Nucleocytoplasmic Trafficking of Dengue Non-structural Protein 5 as a Target for Antivirals

Abstract

Signal-dependent movement of proteins into and out of the nucleus through the importin superfamily of transporters is central to the replication of many viruses in infected cells, including RNA viruses such as the flavivirus Dengue virus (DENV). DENV non-structural protein 5 (NS5) traffics into and out of the host cell nucleus/nucleolus, being observed in the nucleus, although to differing extents, very early in infection in the case of all 4 DENV serotypes; with results from both reverse genetics and inhibitor studies indicating that this trafficking is critical to DENV infection. Knowledge of the transporters and targeting signals responsible for nuclear trafficking of NS5 has enabled inhibitors of DENV NS5 nuclear import to be identified using a novel screening/counterscreen approach. N-(4-hydroxyphenyl) retinamide (4-HPR) is of particular interest as a specific, non-toxic inhibitor able to protect against infection by all four serotypes of DENV, as well as the severe, antibody-enhanced form of DENV infection, in a lethal mouse model. Since 4-HPR can also inhibit DENV-related flaviviruses of medical significance such as West Nile Virus and Zika virus, it is of great interest for future commercialisation. Targeting nucleocytoplasmic trafficking of flavivirus proteins promises to be a powerful strategy to counter flaviviruses, for which the development of protective vaccines has thus far proven problematic.

Discussion of Chapter 15 at Dengue and Zika: Control and Antiviral Treatment Strategies

This discussion was held at the 2nd Advanced Study Week on Emerging Viral Diseases at Praia do Tofo, Mozambique.Transcribed by Hilgenfeld R and Vasudevan SG (Eds); approved by Dr. David Jans.

• Katja Fink: So at least for the mouse model studies with 4-HPR, was that prophylactic treatment or post-infection?

• David Jans: It was co-treatment.

• Katja Fink: So the next step will be to see whether it works post-infection.

• David Jans: Yes, we still need to do all those experiments in mice.

• Laura Rivino: So just a thought: If you block NS5 entry in the nucleus, you will have accumulation in the cytoplasm, right? And also, there will be rapid degradation. So would this lead to increased presentation of NS5 peptides and could this lead to excessive T-cell activation? NS5 is one of the main targets of T-cells, but you may not see this in the AG129 mouse model, because it is more impaired in T-cell responses.

• David Jans: Yes, we can speculate about that. The way I think about it is at least in the first line, that you will not have the effects on T-cell activation, and that the antiviral response will be much more robust.

• Siew Pheng Lim: Does the 4-HPR affect the polymerase activity?

• David Jans: No.

• Siew Pheng Lim: Have you tried to crystallize this compound with importin-alpha?

• David Jans: We have tried that, but failed. It does not crystallize and I don’t think it binds 4-HPR. I suppose, it may seem counter-intuitive, but it looks like most of the 4-HPR binding is on NS5 and of course we would like to get a co-crystal of this.

• Félix Rey: I am not a specialist in this localization-by-fluorescence assay. So I was wondering what would you expect if NS5 was localized at the outer nuclear membrane, in invaginations where there is replication.

• David Jans: So you mean the nuclear envelope?

• Félix Rey: Yes, the outer nuclear membrane which is continuous between the ER and the nucleus. If you have invaginations containing replication complexes all around the nuclear membrane, what would you expect to see by fluorescence?

• David Jans: You saw the images that we get in an infected cell and I do not see very much fluorescence. But we would be able to see differences for sure depending on how much proteins is there, I suppose. So we are limited always by the amount of protein in the infected cell. If we use the GFP protein, then we can visualize it much better, may be because of the dynamics etc.

• Félix Rey: I would expect it to be localized in the cytoplasm, right, where NS5 is supposed to be located for replication.

• David Jans: You saw the pictures: There are huge amounts of NS5 in the nucleus.

• Félix Rey: You mean in the nucleus and not in the nuclear membrane?

• David Jans: Correct. And in the nucleolus as well.

• Aruna Sampath: So this is focused on NS5. I just want to make a comment that in one of the screens where you look at the importance of alpha/beta inhibitor, there is potential for broad-spectrum activity. The exportins are actually more prominent, there are molecules with broad-spectrum activity. There are molecules that are actually in clinical trials right now.

• David Jans: We are working on them. They certainly seem to work for flu.

• Subhash Vasudevan: David, one comment that has to be made is that, as you pointed out in your response to Siew Pheng’s question, 4-HPR does not affect RdRp activity. There is still a lot to be learned about how 4-HPR works. One possible scenario is that if you replace NS5 with a host protein that is required in the replication process and that is probably shuttling in and out of the nucleus, you will get the same antiviral response. Two papers have been published for Zika recently, where they picked up the molecule that you discovered, ivermectin, through cell-based infection assays. I guess it is the same probably for most of the flaviviruses – they require this critical unknown host factor that is cycling in and out the nucleus.

• David Jans: I don’t think we need to go there. These are repurposed drugs, so 4-HPR has been used before for other indications and Ivermectin is the same. But clearly, host factors are required in replication and there is no question about that. Is it that the key thing in the replication complex that is being blocked or is it something else? I think we can speculate on it, but I don’t think there is any evidence for it. What is clear is that 4-HPR binds NS5 directly.