PUM2 binds SARS-CoV-2 RNA and PUM1 mildly reduces viral RNA levels, but neither protein affects progeny virus production

Figures

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  Figure 1.. PUM2 binds conserved PREs on SARS-CoV-2 transcripts. (A) Top: Consensus motif of the Pumilio response element (PRE). Below: List of all PREs and their location in the SARS-CoV-2 genome. (B) Most of the PREs within the SARS-CoV-2 genome are genetically stable. Mutation rates for 8-nt sequences across the genome were calculated using diversity statistics from GISAID data. (C) SARS-CoV-2 transcripts are preferentially bound by PUM2. The graph shows mean values from RNA immunoprecipitations of PUM1 and PUM2 from Calu-3 cells infected with SARS-CoV-2 WA1 for 48 h at MOI 0.05 (n = 3; error bars indicate SD). Significance was calculated using a ratio paired t-test by comparing values to the negative control: GAPDH for the host transcripts and N for the viral RNAs. * indicates a p-value of < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. CDKN1B is a known PUM target, while NORAD contains ~20 PREs.

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  Figure 2.. SARS-CoV-2 replication is largely unaffected by PUM depletion. (A) Validation of successful PUM depletion. A representative Western blot from uninfected and SARS-CoV-2-infected cells at 48 hpi. Clonal cell lines derived from A549-hACE2, either control (KO-ctl) or single knockouts (KO) of PUM1 or PUM2, were treated with siRNAs (siPUM1, siPUM2, or non-targeting control). 48h post siRNA treatment, cells were either infected or mock-infected with SARS-CoV-2 WA1 at an MOI of 0.5. (B) Cells with PUM depletion are viable. Viability was assessed by EdU incorporation into newly synthesized DNA. Data represent the average of two experiments performed in three biological replicates. (C) PUM double depletion de-represses known PUM targets, CDKN1B and TLK1. *p < 0.05 and **p < 0.01 (ratio paired t-test, n =3). (D) PUM depletion modestly increases NDV replication. A549-hACE2 cells, either controls or with single or double PUM depletion, were infected with the NDV LaSota strain at MOI 0.1. Viral replication was measured by plaque assay at 24 hpi. ** – p < 0.01 (paired t-test, n = 6). (E) SARS-CoV-2 progeny virus production is unaffected by PUM depletion. A549-hACE2 cells, either controls or…

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  Figure 3.. PUM overexpression does not affect SARS-CoV-2 replication. (A) Schematic representation of PUM proteins. (B) Representative Western blot from A549-hACE2 cells transiently transduced with the indicated FLAG-tagged constructs. (C) PUM overexpression represses known PUM targets, CDKN1B and TLK1 (n = 2). (D) PUM overexpression does not impact SARS-CoV-2 replication. A549-hACE2 cells were transduced with the indicated constructs and infected with SARS-CoV-2 WA1 at MOI 0.5 for 48 h. Viral replication was measured by plaque assay (n = 5). Error bars represent SD. hpi – hours post-infection; PFU – plaque-forming units.

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  Figure 4.. PUM1 negatively regulates innate immunity genes, but this activity is not enhanced by type I IFN treatment. (A) PUM1 depletion negatively regulates expression of innate immunity genes during steady state and SARS-CoV-2 infection. A549-hACE2 cells, either control or with single PUM depletions, were infected with SARS-CoV-2 WA1 at an MOI of 0.5, and mRNA levels of CXCL10, IFN-β, ISG15, and IFIT2 were measured by RT-qPCR. Results were normalized to GAPDH and then to mock. *p < 0.05, **p < 0.01, (paired t-test, n = 4). (B) PUM1 depletion does not enhance the antiviral state induced by IFNα treatment. A549-hACE2 cells, either control or with single PUM depletions, were treated with 103 U/ml of IFNα for 24 h, after which RT-qPCR was performed (n = 3). (C) Single PUM depletion does not affect the impact of IFNα pretreatment on SARS-CoV-2 replication. A549-hACE2 cells, either control or with single PUM depletions, were pre-treated overnight with 103 U/ml of IFNα and infected at an MOI of 0.5 with SARS-CoV-2 WA1. Viral replication was assayed 48 h post-infection. Error bars represent SD. hpi – hours post-infection; RdRp – RNA-dependent RNA polymerase.