PERLs look promising in fight against viruses
Human cells stained with a blue marker to show the nuclei (left upper panel) and a green marker to show the ER membranes (right upper). The cells were incubated with ER targeting liposomes which are stained red (left lower). When analysed by confocal fluorescent microscopy, the liposomal lipids and the ER membrane co-localise (right lower)
A group of scientists in the Biochemistry department together with colleagues in Romania have discovered a novel approach to developing treatment against HIV, hepatitis B and hepatitis C infections. The approach is particularly promising because it targets the host cells rather than viral pathways, suggesting that it could potentially be directed against all strains of the viruses.
The study from group leader Nicole Zitzmann, postdoctoral researcher Stephanie Pollock, and colleagues in Oxford and Romania, is published in a recent issue of Proceedings of the National Academy of Sciences (1).
Current anti-viral treatments have limited use because of the mutational evasion tactics of many viruses. HIV and hepatitis C are the fastest mutating viruses known and are capable of mutating around most of the treatments thrown at them. Researchers are continuously looking for new approaches to developing antiviral compounds.
Dr Zitzmann's work has focused on developing antiviral compounds against viral glycoprotein targets. Glycoproteins are sugar-coated proteins and are synthesised in the cell's endoplasmic reticulum (ER), an interconnected network of membrane-covered vesicles and tubules. In the ER, glycoproteins and other proteins are packaged and sent out to other parts of the cell or externally to carry out their role.
"We're not sure about the exact mechanism by which this works yet. All we can say is that we lower the cholesterol and that this makes life for these three viruses very difficult"
Having shown that the effects of the antiviral compounds they were developing can be enhanced if they are delivered inside the cell using lipid vesicles known as liposomes, Dr Zitzmann and her colleagues were spurred to go one step further. 'We had an idea,' she explains. 'What if you want to target the drugs to the ER, shouldn't we use a liposome that looks like the ER? As an undergraduate I went to an advanced lecture about lipids which went straight over my head at the time, but I remembered one thing that was said: like likes to fuse with like.'
So she and her colleagues went back to the literature to find out what lipids the ER membrane is made of, with the intention of recreating this in liposomes. They identified a composition that targeted the liposomes to the ER where they fused with the membrane. But to their surprise, the liposomes exhibited a completely unexpected effect. 'Although we didn't get any real enhanced delivery of drugs over and above what we'd seen with the non-ER targeting liposomes, we found that even empty ER targeting liposomes were anti-viral,' says Dr Zitzmann.
Intrigued by this finding, the group wanted to understand what was happening. In the absence of anti-viral drugs, the liposomes alone were capable of affecting both the secretion and infectivity of three viruses tested - HIV, hepatitis B and hepatitis C. By tweaking the lipid ratios of the liposomes, the researchers could optimise the reduction in both secretion and infectivity. They found that the optimal liposome composition was rich in polyunsaturated lipids, and consequently named these PERLs (poly-unsaturated ER-targeting liposomes).
To follow the molecular events inside the cells, the researchers gave the cells PERLs and then observed which proteins were up or down-regulated. An enzyme involved in cholesterol biosynthesis jumped out at them, so they immediately measured the cholesterol inside the treated cells and found that it was reduced. They also found that cholesterol levels in the plasma membrane were disrupted, as were cholesterol levels in the membrane coating of secreted virus.
Researchers know that cholesterol is important in the lifecycle of these three viruses. The viruses piggy-back onto cholesterol-dependent cellular processes to get into and out of cells. In addition, the cholesterol they incorporate into the viral coat as they exit the cell is required to help them infect other cells. Depleted cholesterol levels in the cell will therefore reduce both virus secretion and infectivity.
The ways in which PERLs cause reduction in cellular cholesterol are not clear at this stage, says Dr Zitzmann, although she suspects that a number of pathways could be targeted in complex and subtle ways. 'We're not sure about the exact mechanism by which this works yet. All we can say is that we lower the cholesterol and that this makes life for these three viruses very difficult.'
She is excited about the potential of this new treatment as it could offer substantial advantages over current ones. The virus is much less likely to find a way around it since the approach targets host cell pathways that the virus subverts for its own use. The work also suggests that a single approach could be effective against all three viruses. 'Lots of patients are co-infected with multiple viruses,' comments Dr Zitzmann. 'If this worked, you could hit all of them at the same time.' There is also the possibility of engineering a 'two-pronged attack' by encapsulating other antiviral therapies within the liposomes to increase their efficacy.
For this approach to be developed further, one of the first things that the group will have to show is that the liposomes are not toxic in the body. Their preliminary work in this area looks promising. Alongside this, the group is putting all its efforts into understanding more about what is happening inside the liposome-treated cells.
'Of course we are incredibly interested in exactly how the liposomes work,' says Dr Zitzmann who has a large part of her group working on the project now. Her research is supported by Professor Raymond Dwek, Director of the Glycobiology Institute where Dr Zitzmann is based, with the biotechnology company United Therapeutics supporting the clinical development. 'We want to find out how they get to the ER and how they interfere with the cholesterol metabolism. If further toxicology studies show any side effects, then we may need to improve them by adjusting the composition. We want to get these into clinical trials.'