PerspectiveVirology

Delineating Ebola entry

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Science  27 Feb 2015:
Vol. 347, Issue 6225, pp. 947-948
DOI: 10.1126/science.aaa8121

The means by which Ebola virus enters a cell are becoming less mysterious. Although a definitive cell surface receptor for the virus, if there is one, remains to be identified, the mechanism of gaining entry is beginning to be fleshed out. Once inside the cell, the importance of numerous sequential processes is becoming better understood. On page 995 of this issue, Sakurai et al. (1) add another element to the viral entry pathway by showing that a calcium channel called two-pore channel 2 (TPC2) is required for release of the viral genome into the host cell.

Ebola's entry.

A model of infection shows that once the virus is internalized into an endosome, the viral glycoprotein is cleaved and binds to NPC1. The calcium channel TPC2 is then activated prior to a fusion event that releases the viral genome into the cell. The drug tetrandrine blocks TPC2.

ILLUSTRATION: ADAPTED BY C. BICKEL/SCIENCE

After Ebola's surface glycoprotein binds to receptors, which may be nonspecific and possibly comprise numerous carbohydrate proteins (2), the virus enters the cell via macropinocytosis, a nonselective process of engulfment (3, 4) (see the figure). Once internalized into a membrane-bound vesicle (endosome), Ebola glycoproteins are cleaved (5) while being exposed to an increasingly acidic and reducing environment. All this occurs in the endosome where an essential interaction with a protein called Niemann-Pick C1 (NPC1) was proposed to result in the release of the virus's genetic material in a process known as fusion (6, 7).

A small interfering RNA screen identified calcium signaling in the host cell, among other events, as necessary for Ebola virus entry (8). L-type calcium channels were initially pinpointed as the key element involved. However, one of five compounds that block these channels did not prevent Ebola infection, suggesting that another mechanism is also involved. The effective inhibitors also block nicotinic acid adenine dinucleotide phosphate (NAADP)–stimulated intracellular calcium channels—known as the TPCs. These channels are mainly localized to endosomes and lysosomes (acidic compartments where contents are degraded). Through the use of cells lacking TPC2, small interfering RNA, and small-molecule inhibitors, Sakurai et al. determined that TPC2 is required for Ebola entry. Moreover, the requirement for TPC2 was shown to be specific to the glycoprotein of Ebola virus, suggesting a highly specific endosomal processing pathway. Previous work demonstrated that NPC1 is also essential for Ebola virus entry and proposed that it may function as an endosomal receptor that triggers fusion (7). However, the findings of Sakurai et al. suggest that NPC1 is not likely required for fusion itself because late endosomes expressing TPC2, but not NPC1, correlated with productive infection in cultured cells. This indicates that interaction of the cleaved glycoprotein with NPC1 occurs before the glycoprotein interaction with TPC2 and prior to fusion.

Sakurai et al. further demonstrate the relevance of TPC2 by blocking the channel's activity with the drug tetrandrine, which improved survival of Ebola-infected mice. This supports the important role of TPC2 for Ebola virus infection; however, it does not indicate that a viable treatment is close at hand. The partial effectiveness of the drug in mice was further reduced by delaying treatment by 1 day. This questions whether any protection would be observed in a macaque model (the “gold standard” for Ebola drug efficacy testing) (9). Tetrandrine, originally a traditional Chinese medicine (found in the plant Stephania tetrandra), is not approved for use in humans (except in China). In addition, the dose given to mice by Sakurai et al. was many times the half-maximal inhibitory dose observed in tissue culture, and likely exceeds doses that would be considered safe for humans. Given the mode of action, it seems unlikely that tetrandrine treatment would be superior to the most advanced Ebola post-exposure treatments (including a cocktail of three monoclonal antibodies and an approach based on RNA interference).

Nearly all of the 65 or so antiviral drugs approved for clinical use in the United States are for treating chronic virus infections (including human immunodeficiency virus, hepatitis B virus, and hepatitis C virus). Drugs for more acute viral infections (such as influenza viruses, poxviruses, and herpesviruses) tend to be less effective overall, and translating data from cell culture studies into clinical use is challenging. As Ebola infection typically progresses quickly, the standard metric of the effective concentration values that reduce virus titers by 50% should probably be discarded for this virus. Reducing virus concentrations by 50% is highly unlikely to provide any clinical benefit; rather, aiming for a 90 or 99% reducwtion is more applicable for Ebola. Interestingly, tetrandrine functions in this range in vitro, but given its mode of action as a viral entry inhibitor, it may be better as a prophylactic than as a treatment unless used in combined therapy.

Currently, there are over 60 compounds that have been suggested to be effective against Ebola and/or Marburg virus infections (10), but most of these compounds do not have a clear mechanism of action, whereas there is some such understanding for tetrandrine. A few of the compounds have advanced to clinical trials for Ebola (e.g., brincidofovir and favipiravir) largely in the absence of convincing preclinical data. As one may find ethical arguments to support such trials in the current situation, one needs to be concerned about lack of efficacy that may do more harm than good.

Deriving improved versions of drugs such as tetrandrine may eventually lead to a therapeutic approach, but as it stands (the same hold true for other anti-Ebola compounds), that day is not just around the corner.

References

  1. Acknowledgments: This work was supported in part by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

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