Analysis of Coronavirus Temperature-Sensitive Mutants Reveals an Interplay between the Macrodomain and Papain-Like Protease Impacting Replication and Pathogenesis

Abstract

Analysis of temperature-sensitive (ts) mutant viruses is a classic method allowing researchers to identify genetic loci involved in viral replication and pathogenesis. Here, we report genetic analysis of a ts strain of mouse hepatitis virus (MHV), tsNC11, focusing on the role of mutations in the macrodomain (MAC) and the papain-like protease 2 (PLP2) domain of nonstructural protein 3 (nsp3), a component of the viral replication complex. Using MHV reverse genetics, we generated a series of mutant viruses to define the contributions of macrodomain- and PLP2-specific mutations to the ts phenotype. Viral replication kinetics and efficiency-of-plating analysis performed at permissive and nonpermissive temperatures revealed that changes in the macrodomain alone were both necessary and sufficient for the ts phenotype. Interestingly, mutations in the PLP2 domain were not responsible for the temperature sensitivity but did reduce the frequency of reversion of macrodomain mutants. Coimmunoprecipitation studies are consistent with an interaction between the macrodomain and PLP2. Expression studies of the macrodomain-PLP2 portion of nsp3 indicate that the ts mutations enhance proteasome-mediated degradation of the protein. Furthermore, we found that during virus infection, the replicase proteins containing the MAC and PLP2 mutations were more rapidly degraded at the nonpermissive temperature than were the wild-type proteins. Importantly, we show that the macrodomain and PLP2 mutant viruses trigger production of type I interferon in vitro and are attenuated in mice, further highlighting the importance of the macrodomain-PLP2 interplay in viral pathogenesis.IMPORTANCE Coronaviruses (CoVs) are emerging human and veterinary pathogens with pandemic potential. Despite the established and predicted threat these viruses pose to human health, there are currently no approved countermeasures to control infections with these viruses in humans. Viral macrodomains, enzymes that remove posttranslational ADP-ribosylation of proteins, and viral multifunctional papain-like proteases, enzymes that cleave polyproteins and remove polyubiquitin chains via deubiquitinating activity, are two important virulence factors. Here, we reveal an unanticipated interplay between the macrodomain and the PLP2 domain that is important for replication and antagonizing the host innate immune response. Targeting the interaction of these enzymes may provide new therapeutic opportunities to treat CoV disease.

Keywords: coronavirus; innate immunity; interferon; macrodomain; papain-like protease; temperature sensitive; viral replication.

FIG 1

Evaluation of the replication kinetics of coronavirus temperature-sensitive mutants at permissive and nonpermissive temperatures. (A) Schematic diagram of the MHV genome and the domains of nsp3. Abbreviations: Ubl1, ubiquitin-like domain 1; Ac, acidic region; PLP1, papain-like protease 1; MAC, macrodomain; DPUP, domain preceding Ubl2 and PLP2; Ubl2, ubiquitin-like domain 2; PLP2, papain-like protease 2; NAB, nucleic acid-binding domain; G2M, coronavirus group 2 marker domain; TMDs, transmembrane domains; Y, coronavirus highly conserved domain. Representative structures of the macrodomain with ribose (229E; PDB 3EWR) and PLP2 (MHV; PDB 4YPT) are shown in cyan and green with catalytic pockets circled, and the residues involved in catalysis are shown in magenta. The mutations described in this study are shown in red. (B) Growth kinetics of MHV and mutants at three temperatures. DBT cells were inoculated with the indicated virus (at a multiplicity of infection [MOI] of 5) for 1 h at 37°C and then shifted to the indicated temperatures. Culture supernatants were collected at the indicated hours postinfection and titrated in DBT cells at 32°C. The data are representative of two independent experiments. Error bars indicate ± the standard deviation (SD).

FIG 2

Analysis of plaque size and efficiency of plating at permissive and nonpermissive temperatures. (A) Representative plaque assays at 32 and 40°C for icWT, tsNC11, and engineered mutant viruses. The dilution of the viral stock is indicated and selected to visualize ca. 20 to 50 plaques per plate. (B) EOP data. EOP = average titer at 40°C/average titer at 32°C.

FIG 3

Analysis of small- and large-plaque variants in the MACmut virus population. (A) MACmut isolates with distinct plaque sizes were evaluated for a ts phenotype. (B and C) Sequence analysis of individual plaque-purified revertant isolates identified mutations in the macrodomain and the adjacent downstream region in the large-plaque variants of the MACmut (B) and tsNC11 (C) viruses.

FIG 4

Evaluation of the coimmunoprecipitation of the macrodomain and the PLP2 domain. (A) Schematic diagram of the individual constructs used to evaluate potential interactions between the macrodomain and PLP2. (B) Western blots to identify expression and coimmunoprecipitation of HA-MAC and PLP2-V5. HEK-293T cells were transfected with the indicated plasmid DNAs, lysates were prepared at 18 h posttransfection and subjected to immunoprecipitation with the indicated antibody, and the products were analyzed by SDS-PAGE and immunoblotting. The data represent the results of three independent experiments. Asterisks indicate the cross-detection of IgG chains by secondary antibody.

FIG 5

Mutations in the macrodomain and PLP2 enhance degradation of the polypeptide. (A) Schematic diagram of constructs used to evaluate protein stability. (B and C) Western blotting was used to detect wild-type or mutant forms of MAC-PLP2 polypeptide in the presence of CHX or a combination of CHX and proteasome inhibitor MG132. HEK-293T cells were transfected with the indicated expression plasmid of wild-type (WT) or mutant forms of MAC-PLP2. At 16 h posttransfection, cells were treated with 20 μg/ml of CHX or a combination of 20 μg/ml CHX and 10 μM MG132 and then harvested at the indicated time points. Equal amounts of cell lysate were subjected to immunoblotting with anti-V5 or anti-β-actin antibodies. The intensities of the MAC/PLP2 bands relative to that of β-actin were measured and calculated with AlphaView software. The experiment was repeated two times, and representative immunoblots (B) and curves of relative intensities (C) are shown. The slope parameters of the decay curves were evaluated using nonlinear regression and two-sided t tests in comparison to WT. **, P < 0.005; ****, P < 0.0001.

FIG 6

Mutations in the macrodomain and PLP2 alter the stability of replicase protein nsp3. HeLa-MHVR cells were infected with either icMHV-WT or MAC/PLP2mut virus (MOI of 5) and incubated at 32°C for 9.5 h. Then 20 μg/ml of CHX was added, and the cells were shifted to the nonpermissive temperature. Lysates were prepared every 30 min, the proteins were separated by SDS-PAGE, and nonstructural proteins nsp2-3 and nsp3 were visualized by immunoblotting. (A) Schematic diagram of MHV replicase polyprotein indicating the processing pathway and the region identified by the anti-nsp2-3 antibody. (B) Outline of the experiment. (C) Western blot evaluating the level of nsp2-3 and nsp3 proteins detected after a shift to the nonpermissive temperature. These are representative data from two independent experiments. The arrowhead indicates detections of cellular protein in all lysates. The asterisk indicates degradation products detected by anti-nsp2-3 antibody in the MAC/PLP2mut virus-infected cells.

FIG 7

Macrodomain mutant viruses induce type I interferon in primary macrophages and are attenuated in mice. (A) Mouse bone marrow-derived macrophages were infected with the indicated virus (MOI of 1) at 32°C. Total RNA was extracted at the indicated time points and subjected to RT-qPCR. The mRNA levels of IFN-α (left) and the N gene (right) are presented relative to that of β-actin. The results are representative of three independent experiments and were subjected to a two-tailed, unpaired t test. Error bars indicate ± SD. ***, P < 0.001; ****, P < 0.0001; n.s., not significant; N.D., not detected. (B) Six-week-old mice were injected intracranially with either icWT or the indicated ts mutant virus (600 PFU/mouse) and monitored for weight loss. Viral pathogenicity was evaluated by body weight loss (left) and percent survival (right). The number (n) of infected mice is indicated in parentheses. Error bars indicate ± standard error of the mean. Differences in survival rates were calculated using a log-rank test.