Scientists have finally recreated the early stages of HIV infection in vitro, providing an incredibly magnified view of the effects of the virus.
The amazing image shows a conical shell of geometric tiles called capsids. It is located at the center of the virus and contains the following genetic material: RNA.. Before infiltrating the cell, the capsid is surrounded by an envelope of fat molecules. This envelope fuses with the host cell to put the capsid inside, where it carries RNA to the cell’s nucleus. RNA is replicated along the way, and when it enters the nucleus, it invades the host DNA.
By scrutinizing this replication process, new studies emphasize that capsids themselves play an important role in infection and must meet certain criteria. Virus Interweaving the genome with the host cell.
Know how to reproduce the first step of HIV Infectious diseases “mean that there are more tools to analyze the process of replication,” said research author Wesley Sandquist, a prominent professor of biochemistry at the University of Utah.In particular, a study published in the journal on October 8th. ScienceDescribes a cell-free system that can be used to study how HIV invades the host genome. Such a system has the potential to “revolutionize HIV testing in many laboratories.” He was involved in the research and told Live Science by email.
“Achieving all this is the real power,” James said. In addition to basic research, this system may also help explain how capsid-targeted drugs work to limit HIV replication, Sundquist. Said in a statement..
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Test tube test
Test tube experiments capture the aspects of HIV infection in great detail, but cannot be reproduced. all A step in the process, Sundquist pointed out. Infection usually begins when the outer membrane of the virus fuses with the membrane of the host cell, allowing the capsid and its interior to invade the interior. But in a cell-free system, the author had to bypass this first step.
Instead, they used a compound called meritin in the honeybee venom to make the viral membrane “permeable” and release the capsids retained in it.
According to a 2017 report from the journal, HIV capsids have small pores that normally present a cell-building block of DNA called deoxynucleotide triphosphate that is already present when viral particles float in the cytoplasm of human cells. Pick up. Nature.. When the viral particles move to the nucleus, they use these components to make a complete strand copy of the DNA, thanks to a special enzyme contained within the capsid. This is how the virus copies its genetic material and later inserts it into the host genome. While it is still somewhat strange how the virus “knows” when this so-called reverse transcription begins, research suggests that the biochemical properties of the host cell serve as clues to initiate the reaction.
However, the test tubes do not automatically contain DNA building blocks in solution, so the author added them to initiate reverse transcription quickly. “This method has been around for some time, but it’s difficult to get it going until the reaction is complete,” James said. However, the authors of the study were able to perform reverse transcription smoothly. To do so, they learned that capsids must remain almost intact throughout the process.
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“The capsid must be largely intact and must have adequate stability or flexibility to support reverse transcription.” That is, the capsid is sufficient to prevent it from falling apart during reverse transcription. It needs to be tightly bound, but the capsid enters the nucleus to release the copied DNA, Sandquist said. Fortunately, scientists have recently discovered a way to keep capsids stable enough.
In 2018, research author Owen Polnijos, an associate professor of molecular physiology and biophysics at the University of Virginia, and his colleagues discovered that a compound called IP6 binds to the tiled surface of capsids. Reported in the journal .. IP6 is negatively charged, but each tile is positively charged on the side pointing to the center of the capsid. As the opposite side is attracted, the IP6 binding to the capsid helps to pull the tile into a tighter, more stable placement.
“Before IP6 was discovered, someone [viral] envelope In vitro And everything collapsed and nothing was visible. ” Joan Mamede, an assistant professor at the Department of Microbial Pathogen Immunology at Rush University and not involved in the study, said.
Because IP6 is “quite abundant” in cells, the authors added compounds in in vitro experiments at concentrations similar to those found in cells, Sandquist said. “It was really a trick,” he added. “Until we knew it, we were dealing with capsids that were too volatile.”
seeing is believing
Using a computer model of the molecule and an electron microscope, the author was able to confirm that the 240 tiles that make up the capsid literally maintain a stable lattice structure through reverse transcription. As the DNA strands grew, their ends could penetrate small gaps in the lattice weave, and a single tile could be seen coming off while the rest of the capsid remained intact. ..
Christopher Aiken, a professor of pathology, microbiology, and immunology at Vanderbilt University who was not involved in the study, said capsids may need to remain stable to bring RNA and transcription enzymes closer to each other. Said. “Enzymes can be encapsulated to rebind templates and continue DNA synthesis,” Aiken told Live Science via email, as enzymes tend to shed from RNA during transcription.
Once reverse transcription is complete, the authors move on to the next step in infection, integration, in which viral DNA infiltrates the host genome. They introduced a DNA strand called a plasmid into a test tube to act as a proxy for DNA in the human nucleus, but integration did not begin without additional components. Only a “whole cell extract”, a mixture of proteins and molecules drawn from cells, allows viral DNA to penetrate the plasmid.
In the future, the team wants to pinpoint exactly which component of the cell extract causes integration, Sundquist said. “It’s likely to be more than one,” he said. One challenge is that in test tube experiments, it’s always difficult to know if something is missing.
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One limitation of this study is the inability to completely reproduce the state of the cell, James said.
“Any In vitro No matter how powerful the system is, it can only be used to test the ingredients we know and can be added to the reaction, “said James. For example, in a real cell, the capsid needs to move to the cell nucleus where the DNA is held. It then slips through a portal known as the nuclear pore. Sundquist pointed out that there may be unknown factors that change the capsid during this journey.
That said, the new cell-free system could help uncover the identity of these unknown factors, Mamede added. Scientists can now make observations in a cell-free environment to see if the same behavior appears in real cells, he said.
In addition, this system may also be useful in drug development. “You can test [new drugs] “Using one of these simplified systems makes it easier than using cells,” Mamede told Live Science. This is what is really being done against the virus. You can check it mechanically. ”
The pharmaceutical company Gilead Sciences is currently holding a new drug in human clinical trials that specifically target HIV capsids. ClinicalTrials.gov..based on The drug appears to alter the capsid at various points of infection, including during reverse transcription. Sundquist said cell-free studies emphasized that capsids are a “key factor” in HIV infection and that disruption of capsids can limit the ability of the virus to grow.
Originally published in Live Science.
