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CMPK2 is a host restriction factor that inhibits infection of multiple coronaviruses in a cell-intrinsic manner [1]

['Mingjun Zhu', 'Key Laboratory Of Veterinary Biological Engineering', 'Technology Ministry Of Agriculture', 'Institute Of Veterinary Medicine', 'Jiangsu Academy Of Agricultural Sciences', 'Nanjing', 'Jiangsu', 'Jiangsu Key Laboratory For Food Quality', 'Safety-State Key Laboratory Cultivation Base Of Ministry Of Science', 'Technology']

Date: 2023-03 

Coronaviruses (CoVs) comprise a group of important human and animal pathogens. Despite extensive research in the past 3 years, the host innate immune defense mechanisms against CoVs remain incompletely understood, limiting the development of effective antivirals and non-antibody-based therapeutics. Here, we performed an integrated transcriptomic analysis of porcine jejunal epithelial cells infected with porcine epidemic diarrhea virus (PEDV) and identified cytidine/uridine monophosphate kinase 2 (CMPK2) as a potential host restriction factor. CMPK2 exhibited modest antiviral activity against PEDV infection in multiple cell types. CMPK2 transcription was regulated by interferon-dependent and interferon regulatory factor 1 (IRF1)-dependent pathways post-PEDV infection. We demonstrated that 3′-deoxy-3′,4′-didehydro-cytidine triphosphate (ddhCTP) catalysis by Viperin, another interferon-stimulated protein, was essential for CMPK2’s antiviral activity. Both the classical catalytic domain and the newly identified antiviral key domain of CMPK2 played crucial roles in this process. Together, CMPK2, viperin, and ddhCTP suppressed the replication of several other CoVs of different genera through inhibition of the RNA-dependent RNA polymerase activities. Our results revealed a previously unknown function of CMPK2 as a restriction factor for CoVs, implying that CMPK2 might be an alternative target of interfering with the viral polymerase activity.

Funding: This work was supported by the National Key Research and Development Program (2021YFD1801104 to BL), the National Natural Science Foundation of China (Grant No. 32202787 to MZ; 32272996 and 32202823 to BL), the Jiangsu province Natural Sciences Foundation (BK20190003 and BK20210158 to BL), the Jiangsu Agricultural Science and Technology Innovation Fund (CX(21)3139 to BL), the Innovation Foundation of Jiangsu Academy of Agricultural Sciences (ZX(21)1217 to BL), the China Postdoctoral Science Foundation (2022M711399 to MZ), the startup fund of Washington University in St. Louis (SD), and a DDRCC T32 fellowship (DK007130) (AA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we employed a transcriptome sequencing approach to identify host factors that confer protection against CoV infection and demonstrated that CMPK2 was both up-regulated in vitro and in vivo following PEDV infection. Although multiple proteins of CoVs including PEDV restrain antiviral innate immune response via various mechanisms [ 20 , 21 ], up-regulation of CMPK2 expression occurred in host cells at least partially independent of the IFN-I pathway. Mechanistically, we found that via the catalytic residues and an antiviral key domain (AKD), CMPK2 inhibits PEDV replication through increasing ddhCTP production and inhibiting the RdRp activities. Collectively, our results implicate CMPK2 as an effective host defense factor during virus infection and provided a novel cellular target for controlling CoV infections.

Cytidine/uridine monophosphate kinase 2 (CMPK2, also known as TYKi/TMPK2) is a nucleoside monophosphate kinase and is implicated in the synthesis of mitochondrial DNA (mtDNA) and maintaining intracellular UTP/CTP [ 15 , 16 ]. CMPK2 was originally identified as one of the interferon-stimulated genes (ISGs) induced by type I interferon (IFN-I) and was confirmed for its ability to suppress human immunodeficiency virus (HIV), spring viremia of carp virus (SVCV) as well as dengue virus (DENV) replication [ 17 – 19 ]. However, many CoVs are propagated in Vero cells that naturally lack genes encoding IFN-I, and multiple proteins of CoVs suppress IFN production via various mechanisms [ 20 – 22 ]. Therefore, the relationship between CoVs, CMPK2, and IFN-I still needs further clarification. Furthermore, in the human genome, CMPK2 was found adjacent to and inverted with respect to the RSAD2 (encoding Viperin, a well-characterized ISG with antiviral activity; [ 23 ]). Intriguingly, CMPK2 and Viperin are often coexpressed after IFN-I stimulation or viral infection. CMPK2 catalyzes the phosphorylation of CDP to produce CTP, and Viperin mediates the conversion of CTP to 3′-deoxy-3′,4′-didehydro-cytidine triphosphate (ddhCTP) [ 24 , 25 ], resulting in an approximately 4-fold increase in ddhCTP production [ 24 , 26 ]. More evidence showed that ddhCTP acts as a chain terminator for RdRp and directly prevents the replication of many viruses including Zika virus and West Nile virus [ 25 , 27 ]. CMPK2 was identified in a recent ISG screen to potentially inhibit SARS-CoV-2 replication [ 28 ]. Nevertheless, whether CMPK2 broadly inhibits CoV replication and the underlying molecular mechanisms are still not clear.

CoVs consist of 4 genera: α, β, γ, and δ, represented by porcine epidemic diarrhea virus (PEDV, α), SARS-CoV-2 (β), infectious bronchitis virus (IBV, γ), and porcine delta-coronavirus (PDCoV, δ), which are common pathogens for birds and mammals [ 13 , 14 ]. Here, we use PEDV as a model virological system to probe the molecular mechanisms of host defense against CoV infection, which will pave the path for the development of effective broad-spectrum anti-CoV pharmacological inhibitors.

CoVs are positive-sense single-stranded RNA viruses and possess the largest genome among RNA viruses, which encodes the standard set of 4 structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) and 16 (mostly) functional nonstructural proteins (nsp1-nsp16) [ 3 – 5 ]. These proteins are relevant for CoV replication and pathogenesis in vivo, by either interfering with the host immune response or directly assisting steps of the viral replication cycle. Nsp12, a major component of RNA-dependent RNA polymerase (RdRp), is the powerhouse of CoV replication [ 6 – 8 ]. Although RdRp is an obvious target of antiviral therapeutics development, Gilead’s remdesivir and Merck’s molnupiravir represent the only 2 FDA-authorized antiviral drugs [ 9 , 10 ]. In addition, there is significant concern that monotherapy would rapidly result in the emergence of resistance [ 11 , 12 ].

Coronaviruses (CoVs) are important pathogens that pose serious threats to human and animal health. Research on CoVs has largely been lagging, with sporadic attention during the outbreaks of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) [ 1 ]. The ongoing SARS-CoV-2 pandemic has infected more than 600 million and caused the death of more than 6.6 million people worldwide [ 2 ]. Even in the presence of effective vaccines of various platforms, there is an urgent need to understand SARS-CoV-2 biology and develop specific and broad-spectrum antiviral inhibitors.

Results

CMPK2 inhibits PEDV replication in Vero and IPEC-J2 cells To investigate the potential antiviral activity of CMPK2 toward PEDV replication, we transfected Vero and IPEC-J2 cells with empty vector or CMPK2 encoding plasmids and then infected with PEDV at MOI of 1. Cells were harvested at indicated time points and analyzed by qRT-PCR and western blotting. We found that the transcript and protein levels of PEDV N were significantly lower in the IPEC-J2 cells overexpressing CMPK2 than those transfected with the empty vector (Fig 4A and 4B). Cell culture supernatants were also collected, and viral titers were determined by limiting serial dilutions. The amount of infectious PEDV in the supernatants of IPEC-J2 cells overexpressing CMPK2 was significantly lower than those in the cells transfected with empty vector (Fig 4C). Similar findings were validated in Vero cells that are traditionally known to be IFN-insensitive. The transcript and protein levels of PEDV N were significantly inhibited (S9A and S9B Fig), and the viral titers in the supernatants were decreased approximately 10-fold in Vero cells with CMPK2 overexpression (S9C Fig). We also sought to determine the physiological relevance of endogenous CMPK2. We transfected Vero cells with siRNAs targeting simian CMPK2 and infected with PEDV at MOI of 1 and harvested at 20 hpi. Total RNA of the cells was extracted and analyzed by qRT-PCR, and the cell lysates were analyzed by western blotting. We found that the transcript and protein levels of PEDV N were elevated in the Vero cells transfected with siRNA targeting CMPK2 than in cells transfected with siRNA-negative control (S9D and S9E Fig). Moreover, the viral titers in the supernatants were approximately 10-fold higher than in Vero cells with CMPK2 siRNA knockdown (S9F Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 4. CMPK2 inhibits PEDV replication in IPEC-J2 cells. (A-C) IPEC-J2 cells were transfected with empty vector (pEmpty) and pCMPK2 for 24 h and then infected with PEDV at MOI of 1. The cells and culture supernatants were harvested at indicated time points. Total RNA of the cells was extracted and analyzed by qRT-PCR (A), and the cell lysates were analyzed by western blotting (B). PEDV titers in the culture supernatants were measured as TCID 50 (C). (D) CMPK2 knockout (CMPK2#KO) IPEC-J2 cells were constructed and then infected with PEDV as above. The cells were harvested at indicated time points, and the lysates were analyzed by western blotting. (E) CMPK2 knockout IPEC-J2 cells were transfected with pEmpty and pCMPK2 for 24 h and then infected with PEDV as above. Total RNA of the cells was extracted and analyzed by qRT-PCR (upper panel), and the cell lysates were analyzed by western blotting (lower panel). (F) PEDV N antigen was detected in infected cells by indirect IFA. PEDV N (green), DAPI (blue). Scale bars, 100 μm. Data are means ± SD of triplicate samples; statistical analysis was conducted using one-way ANOVA followed by Dunnett’s multiple comparison test or two-way ANOVA followed by Tukey’s multiple comparison test; only the p-value for the most relevant comparisons are shown for simplicity. The intensities of bands were quantified by ImageJ. **p < 0.01, ***p < 0.001, ****p < 0.0001. Data underlying this figure can be found in S1 Data and S1 Raw Images. CMPK2, cytidine/uridine monophosphate kinase 2; IFA, immunofluorescent assay; MOI, multiplicity of infection; PEDV, porcine epidemic diarrhea virus; qRT-PCR, quantitative real-time PCR. https://doi.org/10.1371/journal.pbio.3002039.g004 We further confirmed the inhibition of PEDV replication by CMPK2 by generating single clonal CMPK2 knockout IPEC-J2 cells via CRISPR-Cas9. The expression of PEDV N was significantly increased in CMPK2 knockout IPEC-J2 cells and reversed after complementing with exogenous expression of wild-type full-length CMPK2 (Fig 4D and 4E). Indirect immunofluorescent assay (IFA) also confirmed that PEDV N expression was decreased with CMPK2 overexpression and increased in CMPK2 knockout IPEC-J2 cells (Fig 4F). Taken together, these results indicate that CMPK2 is a potent host antiviral factor of PEDV replication.

CMPK2 inhibits PEDV infection at a post-entry step We next sought to define the step of the PEDV replication cycle that was restricted by CMPK2. IPEC-J2 cells with or without CMPK2 overexpression were incubated with PEDV at MOI of 5 (4 °C for 1 h), washed extensively with phosphate-buffered saline (PBS), and the genome copies of bound viruses was measured by qRT-PCR. In parallel, CMPK2 transfected IPEC-J2 cells were incubated with PEDV at MOI of 1 (4 °C for 1 h) and shifted to 37 °C for 1 h. The cells were then washed to remove all unbound and unendocytosed virus, and the intracellular viral RNA was quantified using qRT-PCR (S10A Fig). The results showed that there was no significant difference in the amount of PEDV bound or internalized between empty vector and CMPK2 transfected cells (S10B Fig), suggesting that overexpression of CMPK2 does not act to block the process of viral binding or entry. Kinetic studies were further performed to investigate the antiviral action of CMPK2 on PEDV replication. Transcriptional levels of PEDV in IPEC-J2 cells transfected with CMPK2 were analyzed by qRT-PCR at 1, 2, 3, and 4 hpi, respectively, and PEDV N expression was detected by western blotting. Despite no significant differences in intracellular viral RNA levels at 1 hpi (S10C Fig), the amount of viral RNA levels was readily reduced at later time points in CMPK2 expressing IPEC-J2 cells (S10C and S10D Fig), suggesting that CMPK2 likely restricts PEDV replication at the stage of transcription or genome replication.

ddhCTP catalysis by Viperin is required for the antiviral activity of CMPK2 It has been reported that Viperin inhibits viral replication by converting CTP into ddhCTP and that CMPK2 provides enough CTP as a substrate in this process [25,34]. To investigate the molecular mechanisms by which CMPK2 suppresses PEDV replication, we tested the effect of siRNAs targeting porcine Viperin (siViperin) on PEDV infection. IPEC-J2 cells with or without CMPK2 overexpression were transfected with siRNA targeting Viperin or the negative control and then infected with PEDV at MOI of 1. The expression levels of PEDV N were significantly decreased in IPEC-J2 cells overexpressing CMPK2, and the reduction was partially prevented by Viperin knockdown (S11A and S11B Fig). To further tease apart the upstream–downstream relationship and investigate the molecular mechanisms by which CMPK2 suppresses PEDV replication, Viperin knockout and Viperin/CMPK2 double knockout IPEC-J2 cells were constructed and changes in PEDV replication levels were analyzed after supplementing Viperin in CMPK2 knockout cells or supplementing CMPK2 in Viperin knockout cells. We found that Viperin supplementation in CMPK2 knockout cells had a more significant effect on inhibiting PEDV replication than the reciprocal add-back (Fig 5A and 5B). Meanwhile, we also complemented Viperin and CMPK2, respectively, in the double knockout cells. Consistently, the effect of Viperin on PEDV replication appears to be more direct (Fig 5C). Of note, Viperin and CMPK2 are not entirely dependent on each other. Previous studies showed an independent transcriptional regulation and that these 2 factors may restrict viral replication either together or individually in different cellular contexts [17,24]. Taken together, these results confirmed that CMPK2 is an important host factor upstream of Viperin. PPT PowerPoint slide

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TIFF original image Download: Fig 5. ddhCTP catalyzed by Viperin is crucial for CMPK2 inhibition of PEDV replication. (A) CMPK2#KO IPEC-J2 cells were transfected with pEmpty and pViperin-HA for 24 h and then infected with PEDV at MOI of 1. The cells were harvested at 20 hpi, and the lysates were analyzed by western blotting (left). Total RNA of the cells was extracted, and viral copy numbers were analyzed by qRT-PCR (right). (B) Viperin knockout (Viperin#KO) IPEC-J2 cells were constructed and then transfected with pEmpty and pCMPK2-HA for 24 h. Subsequently, Viperin#KO IPEC-J2 cells were infected with PEDV as above. The lysates were analyzed by western blotting (left). Total RNA of the cells was extracted, and viral copy numbers were analyzed by qRT-PCR (right). (C) CMPK2/Viperin double KO (Viperin/CMPK2#KO) IPEC-J2 cells were constructed and, respectively, transfected with pEmpty, pViperin-HA, and pCMPK2-HA for 24 h to rescue Viperin or CMPK2 expression and then infected with PEDV as above. The lysates were analyzed by western blotting (left). Total RNA of the cells was extracted, and viral copy numbers were analyzed by qRT-PCR (right). (D) ddhCTP production in IPEC-J2 cells with or without CMPK2 expression was determined by LC–MS. (E) The detection of ddhCTP production in CMPK2 knockout or wild-type IPEC-J2 cells by LC–MS. (F and G) IPEC-J2 cells were transfected with pCMPK2-HA and empty vector, and a range of concentrations of NTPs were added to compete with ddhCTP. The cell lysates were analyzed by western blotting (F), and PEDV titers in the culture supernatants were measured as TCID 50 (G). Data are means ± SD of triplicate samples; statistical analysis was conducted using one-way ANOVA followed by Dunnett’s multiple comparison test or two-way ANOVA followed by Tukey’s multiple comparison test; only the p-value for the most relevant comparisons are shown for simplicity. The intensities of bands were quantified by ImageJ. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data underlying this figure can be found in S1 Data and S1 Raw Images. CMPK2, cytidine/uridine monophosphate kinase 2; ddhCTP, 3′-deoxy-3′,4′-didehydro-cytidine triphosphate; hpi, hours post-infection; LC–MS, liquid chromatography followed by mass spectrometry; MOI, multiplicity of infection; NTP, nucleotide triphosphate; PEDV, porcine epidemic diarrhea virus; qRT-PCR, quantitative real-time PCR. https://doi.org/10.1371/journal.pbio.3002039.g005 We next determined production of ddhCTP, the end product of CMPK2 and Viperin, with liquid chromatography followed by mass spectrometry (LC–MS) in IPEC-J2 cells previously transfected with mock or Viperin targeting siRNAs as previously described [35]. Consistent with the previous reports [36], ddhCTP level was significantly decreased with the knockdown of Viperin (S11C Fig). To clarify the relationship between CMPK2 and ddhCTP, we also determined ddhCTP production by LC–MS in IPEC-J2 cells with CMPK2 overexpression or knockout. As expected, ddhCTP level was significantly increased in IPEC-J2 cells with CMPK2 overexpression and substantially decreased with CMPK2 knockout (Fig 5D and 5E). To further analyzed the effect of ddhCTP on CMPK2 antiviral activity, IPEC-J2 cells were transfected with CMPK2 and empty vector, and a range of concentrations of nucleotide triphosphates (NTPs) were added to compete with ddhCTP and complement the loss of CTP. Even in the presence of CMPK2, expression levels of PEDV N and viral titers were restored with exogenous NTP supplementation (Fig 5F and 5G). Collectively, these results suggested that ddhCTP catalyzed by Viperin is required for CMPK2 restriction of PEDV replication.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002039

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