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. 2023 Sep 28;97(9):e0088523.
doi: 10.1128/jvi.00885-23. Epub 2023 Sep 11.

PARP12 is required to repress the replication of a Mac1 mutant coronavirus in a cell- and tissue-specific manner

Catherine M Kerr  1 , Srivatsan Parthasarathy  1 , Nancy Schwarting  1 , Joseph J O'Connor  1 , Jessica J Pfannenstiel  1 , Emily Giri  1 , Sunil More  2 , Robin C Orozco  1 , Anthony R Fehr  1
Affiliations

PARP12 is required to repress the replication of a Mac1 mutant coronavirus in a cell- and tissue-specific manner

Catherine M Kerr et al. J Virol. .

Abstract

ADP-ribosyltransferases (ARTs) mediate the transfer of ADP-ribose from NAD+ to protein or nucleic acid substrates. This modification can be removed by several different types of proteins, including macrodomains. Several ARTs, also known as PARPs, are stimulated by interferon indicating ADP-ribosylation is an important aspect of the innate immune response. All coronaviruses (CoVs) encode for a highly conserved macrodomain (Mac1) that is critical for CoVs to replicate and cause disease, indicating that ADP-ribosylation can effectively control coronavirus infection. Our siRNA screen indicated that PARP12 might inhibit the replication of a murine hepatitis virus (MHV) Mac1 mutant virus in bone-marrow-derived macrophages (BMDMs). To conclusively demonstrate that PARP12 is a key mediator of the antiviral response to CoVs both in cell culture and in vivo, we produced PARP12-/-mice and tested the ability of MHV A59 (hepatotropic/neurotropic) and JHM (neurotropic) Mac1 mutant viruses to replicate and cause disease in these mice. Notably, in the absence of PARP12, Mac1 mutant replication was increased in BMDMs and mice. In addition, liver pathology was also increased in A59-infected mice. However, the PARP12 knockout did not restore Mac1 mutant virus replication to WT virus levels in all cell or tissue types and did not significantly increase the lethality of Mac1 mutant viruses. These results demonstrate that while PARP12 inhibits MHV Mac1 mutant virus infection, additional PARPs or innate immune factors must contribute to the extreme attenuation of this virus in mice. IMPORTANCE Over the last decade, the importance of ADP-ribosyltransferases (ARTs), also known as PARPs, in the antiviral response has gained increased significance as several were shown to either restrict virus replication or impact innate immune responses. However, there are few studies showing ART-mediated inhibition of virus replication or pathogenesis in animal models. We found that the CoV macrodomain (Mac1) was required to prevent ART-mediated inhibition of virus replication in cell culture. Using knockout mice, we found that PARP12, an interferon-stimulated ART, was required to repress the replication of a Mac1 mutant CoV both in cell culture and in mice, demonstrating that PARP12 represses coronavirus replication. However, the deletion of PARP12 did not fully rescue Mac1 mutant virus replication or pathogenesis, indicating that multiple PARPs function to counter coronavirus infection.

Keywords: ADP-ribosylation; MHV; PARP; coronavirus; macrodomain; pathology.

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Conflict of interest statement

Anthony R. Fehr was named as an inventor on a patent filed by the University of Kansas.

Figures

Fig 1
Fig 1
Generation of PARP12−/− mice. (A) Schematic of the LacZ insertion used to create the PARP12 knockout in C57B6/NJ mice. The insertion induces a frameshift mutation, creating a completely null mutation. (B) Ratio of PARP12+/+, PARP12+/−, and PARP12−/− mice following PARP12+/− × PARP12+/− breeding over the course of 1 year. (D and E) PARP12 expression (D) and weights (E) in various organs of PARP12+/+, PARP12+/−, and PARP12−/− mice. The data in D-E are the combined results of two independent experiments. n = 9–13 per group: PARP12+/+ = 11; PARP12+/− = 13; PARP12−/− = 9. (F and G) Immune cells from the spleens of naïve PARP12+/+ mice and PARP12−/− mice. The data in F-G are the combined results of two independent experiments. n = 8 for all groups.
Fig 2
Fig 2
PARP12 is required for the restriction of Mac1-mutant MHV replication in BMDMs, but not BMDCs. (A and B) PARP12+/+ and PARP12−/− BMDMs were infected with MHV-JHM (JHMV) WT or N1347A (A) or MHV-A59 WT and N1348A (B) at an MOI of 0.05 PFU/cell. Cells and supernatants were collected at indicated times post-infection (hpi) and assayed for progeny infectious virus by plaque assay. The data in A–B are the combined results of two independent experiments. n = 6 for all groups. (C and D) PARP12+/+ and PARP12−/− BMDCs were infected with JHMV WT or N1347A (C) or A59 WT and N1348A (D) at an MOI of 0.05 PFU/cell. Cells and supernatants were collected at indicated times post-infection (hpi) and assayed for progeny infectious virus by plaque assay. The data in C–D are from one experiment representative of three independent experiments. n = 3 for all groups.
Fig 3
Fig 3
PARP12 does not contribute to enhanced IFN-I and cytokine levels following BMDM infection with JHMV N1347A. PARP12+/+ and PARP12−/− BMDMs were infected with WT or N1347A JHMV. Cells were collected in Trizol at 12 hpi and RNA levels were determined using RT-qPCR with specific primers for each gene of interest and normalized to HPRT. These data are from one experiment representative of two independent experiments. n = 3 for all groups.
Fig 4
Fig 4
PARP12 is not required for the restriction of JHMV virulence and replication following an intranasal (IN) infection. (A) PARP12+/+ mice and PARP12−/− mice were infected intranasally with 104 PFU of JHMV WT or N1347A. Brains were harvested at 5 dpi and titers determined via plaque assay. The data in A are the combined results of three independent experiments. n = 7–8 per group: PARP12+/+ WT=8; PARP12+/+ N1347A = 8; PARP12−/− WT = 8; PARP12−/− N1347A = 7. (B) Infected brains were harvested at 5 dpi, then forebrain sections were stained for MHV nucleocapsid (N) protein by IHC. The images in B are from one experiment representative of three independent experiments. n = 2–4 per group: PARP12+/+ WT=2; PARP12+/+ N1347A = 3; PARP12−/− WT = 2; PARP12−/− N1347A = 4. Scale bar is 100 µm. (C and D) PARP12+/+ mice and PARP12−/− mice were infected as described above. Survival and weight loss were monitored for 12 days. The data in C–D are the combined results of three independent experiments. n = 4–9 per group: PARP12+/+ WT=5; PARP12+/+ N1347A = 6; PARP12−/− WT = 4; PARP12−/− N1347A = 9.
Fig 5
Fig 5
PARP12 KO mildly increases virus replication and lethality in the brain following an intracranial (IC) infection with N1347A. (A) PARP12+/+ mice and PARP12−/− mice were infected intracranially with 750 PFU of JHMV WT or N1347A. Brains were harvested at 4 dpi and titers were determined via plaque assay. Data in A are combined from more than three independent experiments. n = 7–14 per group: PARP12+/+ WT=7; PARP12+/+ N1347A = 10; PARP12−/− WT = 9; PARP12−/− N1347A = 14. (B and C) Mice were infected as described in (A) and survival (B) and weight loss (C) were monitored for 12 days. Data in B–C are combined from more than three independent experiments. n = 6–22 per group: PARP12+/+ WT=7; PARP12+/+ N1347A = 22; PARP12−/− WT = 6; PARP12−/− N1347A = 16.
Fig 6
Fig 6
PARP12 KO increases N1348A replication in livers but does not impact survival or weight loss. (A) PARP12+/+ and PARP12−/− mice were infected intraperitoneally (IP) with 500 PFU of MHV-A59 WT or N1348A. Livers were harvested at 3 dpi and titers were determined via plaque assay. The results in A are from one experiment representative of three independent experiments. n = 3 for each group. (B and C) PARP12+/+ and PARP12−/− mice were infected IP with 5 × 104 PFU of MHV-A59 WT or N1348A and survival (B) and weight loss (C) were monitored for 12 days. The results in B−C are the combined data from at least three independent experiments. n = 12–16 per group: PARP12+/+ WT=13; PARP12+/+ N1348A = 16; PARP12−/− WT = 14; PARP12−/− N1347A = 12.
Fig 7
Fig 7
PARP12 is required to prevent severe liver pathology following A59 N1348A infection. (A) PARP12+/+ and PARP12−/− mice were infected via IP injection with 5 × 104 PFU of MHV A59 WT or N1348A and livers were harvested at 12 dpi and histological analysis of livers was performed. The results are the combined data from at least three independent experiments. n = 7–9 per group: PARP12+/+ WT=7; PARP12+/+ N1348A = 8; PARP12−/− WT = 8; PARP12−/− N1347A = 9. Dark arrowheads represent inflammation, blank arrowheads represent necrosis, and arrows represent edema/fibrin. (B) Liver pathology was scored based on inflammation, necrosis, and edema/fibrin on a scale of 0–4 (see Materials and Methods). The combined score represents the combined scores for all three categories.
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