The HIV-1 Viral Protease Is Activated during Assembly and Budding Prior to Particle Release.

HIV-1 encodes a viral protease that is essential for the maturation of infectious viral particles. While protease inhibitors are effective antiretroviral agents, recent studies have shown that prematurely activating, rather than inhibiting, protease function leads to the pyroptotic death of infected cells, with exciting implications for efforts to eradicate viral reservoirs. Despite 40 years of research into the kinetics of protease activation, it remains unclear exactly when protease becomes activated. Recent reports have estimated that protease activation occurs minutes to hours after viral release, suggesting that premature protease activation is challenging to induce efficiently. Here, monitoring viral protease activity with sensitive techniques, including nanoscale flow cytometry and instant structured illumination microscopy, we demonstrate that the viral protease is activated within cells prior to the release of free virions. Using genetic mutants that lock protease into a precursor conformation, we further show that both the precursor and mature protease have rapid activation kinetics and that the activity of the precursor protease is sufficient for viral fusion with target cells. Our finding that HIV-1 protease is activated within producer cells prior to release of free virions helps resolve a long-standing question of when protease is activated and suggests that only a modest acceleration of protease activation kinetics is required to induce potent and specific elimination of HIV-infected cells. IMPORTANCE HIV-1 protease inhibitors have been a mainstay of antiretroviral therapy for more than 2 decades. Although antiretroviral therapy is effective at controlling HIV-1 replication, persistent reservoirs of latently infected cells quickly reestablish replication if therapy is halted. A promising new strategy to eradicate the latent reservoir involves prematurely activating the viral protease, which leads to the pyroptotic killing of infected cells. Here, we use highly sensitive techniques to examine the kinetics of protease activation during and shortly after particle formation. We found that protease is fully activated before virus is released from the cell membrane, which is hours earlier than recent estimates. Our findings help resolve a long-standing debate as to when the viral protease is initially activated during viral assembly and confirm that prematurely activating HIV-1 protease is a viable strategy to eradicate infected cells following latency reversal.

Premature Activation of the HIV-1 Protease Is Influenced by Polymorphisms in the Hinge Region.

HIV-1 protease inhibitors are an essential component of antiretroviral therapy. However, drug resistance is a pervasive issue motivating a persistent search for novel therapies. Recent reports found that when protease activates within the host cell's cytosol, it facilitates the pyroptotic killing of infected cells. This has led to speculation that promoting protease activation, rather than inhibiting it, could help to eradicate infected cells and potentially cure HIV-1 infection. Here, we used a nanoscale flow cytometry-based assay to characterize protease resistance mutations and polymorphisms. We quantified protease activity, viral concentration, and premature protease activation and confirmed previous findings that major resistance mutations generally destabilize the protease structure. Intriguingly, we found evidence that common polymorphisms in the hinge domain of protease can influence its susceptibility to premature activation. This suggests that viral heterogeneity could pose a considerable challenge for therapeutic strategies aimed at inducing premature protease activation in the future.

Reducing Unplanned Intubations in the Neonatal Intensive Care Unit After Children's Surgery: A Quality Improvement Project.

BACKGROUND: Unplanned intubation following children's surgery is associated with increased postoperative mortality. In response to being a National Surgical Quality Improvement Program - Pediatric (NSQIP-P) high outlier for postoperative unplanned intubation, we aimed to reduce postoperative unplanned intubation events by 25% in one year. METHODS/INTERVENTION: A multidisciplinary team of stakeholders was assembled in 2018. Most unplanned intubation events occurred in the neonatal intensive care unit (NICU). Based on apparent causes of unplanned intubations identified in case reviews, an extubation readiness checklist and a postoperative pain management guideline emphasizing non-opioid analgesics were implemented for NICU patients in September 2019. Postoperative unplanned intubation events were tracked prospectively and evaluated using quality improvement statistical process control methods. RESULTS: Unplanned intubations in the NICU decreased from 0.27 to 0.07 events per patient in the post-intervention group (September 2019-June 2022, n = 145) compared to the pre-intervention group (January 2016-August 2019, n = 200), representing a 76% reduction. Postoperative opioid administration decreased significantly, while acetaminophen usage increased significantly over time. Balancing measures of postoperative pneumonia rate (1.5% vs 0.0%, p = 0.267) and median hospital length of stay [40 (IQR 51) days vs 27 (IQR 60), p = 0.124] were not different between cohorts. The 30-day mortality rate for postoperative patients in the NICU significantly declined [6.5% (n = 13) vs 0.7% (n = 1), p < 0.001]. CONCLUSIONS: Postoperative unplanned intubation rates for NICU patients decreased following a quality improvement effort focused on opioid stewardship and extubation readiness. TYPE OF STUDY: Prospective Quality Improvement. LEVEL OF EVIDENCE: Level III.