Targeting Polyamines Inhibits Coronavirus Infection by Reducing Cellular Attachment and Entry

Abstract

Coronaviruses first garnered widespread attention in 2002 when the severe acute respiratory syndrome coronavirus (SARS-CoV) emerged from bats in China and rapidly spread in human populations. Since then, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged and still actively infects humans. The recent SARS-CoV-2 outbreak and the resulting disease (coronavirus disease 2019, COVID19) have rapidly and catastrophically spread and highlighted significant limitations to our ability to control and treat infection. Thus, a basic understanding of entry and replication mechanisms of coronaviruses is necessary to rationally evaluate potential antivirals. Here, we show that polyamines, small metabolites synthesized in human cells, facilitate coronavirus replication and the depletion of polyamines with FDA-approved molecules significantly reduces coronavirus replication. We find that diverse coronaviruses, including endemic and epidemic coronaviruses, exhibit reduced attachment and entry into polyamine-depleted cells. We further demonstrate that several molecules targeting the polyamine biosynthetic pathway are antiviral in vitro. In sum, our data suggest that polyamines are critical to coronavirus replication and represent a highly promising drug target in the current and any future coronavirus outbreaks.

Keywords: SARS-CoV-2; binding; coronavirus; polyamines.

Figure 1.. Polyamine synthesis inhibitor DFMO limits coronavirus replication.

(A) Schematic of the polyamine pathway. The polyamines putrescine, spermidine, and spermine are synthesized from ornithine via ornithine decarboxylase (ODC1), which is inhibited by difluoromethylornithine (DFMO). (B) Vero-E6 cells treated with increasing doses of DFMO were infected at MOI 0.1 SARS-CoV-2 for 24h and viral genomes measured and converted to titer equivalents via a standard curve. (C) Vero-E6 cells were treated with escalating DFMO doses and viability and (D) polyamine levels were measured. (E) BHK-R cells were treated with DFMO as indicated and infected with MHV at MOI 0.01 for 48h. (F) Cellular viability levels were measured and (G) polyamine depletion was confirmed by chromatography. (H) BHK-R cells were treated with 1 mM DFMO and infected with MHV at MOI 0.01. Viral titers were determined after several rounds of replication. (I) MHV genomes from samples in (E) were quantified by qRT-PCR and compared to viral titers to quantify the relative number of genomes to PFU (J). (K) HeLa-R cells were treated with escalating doses of DFMO and infected with MHV at MOI 0.01 for 48h. (L) HeLa-R cells exposed to DFMO for four days were analyzed for viability. (M) Depletion of polyamines was confirmed by chromatography. *p<0.05, **p<0.01, ***p<0.001 by Student’s T-test. Data from at least three independent experiments.

Figure 2.. Polyamines facilitate coronavirus replication.

(A) BHK-R cells were treated with escalating doses of putrescine, spermidine, and spermine and cellular viability was measure 24h later. Calculated CC50 values are inset. (B) BHK-R cells treated as in (A) were infected with MHV at MOI 0.01 and titered at 48 hpi (upper). Cell-associated polyamines were measured by chromatography (lower). (C) BHK-R cells were treated with 1 mM DFMO for four days prior to infection with MHV at MOI 0.01. At the time of infection, 1 μM polyamines were added to the cells. Viral titers were determined at 48 hpi (upper). Cell-associated polyamines were measured by chromatography (lower). (D) MHV was directly incubated with DFMO or 1 μM of the polyamines putrescine (put), spermidine (spd), and spermine (spm) for 24h and then directly titered. **p<0.01 by Student’s T-test. Data from at least two independent experiments.

Figure 3.. Polyamine depletion is antiviral prophylactically.

Confluent monolayers of BHK-R cells were treated with DFMO at the time of infection with MHV and overlaid with agarose to form plaques. (A) Plaques were quantified two days later. (B) Representative plaques are shown from (A). (C) HeLa-R cells were infected and treated as in (A) and plaque area measured. (D) BHK-R cells were treated with 1.5 mM DFMO either in one, three, or four doses prior to infection with MHV at MOI 0.01 Viral titers were determined by plaque assay at 48 hpi. (E) Cells were treated with 1 mM DFMO and then subsequently refed with fresh medium not containing DFMO 24h before infection with MHV at MOI 0.01. Titers were determined at 24 hpi. **p<0.01, ***p<0.001 by Student’s T-test. Representative data from at least three independent experiments. Individual plaque sizes are indicated in (A) and (C).

Figure 4.. Polyamines facilitate coronavirus cellular attachment.

Confluent BHK-R cells were treated with DFMO prior to MHV binding assay. Virus was inoculated on cells for 5 min prior to removal, wash, and agarose overlay in media without DFMO. Plaques formed were quantified (A) and representative images from 1 mM DFMO treatment are shown (B). (C) BHK-R cells were treated as in (A), but after washing unattached virus, attached virus and cells were collected for RNA purification, reverse transcription, and viral genome quantitation by qPCR. Attached genomes were normalized to input virus. (D) MHV receptor (CECAM-1) was visualized on untreated and 1 mM DFMO treated cells. (E) HeLa-R cells were treated with 1 mM DFMO, infected with MHV, and processed as in (C). (F) Vero-E6 cells were treated with DFMO and analyzed for binding ability of HCoV-NL63 as in (C). (G) ACE2 expression on Vero-E6 cells was confirmed by immunofluorescence. (H) Pseudotyped viral particles with no viral glycoproteins or the SARS-CoV-2 spike were tested for their ability to bind and enter Vero-E6 cells. (I) Spike protein was confirmed to be present on pseudotyped virus by western blot. (J) Vero-E6 cells were treated with DFMO and infected with SARS-CoV-2 pseudoparticles as in (C). *p<0.05, **p<0.01, ***p<0.001 by Student’s T-test. Data from at least three independent experiments.

Figure 5.. Additional molecules targeting the polyamine pathway quell coronavirus infection.

(A) Schematic of the polyamine and hypusination pathway. (B) HeLa-R cells were treated with escalating doses of DENSpm for 16 h prior to infection with MHV at MOI 0.01. Viral titers were determined at 48 hpi (upper). Polyamine levels were measured by chromatography (lower). (C) Treated cells were assayed for viability after 16 h of treatment with DENSpm. (D) HeLa-R cells were treated with DENSpm at the indicated times before and after infection with MHV at MOI 10. Viral titers were determined at 48 hpi. BHK-R cells were treated with (E) CPX, (F) DEF, and (G) GC7 for 24h prior to infection with MHV at MOI 0.01. Viral titers were determined at 48 hpi. (H) BHK-R cells treated with CPX, DEF, and GC7 were analyzed for cell viability after 24h treatment. *p<0.05, **p<0.01, ***p<0.001 by Student’s T-test. Data from at least three independent experiments.