Protoporphyrin IX-Dependent Antiviral Effects of 5-Aminolevulinic Acid against Feline Coronavirus Type II

Abstract

5-Aminolevulinic acid (5-ALA), a non-proteinogenic amino acid, is an intermediate in the biosynthesis of heme and exerts antiviral effects against feline coronavirus (FCoV); however, the underlying mechanisms remain unclear. In the biosynthesis of heme, 5-ALA is condensed and converted to protoporphyrin IX (PpIX), which is then transformed into heme by the insertion of ferrous iron. Previous research has suggested that the metabolites generated during heme biosynthesis contribute to the antiviral effects of 5-ALA. Therefore, the present study investigated the in vitro mechanisms responsible for the antiviral effects of 5-ALA. The results obtained revealed that 5-ALA and PpIX both effectively reduced the viral titer in the supernatant of FCoV-infected fcwf-4 cells. Moreover, PpIX exerted virucidal effects against FCoV. We also confirmed that 5-ALA increased PpIX levels in cells. While hemin induced heme oxygenase-1 gene expression, it did not reduce the viral titer in the supernatant. Sodium ferrous citrate decreased PpIX levels and suppressed the antiviral effects of 5-ALA. Collectively, these results suggest that the antiviral effects of 5-ALA against FCoV are dependent on PpIX.

Keywords: 5-aminolevulinic acid; feline coronavirus; protoporphyrin IX.

Figure 1

Cytotoxic effects of compounds. fcwf-4 cells were cultured with compounds for 24 h. After the incubation, WST-8 solution was added and cells were incubated for 1 h. The absorbance of formazan produced was measured at 450 nm. Percent cytotoxicity was calculated using the following formula: cytotoxicity (%) = [(OD of compound untreated cells–compound-treated cells)/(OD of compound untreated cells)] × 100. The results obtained are shown as means ± SE. Data represent three independent experiments. (A) 5-ALA-SFC combination. (B) PpIX *: p < 0.05 vs. vehicle. (C) Hemin.

Figure 2

Antiviral effects of 5-ALA. fcwf-4 cells were pretreated with 5-ALA for 24 h. After viral adsorption, cells were cultured in MM without 5-ALA. The results obtained are shown as means ± SE. Data represent three independent experiments. (A) The antiviral effects of 5-ALA 48 h after the virus inoculation. (B) Relative FCoV RNA replication with different concentrations of 5-ALA. The amount of FCoV RNA was measured using RT-qPCR targeting 3’-UTR and normalized by GAPDH. *: p < 0.05 vs. vehicle. (C) Virus growth kinetics in fcwf-4 cells pretreated with 5-ALA.

Figure 3

Antiviral effects of PpIX. (A) Antiviral effects of PpIX on PpIX-treated cell. fcwf-4 cells were pretreated with PpIX for 24 h. After viral adsorption, cells were cultured in MM without 5-ALA. The virus titer was measured 48 h after the virus inoculation. The results obtained are shown as means ± SE. Data represent three independent experiments. n.s.: no significant. (B) Virucidal effects of PpIX against FCoV. PpIX or DMSO was mixed with an equivalent volume of FCoV strain 79-1146 and reacted at 37 °C for 1 h under light shielding. The virus titer in the virus solution was then directly measured using the TCID₅₀ assay. The results obtained are shown as means ± SE. Data represent three independent experiments.

Figure 4

A time of additional assay for PpIX. fcwf-4 cells were infected with the FCoV strain and cultured in medium only. After washing, medium containing PpIX was added at designated times ranging from 0 to 24 h after virus inoculation. Virus titer and FCoV RNA levels were measured 48 h after virus inoculation. (A) Schedule for PpIX treatment. (B) The virus titer in supernatants. Virus titer was quantified using the TCID₅₀ assay. (C) The FCoV RNA levels on supernatants. FCoV RNA level was quantified using the RT-qPCR targeting FCoV 3’-UTR. The results obtained are shown as means ± SE. Data represent three independent experiments. *: p < 0.05 vs. DMSO.

Figure 5

Antiviral effects of hemin. (A) The virus titer in cells pretreated with hemin. fcwf-4 cells were pretreated with hemin for 24 h. After viral adsorption, cells were cultured in MM without 5-ALA. The virus titer was measured 48 h after the virus inoculation. (B) HO-1 gene expression levels. fcwf-4 cells pretreated with hemin for 24 h. After incubation, cells were collected and HO-1 gene expression levels were semi-quantitatively analyzed relative to GAPDH gene expression. The results obtained are shown as means ± SE. Data represent three independent experiments.

Figure 6

Impact of SFC on antiviral effects of 5-ALA. fcwf-4 cells were pretreated with the 5-ALA-SFC combination for 24 h. After viral adsorption, cells were cultured in MM without 5-ALA and SFC. The virus titer was measured 48 h after the virus inoculation.

Figure 7

Evaluation of intracellular levels of PpIX in fcwf-4 cells treated with compounds. fcwf-4 cells were cultured with compounds for 24 h. After incubation, cells were collected and lysed in a buffer containing Benzonase Nuclease and incubated at 37 °C for 1 h. The lysate was centrifuged, and the supernatant was treated with Proteinase K at 37 °C for 1 h. After another round of centrifugation, trichloroacetic acid was added to the supernatant, incubated at room temperature for 10 min, and centrifuged again. The resulting sediment was treated with dimethylformamide, agitated, and centrifuged. The final supernatant’s absorbance was measured at 300–800 nm using a BioSpectrometer. (A) Absorption spectrum of the supernatant extracted from compound-treated cells. (B) Absorbance at 410 nm (A410). Intracellular PpIX levels were quantified by measuring absorbance at 410 nm. The results obtained are shown as means ± SE. Data represent three independent experiments. *: p < 0.05 vs. vehicle.