as the virus is budding, which suggests that tetherin might sense not just HIV, but any virus that buds off of the outer cell membrane.

Given that Trim5 has also been shown to double as a PRR, it “is going to be a recurring theme that antiviral factors, by their nature, can be pattern recognition receptors as well,” said Neil. He suggests that this dual function is advantageous for the organism because it makes infected cells much more visible to the immune system.

Viral evolution
Host cell restriction factors are also an important factor for viral evolution. SIVcpz, the predecessor for the globally dominant pandemic group M HIV-1, for example, switched from chimpanzees to humans about 100 years ago. In its original chimpanzee host, SIVcpz used a protein called Nef to counteract the restriction factor tetherin. Human tetherin, however, has a deletion that makes it resistant to Nef, which is why in its new human host, the virus adapted to use Vpu to counteract human tetherin.

To get an idea of just how difficult these kinds of adaptations are for the virus, Daniel Sauter and Nicola Götz, in Frank Kirchhoff’s laboratory at Ulm University Medical center in Germany, studied what happens when HIV-1 is reintroduced to its original chimpanzee host. To do so, they took advantage of a chimpanzee that was infected with HIV-1 about 25 years ago.

Sauter and colleagues found that in the chimpanzee, HIV-1 Nef acquired about 25 amino acid changes, and that just two of these changes sufficed to restore its anti-tetherin activity. As a result, the chimpanzee now carries an HIV-1 strain that can use both Nef and Vpu to antagonize chimpanzee tetherin.  Or to put it another way: after 100 years outside its original host, it took the virus just a single passage in a chimpanzee and two amino acid changes in Nef to regain its ability to antagonize tetherin. Kirchhoff believes that part of the reason Nef made the switch so easily is that the virus retained Nef while it was in humans because the protein has other important functions. For example, it down-regulates the CD4 receptor in the host cell, which is thought to be an important factor in HIV’s pathogenicity.

“It just takes a few changes to restore the interaction with tetherin because everything else is still there,” Kirchhoff said, adding that this shows how rapidly these viruses can re-acquire once lost activities. It also illustrates, he said, that the multifunctionality of these proteins increases their potential to regain lost function because a gene that’s inactive would likely be eliminated.

Such changes in the viral Nef protein are the result of an evolutionary arms race between the virus and the host. At the meeting, Nicholas Meyerson from the University of Texas at Austin reported how the vestigial traces of this arms race in the host cell genome can help identify new cellular factors that directly interact with HIV.

The parts of the cellular host proteins the virus interacts with most directly, said Meyerson, are constantly evolving mutations as an escape response from the virus. Once the virus responds with escape mutations on its own, “the game starts all over again,” he said. As a result, the parts of cellular host proteins that interact with HIV most directly would be expected to evolve more rapidly than the rest of those proteins.

This has already been shown to be the case for host cell restriction factors like tetherin. Now Meyerson and his colleagues are trying to find footprints from this arms race in the sequences of cellular cofactors—host cell proteins the virus uses to complete its life cycle but that also have other functions in the host cell. Their goal is to identify new cofactors that directly interact with the virus. “We [are looking] for regions that are evolving far more rapidly than they should be,” he said.

As a first proof of principle that the approach works, Meyerson showed evidence for such footprints in the CD4 receptor, the receptor HIV uses to infect CD4+ T cells. A comparison of the amino acid sequences of the CD4 receptor of 24 nonhuman primate species and humans revealed eight amino acids that change much more rapidly than the rest of the protein, and all eight are located in the parts of CD4 that are known to interact with HIV gp120. “The sites that we uncovered are all around that [CD4-gp120] interface,” suggesting that the signatures are due to direct physical contact with retroviruses, Meyerson said. The sequences used for this comparison, he added, did not come from HIV-infected people or SIV-infected monkeys, and therefore reflect the genetic footprints left from millions of years of interaction with primate lentiviruses similar to HIV, which also use CD4 to enter their target cells. 

Meyerson and colleagues have used the approach to screen hundreds of cofactors that might interact with HIV, and have so far found footprints in several that suggest they might be engaged in this kind of arms race, Meyerson said. One of them is called RanBP2, a component of the nuclear pore through which HIV gets into the nucleus, and which recently has indeed been found to directly interact with HIV.

Though the approach seems to work quite well, Meyerson said it’s not successful in all cases: Some cellular cofactors have so many other functions in the host cell that they can’t afford to mutate much.

A new kind of antiviral factor
Researchers are also discovering other strategies cells use to fight HIV-1. One example is a cellular factor called Schlafen 11. It is induced as part of the innate immune response to pathogens, but its function wasn’t known until Manqing Li from the University of California, San Diego took a closer look.

Li reported evidence that the ability of this factor to bind and inactivate tRNAs might enable it to fight HIV. tRNAs are molecules the cells use to translate the nucleic acid sequence of mRNAs into the amino acid sequence of proteins. To do so, tRNAs carry a triplet of nucleic acids—or an anticodon—on one end, and the amino acid encoded by this anticodon on the other. Once a tRNA binds the appropriate codon on the mRNA, the amino acid it carries on the other end is tacked onto the end of a growing protein chain.

HIV-1 tends to use unusual codons to encode its proteins. As a result, Li said, human cells likely have fewer tRNAs that can translate HIV proteins than they have to translate their own proteins. Li found that Schlafen 11 seems to inhibit tRNAs by binding, which led him to hypothesize that inhibition of tRNAs by Schlafen 11 should affect translation of HIV proteins more than translation of host cell proteins.

To test this idea, Li introduced the original HIV gag gene or a modified version of gag that uses human codons into a human HEK293 cell line that expresses Schlafen 11. He found that inhibiting Schlafen 11 expression in these cells led to increased protein levels of HIV Gag, but not the human version of Gag. This suggests that Schlafen 11 only inhibits the levels of the HIV-encoded Gag protein. Li got the same results when he used the gene for green fluorescent protein from jellyfish (which uses similar codons as HIV but is not found in HIV or humans) and compared it with a version of GFP that’s encoded by human codons: Only the jellyfish version of GFP was affected by Schlafen 11.

To Li, this suggests that the inhibitory Schlafen 11 effect on HIV protein levels is not HIV specific and is more likely related to the retrovirus’s unusual codon usage.

HIV's hiding place in the nucleus
Researchers have known for some time that HIV DNA tends to integrate into actively transcribed genes, and that it favors certain parts of the genome more than others. Where these spots are in the nucleus, however, hasn’t been clear. But at the conference, researchers reported that they’ve been able to visualize for the first time where in the nucleus integrated HIV DNA is located.

One report came from Bruna Marini, a PhD student at the International Centre for Genetic Engineering and Biotechnology (ICGEB) in Trieste, Italy, who with her colleagues cultured activated primary CD4+ T cells, infected them with HIV, and quick-froze them with liquid nitrogen to preserve the structure of the nucleus. She then added fluorescently tagged DNA probes specific for the HIV genome to the infected frozen cells, and took a three-dimensional look at the nucleus with a fluorescent confocal microscope. This revealed that the integrated HIV DNA was at the periphery of the nucleus, close to the nuclear pore, which she had also visualized with a fluorescently tagged antibody (see image below).

HIV's Hiding Place  

Nucleus of an HIV-1 infected CD4+ T-cell that shows where the HIV DNA (green) is integrated into the host cell genome. The researchers visualized the HIV DNA by adding fluorescently tagged DNA probes specific to the HIV genome, and they visualized the nuclear membrane (red) by adding fluorescently tagged antibodies to a nuclear pore protein. The image is an optical section through the nucleus taken with a fluorescent confocal microscope. Image courtesy of Bruna Marini, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.


Initially, Marini said, this seemed to contradict previous observations that HIV tends to integrate next to actively transcribed genes, since the periphery of the nucleus is thought to primarily harbor inactive genes. But further analysis showed that RNA Polymerase II, one of the main enzymes involved in gene transcription, was bound to the integrated HIV genome, suggesting that the integrated HIV DNA was indeed being actively transcribed. 

This strategy makes sense for the virus, the researchers said. “[HIV] finds the first [active] genes that are available [next to] this pore,” said Marina Lusic, a research scientist at the ICGEB and one of the supervisors of Marini’s project. “The whole philosophy of the virus is to replicate as quick[ly] as possible and to infect as [many] cells as possible.”

Marini and colleagues also found that cells that had been infected with low HIV titers carried just one and, on rare occasions, two or three integrated copies of HIV close to the periphery. This suggests that the virus doesn’t integrate more often than necessary. 

Using the same approach, the researchers also found typically one HIV DNA copy integrated into the genome of latently HIV-infected CD4+ T cells. In such cells, they found integrated HIV DNA closely associated with a nuclear structure called PML nuclear body, which they showed plays a role in silencing transcription of HIV DNA. 

Cristina Di Primio, a postdoctoral researcher at the Scuola Normale Superiore in Pisa, Italy, also reported evidence that HIV DNA integrates into sites at the periphery of the nucleus. But instead of visualizing the integrated DNA directly, Di Primio and colleagues used a more indirect approach. They used fluorescently labeled antibodies to visualize proteins involved in the cellular repair response to a cut in the integrated HIV DNA. That cut doesn’t normally occur, but Di Primio and colleagues made modified HIV particles that carried a short yeast sequence in their genome that can be recognized by a yeast enzyme that cuts DNA; they then used these modified HIV particles to infect a human T cell line they had modified to express this yeast enzyme. 

Like Marini and colleagues, Di Primio’s team found the cuts in the integrated HIV DNA mostly at the periphery of the nucleus, close to the nuclear pore. She added that the cuts are unlikely to be in unintegrated HIV DNA; for example, the DNA cuts could still be observed two weeks after the cells got infected, at which time most of the unintegrated DNA should have degraded. The cuts also disappeared in cells treated with antiretrovirals that are known to inhibit DNA integration.

Currently, Di Primio and colleagues are using their approach to identify host cell proteins or drug candidates that can affect nuclear import or integration of HIV DNA into the host cell. “We can now use this tool to search for new targets for therapy [and] to find new cofactors or new inhibitors for HIV integration,” she said. —Andreas von Bubnoff

1. Proc. Natl. Acad. Sci. 109, 7475, 2012
2. Nature 472, 361, 2011
3. Oncogene 22, 3307, 2003