ABOVE: To establish infection, HIV must deliver its genome to the host’s nucleus. Now, researchers know how the virus achieves this tricky feat, adding another piece to the HIV replication puzzle. ©iStock, vdvornyk

Smuggling its genome into the nucleus is essential for HIV to infect its host, but entering the cell’s control center is no easy feat. Molecules must pass through tightly-regulated nuclear pores on the backs of specialized receptor chaperones. How the bulky HIV-1 virus manages to squeeze through this portal has baffled scientists for decades.

In a paper published in Nature, researchers revealed new evidence in this long-standing mystery by reporting that HIV mimics a common transport mechanism used by cells to shuttle large molecules to and from the nucleus.1 Their findings demonstrate how the HIV-1 capsid effectively targets the viral genome to the nucleus and provide novel targets for antiviral therapies. 

“This had been a black box for a very long time,” said Dirk Görlich, a biochemist at the Max Planck Institute for Multidisciplinary Sciences and coauthor of the study. 

Nuclear pore complexes (NPC), massive 100-megadalton structures, oversee the comings and goings at the nucleus. For a long time, scientists thought that the NPC pore was approximately 40 nanometers (nm) wide, a diameter too small to accommodate the 60 nm wide, cone-shaped HIV-1 capsid that envelops the viral genome. Instead, they reasoned that the bulky viral capsid must disassemble in the cytoplasm, where it prepares the viral genome for transport into the nucleus. However, in 2020, evidence emerged showing intact HIV-1 capsids inside the nucleus, casting doubt on this theory.2 “This was sort of going against the dogma at the time,” said Vinay Pathak, a virologist at the National Cancer Institute who was not involved in the research.

Around the same time, Thomas Schwartz, a structural biologist at Massachusetts Institute of Technology and coauthor on the study, demonstrated that the NPC architecture was flexible and could expand beyond 40 nm, potentially accommodating larger cargoes like HIV-1.3 Then another team led by biophysicist Martin Beck at the Max Planck Institute of Biophysics used a high-resolution, three-dimensional imaging technique called cryoelectron tomography to catch HIV-1 in the act of traversing the NPC, which was wedged open to about 64 nm, just large enough for the viral capsid.4

These studies confirmed that intact HIV-1 capsids traversed the NPC, but how the virus pulled this off mechanistically was unclear. Görlich and Schwartz teamed up to answer this question. 

The NPC is not simply a hole; instead, it is filled with nuclear pore proteins that create a dense gel. Some of these proteins are intrinsically disordered, meaning that they lack a fixed three-dimensional structure, and they harbor segments of repeating phenylalanine and glycine amino acids known as FG repeats. 

The FG repeats are hydrophobic elements in an otherwise hydrophilic sea, and the interactions of proteins with these two properties creates a sieve-like barrier that allows tiny molecules to diffuse through but requires larger cargoes to use a different approach.5 Any molecule that is larger than 40 kilodaltons needs help to breach the FG barrier and enter the nucleus. Importins—molecules that transport larger proteins into the nucleus—are decorated with binding pockets that interact with FG repeats in a way that allows them to rapidly diffuse through the NPC to the nucleus.

Görlich and Schwartz considered the possibility that the HIV-1 capsid might hitch a ride on an importin, but the capsid alone is larger than the transport proteins’ nearly 40 nm size limit.6 Schwartz started to wonder why no one had tested whether the capsid, which scientists had previously shown binds specific FG repeats, actually had a broader FG binding profile similar to an importin.7 “This seemed to me so obvious that it should work this way,” Schwartz said.

To test this hypothesis, they turned to a method previously developed by Görlich’s team for reconstituting the jelly-like NPC permeability barrier in vitro.8 For this, FG repeat domains self-assemble into an FG phase that retains key features of an NPC, including importin-based transport. In the present study, they filled HIV-1 capsids with fluorescent proteins and tracked their movements across the recapitulated FG barrier. The researchers found that the capsids efficiently targeted the NPC-like structures and interacted directly with several types of FG repeats. Mimicking an importin, the giant HIV-1 capsid fragments melted into and through the FG barrier. At the same time, the barrier excluded other, much smaller, macromolecules that were not linked to the capsid.

“This is a masterpiece of evolution,” said Görlich.

Next, the researchers tracked the capsids inside cells. Görlich and his team found that GFP-filled HIV-1 capsids efficiently targeted human NPC in vitro without the help of cellular transport receptors. Furthermore, capsids injected into the cytoplasm of mouse oocytes traversed NPC and entered the nucleus.

Pathak noted the importance of Görlich and Schwartz’s findings. “It pretty much cements the idea that the intact cores get into the nucleus,” he said. “This will become a new model for how HIV replicates in infected cells.” 

In the same issue of Nature, a research team at the University of New South Wales led by David Jacques published similar findings.9 They showed that sections of the HIV capsid interacted with FG domains in a manner akin to transport proteins, which allowed the viral capsid to cross the NPC permeability barrier. 

“The two papers together make a very convincing argument,” said Pathak. 

Also noting the synergy between the two studies, Schwartz said, “In combination, I think it's very powerful. You have two groups that don't know about each other, and they don't even use the same assays and come to a—let's say 90 percent—overlapping conclusion.”

The researchers are considering how they can exploit this newly revealed HIV replication stage for therapeutic intervention. “That's a very promising approach to add to the already powerful and potent inhibitors of HIV that we have,” said Pathak. He also noted that having a full arsenal of antiviral weapons is critical as the virus develops resistance to drugs.

“It's a new element of the HIV life cycle that previously nobody targeted therapeutically, and now you have a whole new aspect of this life cycle that you may be able to actually target,” said Schwartz.

References 

  1. Fu L, et al. HIV-1 capsids enter the FG phase of the nuclear pores like a transport receptor. Nature. 2024;626(8000):843-851.
  2. Burdick RC, et al. HIV-1 uncoats in the nucleus near sites of integration. Proc Natl Acad Sci USA. 2020;117(10):5486-5493.
  3. Schuller AP, et al. The cellular environment shapes the nuclear pore complex architecture. Nature. 2021;598(7882):667-671.
  4. Zila V, et al. Cone-shaped HIV-1 capsids are transported through intact nuclear pores. Cell. 2021;184(4):1032-1046.
  5. Frey S, et al. FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. Science. 314(5800):815-817.
  6. Panté N, Kann M. Nuclear pore complex is able to transport macromolecules with diameters of about 39 nm. Mol Biol Cell. 2002;13(2):425-434.
  7. Matreyek KA, et al. Nucleoporin NUP153 phenylalanine-glycine motifs engage a common binding pocket within the HIV-1 capsid protein to mediate lentiviral infectivity. PLoS Pathog. 2013;9(10):e1003693.
  8. Ng SC, et al. Recapitulation of a selective nuclear import and export with a eprfectly repeated 12mer GLFG peptide. Nat Commun. 2021;12(1):4047.
  9. Dickson CF, et al. The HIV capsid mimics karyopherin engagement of FG-nucleoporins. Nature. 2024;626(8000):836-842.