ABOVE: Researchers discovered the key role of a host lipid-modifying enzyme in the formation of the HIV envelope. ©ISTOCK, Artem_Egorov

Since HIV’s discovery in the 1980s, scientists have come a long way in understanding the different steps required for its assembly and maturation. Researchers knew, for instance, that HIV wraps its core viral components in a lipid envelope formed from the host plasma membrane. However, they were uncertain about how HIV controls the remodeling of the cell membrane as it exits the cells and what role the lipid envelope plays in HIV replication.

The image shows round HIV particles on the left and misshaped viral particles on the right. 
By using electron microscopy, researchers found that HIV-1-infected cells treated with the nSMase2 inhibitor PDDC generated oddly shaped viral particles (right) compared to virions from untreated cells (left). 
Abdul Waheed and Ferri Soheilian, National Cancer Institute.

In a paper published in the Proceedings of the National Academy of Sciences (PNAS), researchers described the intracellular enzyme neutral sphingomyelinase-2 (nSMase2) as a key player in HIV replication.1 By blocking nSMase2, the team disrupted the formation of HIV type 1 (HIV-1) viral particles and prevented the virus from becoming infectious. Inhibition of nSMase2 also reduced plasma viral levels in humanized mouse models and prevented or delayed viral rebound. These findings shed light on a new mechanism used by HIV and could point to a new target for antiretroviral therapies.

“Viruses in general, because they are quite limited in their coding capacity, have to rely very heavily on host cell machinery to replicate,” said Eric Freed, a cellular and molecular biologist at the National Cancer Institute and author of the study. “This study reveals that this enzyme [nSMase2] is such an enzyme that the virus has evidently hijacked to use in its replication strategy.” 

Previous research suggested that HIV infection increased the levels of ceramides, a family of lipids found in high concentrations in the cell membrane, and that people infected with HIV showed elevated levels of those lipids in the brain tissue and the cerebrospinal fluid.2 

For the study, the researchers focused on nSMase2 because this enzyme produces ceramides from the sphingomyelin lipid. “This enzyme is also in the inner leaflet of the plasma membrane, which puts it in a very good location to be able to control cellular signaling as well as aspects of viral replication,” explained Norman Haughey, a pharmacologist at the Johns Hopkins University and coauthor of the study. 

To test whether nSMase2 is essential for viral replication, the team inhibited the enzyme by using an antiviral compound, [phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2, 6-dimethylimidazo[1,2-b]pyridazin-8-yl) pyrrolidin-3-yl)-carbamate] (PDDC), which they synthesized in the lab, or by knocking down nSMase2 expression using RNA interference. In human cell cultures, interfering with the enzyme’s activity led to misshapen viral particles with reduced ceramide content that did not mature and were not infectious. 

“These findings show—probably more compellingly than most, if not all, papers that have gone before—that the lipid composition of the HIV envelope is crucial for generating an infectious virus,” said Paul Bieniasz, a virologist at Rockefeller University who was not involved in the research.

Next, the team evaluated the effects of inhibiting nSMase2 activity in mice with humanized immune systems. They infected mice with HIV-1 and then treated half of the animals with a combination of antiretroviral drugs and the other half with the nSMase2 inhibitor. By monitoring the plasma viral loads, they found that both treatments effectively reduced HIV levels. However, after stopping both treatments, mice given the antiretrovirals showed viral rebound, while mice treated with the nSMase2 inhibitor showed no or delayed viral rebound.

The researchers then tested the effect of the nSMase2 inhibitor on cell viability by exposing human cells to different concentrations of PDDC. Blocking nSMase2 activity induced an apoptotic phenotype in cells in which HIV-1 was actively replicating. According to Haughey, these findings differ from the known effects of all currently available antiretrovirals, which suppress HIV replication but do not promote apoptosis in infected cells, so as soon as people stop taking the medication, viral replication starts again. 

Although these results are promising, there are still open questions as to whether the nSMase2 inhibitor could be used as a drug against HIV, Bieniasz noted. “Can animals survive long term with this enzyme being inhibited? That is going to be a key question in terms of going from this very elegant laboratory work into a useful medicine,” he added. 

Freed, Haughey, and their colleagues were also interested in understanding whether other retroviruses also require nSMase2 in their maturation processes. The results, described in a companion paper in PNAS, showed that more distantly related nonprimate lentiviruses did not require nSMase2 to form mature and infectious viral particles.3 According to Freed, understanding why primate and nonprimate lentiviruses differ in their requirements to form mature viral particles is an interesting question that he plans to pursue. 

Freed, who has studied HIV assembly for over 20 years, believes that the findings from both studies help clarify the critical role of the lipid composition of the virus envelope in HIV assembly and potentially, its maturation. “This is really the beginning of a pathway towards exploring nSMase2 and potentially other lipid modifying enzymes as potential therapeutic targets,” he said.

References

  1. Yoo S-W, et al. Inhibition of neutral sphingomyelinase 2 impairs HIV-1 envelope formation and substantially delays or eliminates viral rebound. Proc Natl Acad Sci U S A. 2023;120(28):e2219543120.
  2. Haughey NJ, et al. Perturbation of sphingolipid metabolism and ceramide production in HIV-dementia. Ann Neurol. 2004;55(2):257-267.
  3. Waheed AA, et al. Neutral sphingomyelinase 2 is required for HIV-1 maturation. Proc Natl Acad Sci U S A. 2023;120(28):e2219475120.