The new study, published July 21, 2023 in Science Advances, reveals how changes to the 3D structures of integrase, an HIV protein, can lead to Dolutegravir resistance and how other compounds may be able to overcome this resistance.

“With HIV, one must think two steps ahead of the virus,” says Salk Associate Professor Dmitry Lyumkis, co-senior author and the Hearst Foundation Developmental Chair. “We’ve now determined how the virus could continue evolving against drugs like Dolutegravir, which is important to consider for the development of future therapeutics.”

HIV infection depends on the ability of the virus to paste its own genetic material into the genomes of human cells, essentially hijacking the cells to become virus-producing factories. Dolutegravir and related drugs work by blocking integrase, a protein critical to the ability of the virus to integrate its own DNA into the host genome. Without functioning integrase, HIV can’t effectively infect human cells. However, HIV is a rapidly mutating virus, and an increasing number of HIV strains are resistant to Dolutegravir.

In the past, Lyumkis’ lab discovered the 3D structure of the integrase protein while attached to DNA as well as exactly how drugs like Dolutegravir bind to and block integrase. But researchers weren’t sure how the integrase structure changed when the virus stopped responding to Dolutegravir.

In the new study, Lyumkis and collaborators from the National Institutes of Health created versions of the integrase protein with mutations known to make HIV resistant to Dolutegravir. Then they determined the structure of each mutant integrase, revealing why Dolutegravir could no longer bind to and block each version of the protein. The scientists also evaluated the “fitness” of the virus (its capacity to produce infectious descendants) and the activity of the enzyme to better understand what leads to drug resistance in patients.

“We were quite surprised by the magnitude of resistance that these integrase variants had,” says Lyumkis. “The ability of Dolutegravir to function was completely compromised.”

The researchers also tested the efficacy of an experimental HIV drug, 4d, to block the function of Dolutegravir-resistant integrase proteins. 4d was developed by Lyumkis’ collaborators at the NIH as a next-generation integrase-targeting drug and is currently in pre-clinical animal trials. In all the variants, they discovered that 4d still potently blocked the ability of HIV to integrate its genes into human cells. This suggests that 4d or variants of this compound may be effectively used to treat the virus in patients who have developed resistance to Dolutegravir.

The structural data on how 4d binds to the Dolutegravir-resistant integrase proteins also hinted at how new drugs could overcome drug resistance.

“4d is really just an example of how to combat drug resistance, but it provides us with some basic principles that we can learn from to design other therapeutics,” says co-senior author Robert Craigie of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), part of the National Institutes of Health. “The way a section of the 4d molecule stacks like a flat sheet on top of a section of the integrase protein-DNA assembly could be replicated in other compounds.”

Next, the scientists will study how integrase variants evolve — including those not yet seen in patients but possible in the future — and how they impact response to the best clinically used drugs as well as the ability of HIV to infect humans.

Other authors include Dario Oliveira Passos, Zelin Shan, Avik Biswas and Timothy S. Strutzenberg of Salk; Min Li, Zhaoyang Li, Steven J. Smith, Xue Zhi Zhao, Terrence R. Burke, Jr. and Stephen H. Hughes of the National Institutes of Health; Qinfang Sun, Indrani Choudhuri, Allan Haldane, and Ronald M. Levy of Temple University; Nanjie Deng of Pace University; and Lorenzo Briganti and Mamuka Kvaratskhelia of University of Colorado Anschutz Medical Campus.

“The first two years is a really critical period where a lot of development is going on, and sleep is important for health. We wanted to look at the association of mother and infant sleep and whether it changes over time,” said Tianying Cai, now a postdoctoral researcher at Northwestern University. She worked on the research as a doctoral student in the Department of Human Development and Family Studies (HDFS), part of the College of Agricultural, Consumer and Environmental Sciences (ACES) at the U. of I.

“We identified two distinct groups, a low maternal sleep group where the mothers get 5 to 6 hours of sleep per night, and an average maternal sleep group, which meets the national recommended sleep guidelines with 7 to 8 hours per night. Children in the low maternal sleep group also slept less, although the difference wasn’t as large as for the mothers,” Cai stated.

The research team followed parents of 464 infants in the first two years of life. Mothers completed surveys about bedtime routines, their child’s sleep duration, nighttime waking, and sleep problems at 3, 12, 18, and 24 months of age.

The families were part of STRONG Kids 2, a program at the U. of I. that promotes nutrition and healthy habits in families with young children. STRONG Kids 2 co-directors Barbara Fiese, professor emerita of HDFS, and Sharon Donovan, professor of food science and human nutrition, also contributed to the study.

Mothers who fit the low maternal sleep profile got an average of 5.74 hours of sleep per night at 3 months and 5.9 hours at 12 to 24 months, while their children got 9.6 and 10.52 hours, respectively. In the average sleep profile, mothers got 7.31 hours at 3 months and 7.28 hours at 12 to 24 months, while child sleep averaged 9.99 hours at 3 months and 11 hours at 12 to 24 months.

The research team also identified factors that influence the amount of sleep a mother gets. Not surprisingly, one of the strongest predictors is infant-signaled nighttime waking, which means the infant is more likely to alert the parent at night. This could be either because these infants woke more frequently, or because the mothers were more likely to wake up when infants stirred, Cai noted.

Mothers who had longer employment hours were more likely to be in the low sleep group at 3 months, although that was no longer a factor by 12 months. Furthermore, those who breastfed their infant at 12 months were more likely to be in the average sleep group.

Over time, many families transitioned from the low to the average sleep group as infant sleep patterns consolidated. At 3 months, 60% were in the low maternal sleep group and 40% were in the average group, while at 12 months the numbers were reversed. Most of those who were in the average sleep group at 3 months continued to be so throughout the study period.

The researchers found that an earlier bedtime and consistent routines were associated with better sleep patterns, corroborating a previous study from Fiese and Cai.

“If parents can establish early bedtime routines at three months, it improves sleep duration and reduces sleep problems,” Fiese said. “Parents may feel overwhelmed and don’t realize that they have this in their toolkit. Something as simple as setting a regular bedtime early on and having routines, like reading a story to your child before they go to bed. You may not think they’re understanding, but the rhythm of your voice establishes predictability, and you can expand this bedtime routine over the first few years of life.”

The researchers noted they did not observe any significant differences due to demographic characteristics in the sample.

“Maternal education, income, or ethnicity did not predict sleep group memberships across 3 to 24 months; all parents were facing similar challenges. I think having a baby is a great equalizer for a lot of things, although moms who have to go back to work or work longer hours may have more pressures,” Donovan said.

Even so, there are steps everyone can take to improve bedtime habits and sleep patterns.

“Getting kids to bed earlier and trying to meet the American Academy of Pediatrics guidelines is really important because studies have shown that sleep is associated with a lot of neurocognitive outcomes and health in kids. The parents can be quite proactive even early in life to get their kids off on the right foot,” she concluded.