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New HIV "Cure" Strategy
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This strategy led to the clearance of latent HIV-1 in patient CD4+ T cells after viral reactivation. Premature intracellular activation of the viral protease triggered CARD8 inflammasome-mediated pyroptosis of HIV-1-infected cells ....our study identifies CARD8 as an inflammasome sensor of HIV-1, which holds promise as a strategy for clearance of persistent HIV-1 infection.
In Science, full paper below:
Abstract
HIV-1 has high mutation rates and exists as mutant swarms within the host. Rapid evolution of HIV-1 allows the virus to outpace the host immune system, leading to viral persistence. Approaches to target immutable components are needed to clear HIV-1 infection. Here, we report that the CARD8 inflammasome senses HIV-1 protease activity. HIV-1 can evade CARD8 sensing because its protease remains inactive in infected cells prior to viral budding. Premature intracellular activation of the viral protease triggered CARD8 inflammasome-mediated pyroptosis of HIV-1-infected cells. This strategy led to the clearance of latent HIV-1 in patient CD4+ T cells after viral reactivation. Thus, our study identifies CARD8 as an inflammasome sensor of HIV-1, which holds promise as a strategy for clearance of persistent HIV-1 infection.
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Human immune cells have natural alarm system against HIV
Drugs that trigger alarm could lead to new HIV therapies
Date:
February 4, 2021
Source:
Washington University School of Medicine
Summary:
Researchers have identified a potential way to eradicate the latent HIV infection that lies dormant inside infected immune cells. Studying human immune cells, the researchers showed that such cells have a natural alarm system that detects the activity of a specific HIV protein. Rather than attack the virus based on its appearance, this strategy is to attack the virus based on what it is doing -- vital activities that are required for the virus to exist.
Treatment for HIV has improved tremendously over the past 30 years; once a death sentence, the disease is now a manageable lifelong condition in many parts of the world. Life expectancy is about the same as that of individuals without HIV, though patients must adhere to a strict regimen of daily antiretroviral therapy, or the virus will come out of hiding and reactivate. Antiretroviral therapy prevents existing virus from replicating, but it can't eliminate the infection. Many ongoing clinical trials are investigating possible ways to clear HIV infection.
In a study published Feb. 4 in the journal Science, researchers at Washington University School of Medicine in St. Louis have identified a potential way to eradicate the latent HIV infection that lies dormant inside infected immune cells. Studying human immune cells, the researchers showed that such cells have a natural alarm system that detects the activity of a specific HIV protein. Rather than attack the virus based on its appearance, which is the basis of most immunotherapies, this strategy is to attack the virus based on what it is doing -- vital activities that are required for the virus to exist.
HIV is nearly impossible to eradicate because the immune system can't keep up with its unusually rapid mutation rate, constantly changing the way it looks to evade immune attack -- akin to a spy quickly changing appearance to evade authorities. Just as the body's immune cells learn to recognize one manifestation of the virus, it already has changed into multiple new disguises.
"When we identified a part of the immune system that could recognize and attack a core function of the HIV virus -- rather than what it looks like -- it was really exciting," said senior author Liang Shan, PhD, an assistant professor of medicine. "An analogy might be that it's relatively easy to change clothes or hairstyle to go undetected, but it's impossible to conceal running. This is exciting because it raises the possibility of clearing all the dormant virus in a single patient -- no matter how widely mutated -- based on something all the viral variants have in common. For patients, even if they are consistent with therapy and experience no symptoms, having a treatment that could change their HIV status from positive to negative would have a massive impact on their lives."
This strategy relies on detecting the activity of a specific protein -- called HIV protease -- that the virus requires to replicate and spread.
The researchers identified a natural alarm system -- found inside human immune cells and called the CARD8 inflammasome -- that recognizes active HIV protease and triggers a self-destruct program to eliminate the infected cell. Unfortunately, HIV can exist a long time in the cell without ever tripping the alarm. When inside cells, HIV protease is inactive, lying low, and the CARD8 inflammasome can't detect it.
"The virus is smart," Shan said. "Normally, HIV protease doesn't have any function inside infected cells. Viral protease is only activated once the virus leaves the infected cells. Outside the cells, there is no CARD8 to sense the active protease."
Shan and his colleagues showed that certain drugs force HIV protease to show itself prematurely, when the virus is still inside the immune cell. There, active HIV protease triggers the CARD8 inflammasome, setting off a chain of events that destroys the infected cell and the virus along with it.
"We found that our immune system can recognize this key protein function -- not the protein sequence, which the virus changes constantly through mutation," Shan said. "HIV protease can't mutate because its function is required to complete the virus's life cycle. Otherwise, it's a dead virus. HIV protease's specific action for the virus is also the specific action that sets off CARD8. Our findings show that our immune system can recognize a virus's protein function and, under the right circumstances, use that information to kill HIV-infected cells."
One of the drugs that forces HIV protease to become active is called efavirenz (brand name Sustiva). It is part of a class of drugs called non-nucleoside reverse transcriptase inhibitors (NNRTIs), which have been used to treat HIV since the 1990s.
"We've long used this class of drugs to block HIV from inserting its genetic material into new cells," Shan said. "That's their day job. But now, we have learned they have a second job -- activating HIV protease inside the infected cell. When we treat HIV-infected human T cells with this drug, the protease becomes activated before the virus successfully leaves the infected cells. This triggers the CARD8 inflammasome, and the infected cells die within hours. This is a potential route to clearing the virus that we have never been able to completely eliminate."
But the researchers also found that efavirenz and other NNRTIs won't activate HIV protease unless the virus is awake, as Shan puts it. And in patients on HIV therapy, the virus is dormant. Many clinical trials seeking cures for HIV use a shock-and-kill method, waking up the virus with one drug and then attacking it with one of a variety of investigational strategies. Any clinical trial evaluating NNRTIs, or similar drugs that work the same way, as a potential approach for clearing HIV infection would require this shock-and-kill strategy.
The researchers also showed that the CARD8 inflammasome can trigger the death of human immune cells infected with HIV subtypes from around the world, including strains common in North America, Europe, Africa and Asia.
"We would like to identify or develop compounds that do an even better job of activating HIV protease than NNRTIs do and at lower doses," Shan said. "This study serves as a guide for developing new drugs that have the potential to eliminate the dormant HIV reservoir."
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CARD8 is an inflammasome sensor for HIV-1 protease activity
Science 04 Feb 2021
Abstract
HIV-1 has high mutation rates and exists as mutant swarms within the host. Rapid evolution of HIV-1 allows the virus to outpace the host immune system, leading to viral persistence. Approaches to target immutable components are needed to clear HIV-1 infection. Here, we report that the CARD8 inflammasome senses HIV-1 protease activity. HIV-1 can evade CARD8 sensing because its protease remains inactive in infected cells prior to viral budding. Premature intracellular activation of the viral protease triggered CARD8 inflammasome-mediated pyroptosis of HIV-1-infected cells. This strategy led to the clearance of latent HIV-1 in patient CD4+ T cells after viral reactivation. Thus, our study identifies CARD8 as an inflammasome sensor of HIV-1, which holds promise as a strategy for clearance of persistent HIV-1 infection.
Activation of the CARD8 inflammasome clears latent HIV-1 in patient CD4+ T cells
To determine whether HIV-1 protease function in activating the CARD8 inflammasome is conserved, we tested a panel of HIV-1 virus isolates from chronically infected individuals of subtypes A, B, C, and D (39). Subtype B is the dominant subtype in Europe and North America, whereas A, C, and D are more prevalent worldwide. T-20 and Raltegravir (RAL) were used to completely block new infection but had no killing effect or cellular toxicity (fig. S9). The addition of EFV and RPV but not NVP effectively cleared primary CD4+ T cells infected with all HIV-1 subtypes. Additionally, the killing efficiency did not correlate with viral replication fitness (Fig. 6A and fig. S10), suggesting that the enzymatic activity of HIV-1 protease with regard to CARD8 activation is well conserved across major HIV-1 subtypes. Next, we confirmed that EFV and RPV treatment induced CARD8-dependent caspase-1 activation and pyroptosis of primary CD4+ T cells infected with clinical viral isolates (Fig. 6, B and C, and figs. S11 and S12). To test whether strategies involving targeted activation of the CARD8 inflammasome could be used for the clearance of latent HIV-1, we obtained blood CD4+ T cells from patients under suppressive ART to measure the size of viral reservoirs (40). The control antiretroviral (ARV) combination containing T-20, RAL, and NVP had no killing effect. The median IUPM in control and RPV groups was 2.61 and 0.16, respectively, suggesting a rapid clearance of 93.9% of the latent HIV-1 reservoirs (Fig. 6, D and E). Notably, three out of eight patient samples had no detectable viral replication after RPV treatment. In our "shock and kill" assay, cells were only treated with RPV for the first 2-3 days. It is possible that the residual viruses in the RPV group came from delayed virus reactivation which occurred after removal of RPV.
Discussion
Due to rapid viral evolution, it is very difficult for the host immune system to control HIV-1 infection and clear residual viral reservoirs without targeting immutable components of the virus. In this study, we found that CARD8 is a sensor for HIV-1 protease activity to trigger inflammasome activation and pyroptosis of infected cells. This work demonstrates that the CARD8 and NLRP1 inflammasomes share similar mechanisms of activation, which involves their N-terminal cleavage by microbial proteases, followed by proteasome-mediated release of the bioactive C-terminal fragment to trigger inflammasome assembly and CASP1 activation. Interestingly, HIV-1 protease cleaves CARD8 at two different sites. Cleavage of the unstructured N terminus but not the FIIND domain leads to inflammasome activation. We also observed that a deletion (∆51-60) or mutations (F59A or F60A) of the first cleavage site increased the cleavage efficiency at the second site (Fig. 2, C and D), suggesting a competition between the two sites. Cleavage of HIV-1 Gag and Gag-Pol by the viral protease is a sequential process regulated by the rate of cleavage at individual site (41). Since CARD8 activation requires a cleavage within the unstructured N terminus, the cleavage site preference may influence the CARD8 inflammasome activation. In HIV-1-infected cells, the CARD8 inflammasome cannot detect the virus because the viral protease remains inactive as a subunit of unprocessed viral Gag-Pol polyprotein. Surprisingly, some NNRTIs which have been used to treat HIV-1 infection for more than two decades can facilitate CARD8 sensing by mediating premature intracellular activation of HIV-1 protease. NNRTIs bind to HIV-1 RT and act as enhancers of Gag-Pol dimerization to activate Pol-embedded viral protease (32). Additional investigations are needed to better understand the mechanism of the NNRTI-mediated Gag-Pol dimerization process. Although NNRTI-containing treatment regimens cannot eliminate HIV-1 infection in patients because the viral latent reservoirs are rapidly established prior to treatment initiation, inclusion of NNRTIs without protease inhibitors in the initial ARV regimen may partially reduce the seeding of latent viral reservoirs. In addition, inclusion of NNRTIs in HIV-1 cure strategies should facilitate the elimination of infected cells after viral latency reversal. Intriguingly, CARD8 is preferentially and highly expressed in blood and lymphoid tissues (42) as well as in many hematopoietic-derived cells (27), suggesting that targeting the CARD8 inflammasome may be effective in lymphoid tissues, the most important anatomical sites for persistent HIV-1 infection. Notably, the cell-killing IC50 of EFV and RPV is approximately 1-2 μM (Figs. 2C and 3B), which is about 100-fold higher than the infection-blocking IC50. The plasma EFV concentration in patients receiving EFV-containing regiments (1-4 μg/ml or 3-12 μM) is within the therapeutic range for cell killing. This strategy is unlikely to be effective in tissues with markedly lower drug concentration such as central nervous system. Importantly, HIV-1 Pol that confer resistance to NNRTIs also abrogate NNRTI-triggered cell killing (38) likely because the resistant viral variants can avoid drug binding. Thus, the identification of more potent chemical compounds that promote Gag-Pol dimerization regardless of viral inhibition is warranted. Taken together, this work reveals a mechanism of innate sensing of HIV-1 infection that has immediate implications for HIV-1 cure strategies.
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