Amaryllidaceae alkaloids inhibit nuclear-to-cytoplasmic export of ribonucleoprotein (RNP) complex of highly pathogenic avian influenza virus H5N1

Abstract

Background: Few drugs are currently licensed to treat influenza A infection, and new therapies are needed, especially for highly pathogenic strains. Traditional medicinal plants, such as Lycoris radiata, are a potential source of new antiviral agents.

Objective: To test 15 Amaryllidaceae alkaloids isolated from the bulbs of L. radiata in vitro for antiviral activities against influenza virus type A, A/Chicken/GuangDong/178/2004 (H5N1, 178).

Methods: Antiviral activities of the compounds were tested in time-of-addition assays, hemagglutination inhibition (HI) assays, neuraminidase (NA) activity assays, and viral entry inhibition assays using H5N1-HIV pseudoviruses. Effects of the compounds on localization and activity of the viral ribonucleoprotein (RNP) were determined by immunofluorescence and an RNP minigenome assay, respectively.

Results: Among the alkaloids, lycorine (AA1), hippeastrine (AA2), hemanthamine (AA3) and 11-hydroxy vittatine (AA4) exhibited antiviral activities, with EC90 values of 0·52, 82·07, 4·15, and 13·45 μm, respectively. These compounds did not affect the function of the outer membrane proteins or the viral entry process and viral RNP activity. As AA1 and AA3 exhibited stronger antiviral activities, they were further analyzed. Intracellular nucleoprotein (NP) localization showed that AA1 and AA3 inhibited the RNP complex in the nucleus at an early stage of a single-round and multi-round of replication.

Conclusion: Four Amaryllidaceae alkaloids were first determined that could exert anti-influenza activities after virus entry into cells. Furthermore, AA1 and AA3 could inhibit nuclear-to-cytoplasmic export of the RNP complex of virus replication. Thus, these compounds may be developed further as anti-influenza drug candidates.

Keywords: Amaryllidaceae alkaloid; H5N1 influenza A virus; vRNP export.

Figure 1

Chemical structures of alkaloids AA1–AA4.

Figure 2

Experimental design of protocols for testing effect of the alkaloid compounds by HA assay at different time points. Virus mixtures contained 0·01 MOI. 1, negative control; 2, virus only as positive control; 3, mixture of virus at 0·01 MOI and different compounds (AA1:0·52 μm; AA2:82·07 μm; AA3:4·15 μm; AA4:13·45 μm, Oseltamivir: 0·625 μm, 100 μl) were incubated at 4°C for 1 h, inoculated onto cells 1 h and then the media were replaced with drug‐free DMEM for 72 h; 4, same as 3, except the replacement media DMEM contained compounds; 5, after adsorption 1 h, the culture containing Amaryllidaceae alkaloids compounds; 6, mixture of virus at 0·01 MOI and Amaryllidaceae alkaloids compounds were added to cells at 37°C for 1 h, and then replaced with drug‐free DMEM for 72 h; 7, same as 6, except the replacement media contained drugs. Supernatants from the above seven protocols were collected at different time points (12, 24, 36, 48, 60 and 72 h) to test the HA titer.

Figure 3

HA titers of different Amaryllidaceae alkaloid compounds (A, B, C, D corresponding to AA1, AA2, AA3, AA4 and different times of addition were tested. The different protocols (1–7) are illustrated in Figure 2 and described in Methods. Values are shown the means ± SD of the three independent experiments and standardized.

Figure 4

Polymerase activities of combinations of RNP proteins from virus strain 178. 293T cells were transfected in triplicate with the luciferase reporter plasmid polI‐Luc and internal control plasmid Renilla, together with plasmids expressing PB2, PB1, PA and NP from the 178 virus strain. At 8 h p.i. the supernatants were removed and replaced with Optimem containing various concentrations of Amaryllidaceae alkaloids compounds (AA1:4·67 μm; AA2:15·20 μm; AA3:4·90 μm; AA4:16·70 μm, 500 μl) for 24 h, and cell lysates were analyzed to measure firefly and Renilla luciferase activities. Values are shown the means ± SD of the three independent experiments and standardized to those of the influenza virus strain 178 only measured at 37°C (100%).

Figure 5

Effect of the time‐of‐addition of AA1:0·52 μm; AA3:4·15 μm, 100 μl at various times during the replication cycle of influenza virus. Time 0 = post 1 h adsorption period at 4°C. Each value represents the mean ± SEM of three separate assays.

Figure 6

178 H5N1 HPAIV (MOI = 1) and MDCK cells were either untreated or were treated with AA1 or AA3 as follows: (−) AA, normal infection with no AA treatment as control (Figure 6A,B); The location of NP was tested in single replication (8 h p i), cells treated with AA1 (4·67 μm), AA3 (4·90 μm) after 1 h of virus adsorption at 4°C. Infected cells were then incubated in medium with AA1 (d, e, f, j, k, l, p, q, r, v, w, x) or AA3 (g, h, I, m, n, o, s, t, u, y, z, z‐) at different time intervals (1, 2, 4 and 6 h p.i.) until 8 h p.i., and the intracellular amount and localization of viral RNPs (green, e, h, k, n, q, t, w, z), as well as the nuclei (blue, d, g, j, m, p, s, v, y), DAPI + NP (f, I, l, o, r, u, x, z‐) were detected by immunofluorescence. The location of NP protein at muti‐round replication (24 and 48 h p i) was tested in Figure 6C.