Accomplishing a feat that had been a pipe dream for decades, scientists have recreated in a test tube the first steps of infection by HIV (human immunodeficiency virus), the virus that causes Aids (acquired immunodeficiency syndrome). Doing so has provided up-close access to the virus – which is otherwise obstructed from view deep within the cell – and enabled identification of essential components that HIV needs to replicate within its human host.
Specifically, the scientists were able to monitor the virus as it replicated its genome and inserted it into target DNA, mirroring steps that ordinarily take place within the host. These advances yield a new understanding of how HIV works, the authors say, allowing for explorations of early stages of the virus life cycle in unprecedented detail. Such knowledge could lead to improved treatments for Aids, a lifelong disease that can only be kept under control with a continuous medication regimen.
"This is teaching us how HIV infects," says Dr Wesley I Sundquist, distinguished professor of biochemistry at University of Utah Health. He is co-senior author of the study with his former trainee, Dr Owen Pornillos, now an associate professor at the University of Virginia. The co-first authors are Dr Devin Christiansen, and Dr Barbie Ganser-Pornillos. "We are learning new things about one of the most significant pathogens that humans have ever encountered, and that is important."
For all its danger, HIV is deceptively simple in appearance. The virus resembles a rounded ice cream cone, where an outer shell encapsulates the virus' genetic material inside. Previously, it had been thought that the main role of the shell, called the capsid, was to protect its precious cargo. But the investigations by Sundquist and Pornillos' team show that the capsid also plays an active role in infection.
Carrying out initial steps of infection in a test tube allowed the research team to precisely manipulate HIV in ways that had not been possible before. They found that when they used genetic and biochemical methods to destabilise the capsid, HIV could not effectively replicate its genetic material. It was the first direct demonstration that, rather than serving merely as packaging, the capsid is an essential component of the HIV infection process itself.
If seeing is believing, then watching the HIV molecule in action gave credence to the experimental finding. Recent advances in cryo-electron microscopy and molecular modelling have made it possible to see the virus – which, at 130nm, is about 60 times smaller than a red blood cell—in exquisite detail. Using these techniques, the team visualised each of the 240 tiny protein "tiles" that fit together to make the cone-shaped outer shell. With the up-close view, the scientists could literally see that the capsid remained largely intact throughout the replication process, called reverse transcription. "This is different than in the textbooks," Sundquist says. "Our data indicate that the viral capsid plays an active and indispensable role in supporting efficient reverse transcription."
Sundquist says that the discovery may help explain why an investigative HIV drug developed by Gilead Sciences, the first to target the capsid, is a potent inhibitor of the virus. Previous work by Sundquist, Pornillos, and others elucidating the structure and function of the HIV capsid informed the design of the drug, which has performed well in phase 1 clinical trials. Additional insights gained through the test tube system could improve drug design even further.
Advances in microscope technology, coupled with dogged persistence, led to the new view of HIV, which was first discovered as the cause of Aids more than 35 years ago. It took years of trial and error to determine the minimum components required for recapitulating the process in a test tube, outside the cell. Now that the simplified system is up and running, Pornillos says, it opens doors to learning more fundamental truths about a familiar foe.
"For me, there is both the fundamental knowledge aspect of it, but also the translational aspect that could help us come up with better ways to stop HIV," Pornillos says. "That's why it's great research."
The research was published by Sundquist, Pornillos, Christensen, Ganser-Pornillos, and Jarrod Johnson from the University of Utah.
Abstract
Introduction: Reverse transcription and integration are the signature events of retrovirus replication. Reverse transcription creates a double-stranded DNA (dsDNA) copy of the positive-sense viral RNA genome, and integration archives that copy within the genome of the infected cell. Both processes are targets of successful HIV-1 antiretroviral therapies, and the associated enzymatic activities have been characterized by elegant structural, biochemical, and molecular virological analyses. Nevertheless, mechanistic studies of these processes remain challenging because they are performed by viral core particles deep within the infected cell cytoplasm and nucleus. Of particular interest is defining whether the conical capsid that surrounds the viral RNA genome participates in the process of viral replication.
Rationale: In principle, informative mechanistic and imaging analyses of HIV-1 replication could be performed in vitro, but the coupled processes of reverse transcription and concerted integration have not yet been recapitulated outside of the cell. To address this limitation, we reconstituted these processes in a cell-free system, using purified HIV-1 virions as the source of viral genomes and enzymes.
Results: We recapitulated the sequential processes of endogenous reverse transcription (ERT) and integration from viral core particles, which were released from purified HIV-1 virions by gently permeabilizing the viral membrane using a pore-forming peptide. ERT was initiated through addition of deoxynucleotide triphosphates (dNTPs), and the DNA products—early, intermediate, and late transcripts—appeared in high yields and in the expected temporal order, with late dsDNA products accumulating maximally after 8 to 10 hours. Integration of the resulting viral DNA into an exogenous target DNA was also recapitulated, provided that cell extract was added to the reaction mix. Deep sequencing and cloning confirmed that the 3′ and 5′ termini of the viral DNA were integrated in a concerted fashion, with the expected 5–base pair target site duplications in the target DNA, and with target site sequence preferences that resembled those reported for HIV-infected cells.
HIV-1 capsids are composed of hexagonal lattices of the viral CA protein, arrayed in a “fullerene cone” structure. Analyses of the requirements for efficient ERT and integration in our cell-free system revealed that these processes require the presence of capsid lattices of appropriate stability and geometry, as determined by using site-directed CA mutations, capsid-stabilizing factors, and potent new capsid inhibitors. Imaging with electron cryotomography revealed that many viral capsids remained largely intact during the ERT reaction. Capsid uncoating, when observed, did not occur in an all-or-none fashion, as might have been anticipated for a highly cooperative structure. Rather, uncoating proceeded through a continuum of disassembly intermediates in which portions of the capsid wall appeared lost in patches, as revealed through subunit-level lattice mapping. Largely intact capsids in which viral nucleic acid strands extruded through lattice openings were observed after 8 to 10 hours, which was coincident with the maximal accumulation of late ERT products and integration events.
Conclusions: We have reconstituted efficient reverse transcription and integration—the major early steps of the HIV-1 life cycle—in a cell-free system. Our data indicate that the viral capsid plays an active and indispensable role in supporting efficient reverse transcription. Thus, we consider the entire core particle, including the outer capsid shell, to be the true viral “replication complex.” We further found that complete capsid uncoating may be a prerequisite for integration to occur. Thus, the capsid plays essential roles in the reactions that duplicate and archive the viral genome, in addition to previously established roles in protecting the viral genome from innate immune sensor surveillance and in helping the core to traverse the cytoplasm, enter the nucleus, and traffic to integration sites. We anticipate that our cell-free system will enable systematic analyses of the key steps in viral replication and integration and thereby elucidate the transformations that occur as HIV-1 proceeds through the first half of the viral life cycle.
Authors
Devin E Christensen, Barbie K Ganser-Pornillos, Jarrod S Johnson, Owen Pronillos, Wesley I Sundquist
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[link url="https://science.sciencemag.org/content/370/6513/eabc8420"]Science abstract[/link]