Rhinovirus and zinc part 5: Magnesium is not the culprit

If you have been following the results of my experiments on inhibition of rhinovirus replication by ZnCl2, you know that I’ve been trying to determine why concentrations of the salt higher than 0.1 mM are toxic to HeLa cells. I have found that 0.1 mM ZnCl2 does inhibit rhinovirus plaque formation but not sufficiently to be able to select resistant mutants. In today’s set of experiments I asked whether the presence of MgCl2 in the agar overlay potentiates zinc toxicity.

We always include MgCl2 (40 mM) in the agar overlay when assaying rhinoviruses, because it significantly improves plaque size. The following monolayers of HeLa cells were inoculated with 200 plaque-forming units of rhinovirus type 1a, then incubated at 32°C for 5 days. The effect of MgCl2 is remarkable.

hrv_mg_effect

The use of MgCl2 to improve plaque formation of certain picornaviruses dates to the 1962 observation that the salt increases susceptibility of cells to poliovirus infection. It was later shown to enhance plaque formation of rhinoviruses.

Unfortunately, omission of MgCl2 from the agar overlay has no effect on zinc toxicity. As shown below, cell viability was still poor in the presence of 0.2 mM ZnCl2.

hrv_mg_zn

What’s next? One reader suggested that I try selecting HeLa cells in successively higher concentrations of ZnCl2 to obtain cells resistant to the toxic effects of the salt. This approach is under way. I will also attempt to propagate rhinovirus in cells covered with liquid growth medium rather than under agar. If 0.1 mM ZnCl2 is included in the culture medium, a good fraction of the viruses produced might be resistant to the salt. The virus produced in these infected cells will be used to infect fresh cells, also in culture medium with ZnCl2. After 5-10 passages in this manner the majority of the viral population should be resistant to ZnCl2. This is a more time consuming approach that the plaque assay, but might yield zinc resistant rhinovirus mutants.

Wallis, C., & Melnick, J. (1962). Magnesium chloride enhancement of cell susceptibility to poliovirus. Virology, 16 (2), 122-132 DOI: 10.1016/0042-6822(62)90287-8

Fiala M, & Kenny GE (1966). Enhancement of rhinovirus plaque formation in human heteroploid cell cultures by magnesium and calcium. Journal of bacteriology, 92 (6), 1710-5 PMID: 4289358

Rhinovirus and zinc part 4: cell toxicity

My experiments to understand how ZnCl2 inhibits rhinovirus replication have been thwarted by the finding that concentrations of the salt higher than 0.1 mM are toxic for cultured HeLa cells. The cells can tolerate 0.1 mM but not 0.2 mM ZnCl2. Last week I asked whether I could identify a concentration between 0.1 and 0.2 mM that does not harm the cells but inhibits viral plaque formation >99%. Here are the results.

hrv_zinc_4

Unfortunately even 0.125 mM ZnCl2 is toxic to the cells – which is surprising since the cells can tolerate 0.1 mM. The goal of these experiments is to identify Zn-resistant rhinovirus mutants, and this cannot be done with cell monolayers that are not healthy.

I have one more idea for how to get around the ZnCl2 toxicity. To improve the formation of rhinovirus plaques, 25 mM MgCl2 is added to the agar overlay. It is possible that this high amount of magnesium, together with 0.2 mM ZnCl2 or higher, is toxic to cells. Therefore I will determine whether Zn toxicity is reduced if MgCl2 is omitted from the agar overlay. The rhinovirus plaques will be smaller but that is a reasonable trade-off for healthier monolayers.

If omitting MgCl2 doesn’t work, then I will have to select Zn resistant rhinovirus mutants in cells propagated in liquid cell culture medium. I’ll explain that approach next week.

Rhinovirus and zinc part 3

So far in my experiments to understand inhibition of rhinovirus replication by ZnCl2 I’ve found that at a concentration of 0.1 mM, viral plaque formation is inhibited but not sufficiently to be able to select resistant mutants. Attempts to use higher concentrations of the metal have consistently failed.

When I initially I tried higher concentrations of ZnCl2 in the plaque assay (0.2, 0.3, 0.4 mM) the cell monolayers looked poor. I thought one reason for this apparent toxicity was that the HeLa cell monolayers were too sparse. Last week I repeated the experiment using plates of HeLa cells seeded with 2 or 2.25 million cells each the night before. I infected the cells with two different amounts of rhinovirus type 1a, added a semisolid overlay with or without ZnCl2, and incubated for four days. The results are shown below.

rhinovirus_zinc_3

It’s clear that seeding the plates with 2 or 2.25 million HeLa cells made no difference: 0.2 mM ZnCl2 is still toxic to the monolayers. I find it surprising that the cells have no problem with 0.1 mM ZnCl2 but fare poorly at twice the concentration; but that is the observation and I’ll have to work around it.

Tomorrow’s experiment: try intermediate concentrations of ZnCl2 in the plaque assay, between 0.1 and 0.2 mM. It might be possible to identify a concentration that does not harm the cells but inhibits viral plaque formation >99%.

Zinc and rhinovirus replication

hrv1a_zincRecently I began experiments to understand how zinc inhibits rhinovirus replication, and I promised to document my findings on the pages of this blog. Here are the results of the second plaque assay.

In the last experiment I confirmed the finding that 0.1 mM ZnCl2 inhibits plaque formation by rhinovirus type 1A. Based on the results of that plaque assay, shown in the figure at left, I’ve decided that this concentration of zinc isn’t sufficient to completely inhibit viral replication. Although 0.1 mM ZnCl2 blocked plaque formation when 20 or 200 pfu were inoculated on cells, many plaques arose on plates inoculated with 2000 pfu. These cannot be viral mutants resistant to zinc – there are too many of them. If there are 2000 plaques on the untreated plate, and 200 on the plate with zinc, that would mean that resistance to zinc arises at a frequence of 200/2000 = 0.1 or one in ten viruses. I would expect a mutation rate for an RNA virus to be more in the range of 1/1000 – 1/10,000.

I decided to repeat the plaque assay with higher levels of zinc in the agar overlay – 0.2, 0.3, and 0.4 mM. The results of that experiment are shown below.

rhinovirus_zinc_2

Unfortunately, the cells did not like the higher concentrations of ZnCl2! All the plates with zinc had very lightly staining monolayers – compare with the original experiment shown above – and no plaques were visible.

I was surprised that slight increases in the concentration of ZnCl2 would have such a dramatic effect on cell viability. The cells I used are HeLa cells, which are quite sturdy. Two other variables were changed in this experiment. I made a new stock of ZnCl2 – I took the 1 M stock I made for the first experiment and diluted it to 0.1 M. I doubt this made any difference. Second, the cell monolayers were less confluent than in the first experiment – about 70% of the surface of the cell culture dish was covered with cells. In the previous experiment, the monolayers were 100% confluent.

I repeated the experiment with HeLa cell monolayers that are 100% confluent. The results will be posted here in a few days.

Zinc inhibits rhinovirus replication

hrv1a_zincThe title of this post should not come as a surprise to readers of virology blog – it was shown in 1974 that zinc could interfere with replication of rhinoviruses (see “Zinc and the common cold“). I am referring to the result of my first experiment to study the mechanism of zinc inhibition – something I promised I would document on these pages.

I am interested in understanding how zinc inhibits rhinovirus replication. Answering this question could lead to new ways to prevent common colds caused by these viruses. The first step was to reproduce the effect of zinc in my laboratory with my stocks of rhinovirus. I selected rhinovirus type 1a for my initial experiments because we’ve worked with this serotype in the past: we know the genome sequence and how the virus behaves in a mouse model. I started by doing a plaque assay with and without zinc in the medium. I prepared tenfold dilutions of virus and inoculated separate monolayers of HeLa cells with 2000, 200, and 20 plaque forming units. After allowing the virus to attach to cells for 45 minutes, I added an agar overlay to the cells with or without zinc chloride (ZnCl2). I selected 0.1 millimolar ZnCl2 because that is the concentration which had been reported to effectively inhibit plaque formation by rhinovirus type 1a. The plates were incubated for four days at 32°C and then stained. The results are shown in the photo. Plaque assays are typically done in duplicate but for simplicity only one plate of each dilution is shown.

Twenty plaques were observed on the highest dilution of virus plated in the absence of ZnCl2. Ten-fold lower dilutions produced increases in plaque number, although the plaques are too numerous to count. In the presence of ZnCl2, no plaques were observed on cells inoculated with 20 PFU. A few plaques are observed on the intermediate dilution and many more on the lowest dilution. Plaques observed in the presence of ZnCl2 are smaller than those observed in the absence of the metal.

What do you think is going on here, and what should I do next? If you’ve kept up with virology 101 you have all the tools to answer these questions. Please post your thoughts in the comments section.

KORANT, B., KAUER, J., & BUTTERWORTH, B. (1974). Zinc ions inhibit replication of rhinoviruses Nature, 248 (5449), 588-590 DOI: 10.1038/248588a0

Zinc and the common cold

cold-eezeShortly after I developed sore throat, cough, and congestion last week, a package of ‘Cold – Eeze’ materialized on my kitchen counter. The writing on the package of zinc-laden lozenges promised to ‘shorten your cold’, and noted that they were ‘clinically proven to reduce the duration of the common cold’. Do zinc lozenges have any effect on the common cold?

The common cold is the primary cause of doctor visits in the United States, leading to 189 million lost school days each year. But it’s important to point out that the common cold can be caused by a number of different viruses, including rhinovirus, coronavirus, influenza virus, adenovirus, and paramyxovirus. Rhinoviruses are responsible for over half of all common colds.

The idea that zinc could be used to treat the common cold originated from a 1974 paper in Nature which showed that zinc blocks the replication of rhinoviruses in cell culture. Viral plaque formation was inhibited over 99% when 0.1 millimolar zinc chloride was included in the agar overlay. However, this concentration of zinc is too high for therapeutic use, and subsequent studies showed that levels compatible with use of the metal ion in humans minimally inhibited rhinovirus replication in cell culture. Zinc does not readily pass through the cell membrane, explaining why high concentrations are required to produce an antiviral effect.

Many trials have been conducted to determine if zinc – taken as a lozenge, nasal spray, or ointment – has any effect on the common cold in humans. In one study, 200 children were given 15 mg zinc daily by mouth for seven months. The mean number of colds in the treated children was 1.2 compared with 1.7 in the untreated children – statistically significant but not therapeutically useful. Many studies have evaluated the effectiveness of zinc containing lozenges. In one, 65 people took one lozenge containing 23 mg zinc every two hours while awake. After one week, 86% of the treated group were free of cold symptoms, compared with 46% of the placebo group. In another similar study, the duration of cold symptoms was reduced in the zinc group versus the placebo group – 4.5 days compared with 8.1 days. However, as many studies have lead to the conclusion that zinc lozenges – as well as zinc administered intransally or in a gel – have no effect on severity or duration of the common cold. A good summary of many of these trials can be found in the Alternative Medicine Review cited below.

It’s important to note that in these studies the virus responsible for the colds is not identified. Given the prevalence of rhinoviruses it is appropriate to assume that these viruses are involved in over half of the colds observed. Nevertheless, the extreme variability in the trial results may in part reflect the fact that various etiologic agents are involved, some of which might not be susceptible to inhibition by zinc. Other possible explanations for the inconsistent results include differences in the zinc preparations used (zinc gluconate and zinc acetate), the quantity of zinc administered, and the composition of the lozenge.

Although inhibition of rhinovirus replication by zinc was reported in 1974, the mechanism is not understood. It is believed that zinc enters the cell and binds to the rhinovirus protein that will form the capsid. This interaction blocks cleavage of the protein, thereby inhibiting production of infectious virus. Consistent with this proposed mechanism is the observation that zinc ionophores – compounds that allow the uptake of zinc into cells – have recently been shown to inhibit rhinovirus replication. The effectiveness of such compounds, which include pyrithione and hinokitiol, for treating the common cold is currently being investigated.

Although zinc does inhibit rhinovirus replication, this activity might not account for the effect on the common cold. It has been suggested that zinc reduces inflammation in the respiratory tract, which would explain the observed decrease in symptoms observed in some trials.

As for the Cold-eeze on my kitchen counter – the package was never opened. The unimpressive results of clinical trials made the idea of taking 6-8 lozenges a day for several days less appealing than enduring sore throat, cough, and congestion for less than a week. But the lozenges served a different purpose – I am now very interested in the revealing the mechanism of zinc inhibition of rhinovirus replication. I have begun experiments in my lab to solve this problem, and I’ll write about what I discover.

Korant BD, Kauer JC, & Butterworth BE (1974). Zinc ions inhibit replication of rhinoviruses. Nature, 248 (449), 588-90 PMID: 4363085

Geist FC, Bateman JA, & Hayden FG (1987). In vitro activity of zinc salts against human rhinoviruses. Antimicrobial agents and chemotherapy, 31 (4), 622-4 PMID: 3038000

Krenn, B., Gaudernak, E., Holzer, B., Lanke, K., Van Kuppeveld, F., & Seipelt, J. (2008). Antiviral Activity of the Zinc Ionophores Pyrithione and Hinokitiol against Picornavirus Infections Journal of Virology, 83 (1), 58-64 DOI: 10.1128/JVI.01543-08

Roxas M, & Jurenka J (2007). Colds and influenza: a review of diagnosis and conventional, botanical, and nutritional considerations. Alternative medicine review : a journal of clinical therapeutic, 12 (1), 25-48 PMID: 17397266