Mouse hepatitis virus JHMV I protein is required for maximal virulence

Abstract

Betacoronaviruses encode a conserved accessory gene within the +1 open reading frame (ORF) of nucleocapsid called the internal N gene. This gene is referred to as "I" for mouse hepatitis virus (MHV), ORF9b for severe acute respiratory CoV (SARS-CoV) and SARS-CoV-2, and ORF8b for Middle East respiratory syndrome CoV (MERS-CoV). Previous studies have shown ORF8b and ORF9b have immunoevasive properties, while the only known information for MHV I is its localization within the virion of the hepatotropic/neurotropic A59 strain of MHV. Whether MHV I is an innate immune antagonist or has other functions has not been evaluated. In this report, we show that the I protein of the neurotropic JHM strain of MHV (JHMV) lacks a N terminal domain present in other MHV strains, has immunoevasive properties, and is a component of the virion. Genetic deletion of JHMV I (rJHMV^IΔ57-137) resulted in a highly attenuated virus both in vitro and in vivo that displayed a post RNA replication/transcription defect that ultimately resulted in fewer infectious virions packaged compared with wild-type virus. This phenotype was only seen for rJHMV^IΔ57-137, suggesting the structural changes predicted for A59 I altered its function, as genetic deletion of A59 I did not change viral replication or pathogenicity. Together, these data show that JHMV I both acts as a mild innate immune antagonist and aids in viral assembly and infectious virus production, and suggest that the internal N proteins from different betacoronaviruses have both common and virus strain-specific properties.IMPORTANCECoV accessory genes are largely studied in overexpression assays and have been identified as innate immune antagonists. However, functions identified after overexpression are often not confirmed in the infected animal host. Furthermore, some accessory proteins are components of the CoV virion, but their role in viral replication and release remains unclear. Here, we utilized reverse genetics to abrogate expression of a conserved CoV accessory gene, the internal N ("I") gene, of the neurotropic JHMV strain of MHV and found that loss of the I gene resulted in a post replication defect that reduced virion assembly and ultimately infectious virus production, while also increasing some inflammatory molecule expression. Thus, the JHMV I protein has roles in virion assembly that were previously underappreciated and in immunoevasion.

Keywords: coronavirus; encephalitis; innate immunity; mouse hepatitis virus; virion structure.

Fig 1

Loss of JHMV I protein results in attenuated replication. (A) Clustal omega sequence alignment of A59 (cyan) and JHMV I (magenta) proteins. Gray bar marks JHMV I termination codon that results in shortened protein. Black bars mark where L56* and L73* stop codons were introduced into rJHMV^IΔ57-137. Red and blue residues indicate sequence dissimilarity with red representing no similarity and blue representing conserved side chains. (B) AlphaFold predicted structure rank #1 of JHMV I (magenta) and A59 I (cyan). (C) 17Cl-1s or (D and E) BMDM were infected with rJHMV and rJHMV^IΔ57-137 at 0.01 multiplicity of infection (MOI). (C and D) Cell lysates and supernatants were assayed at indicated time points for infectious virus by plaque assay. (E) quantitative reverse transcriptase-PCR (qRT-PCR) for gRNA. (F) 17Cl-1s and (G and H) BMDMs were infected with rA59 and rA59 ^I-KO at 0.01 MOI. (F and G) Cell lysates and supernatants were plaque assayed at the indicated time points for infectious virus. (H) qRT-PCR for gRNA. The data in C–H are shown from one experiment representative of three independent experiments: n = 3 biological replicates. *P < 0.05 and **P < 0.01.

Fig 2

JHMV I has immunoevasive properties. BMDMs were infected at MOI of 1 with (A) rJHMV and rJHMV^IΔ57-137 or (B) A59 and A59 ^I-KO. Cell lysates were collected at 8 hpi for measurement by qRT-PCR of mRNA levels of immune mediators IFN-α, IFN-β, ISG15, CCL2, CXCL10, TNF, and IL-6. The data from one experiment representative of two independent experiments are shown: n = 6 biological replicates. *P < 0.05 and **P < 0.01.

Fig 3

Pathogenicity is attenuated in rJHMV^IΔ57-137-infected mice. (A and B) Five- to seven-week-old C57L/B6 mice were infected intranasally with 60,000 PFU/12 µL rJHMV or rJHMV^IΔ57-137. Animal weights, survivability, and clinical scores were evaluated. (B) Whole brain and spinal cord were harvested at indicated time points and homogenized, and infectious virus titers were measured by plaque assay. (C and D) Five- to seven-week-old C57L/B6 mice were infected with 30,000 PFU/30 µL of rA59 and rA59 ^I-KO intracranially. Animal weights and survival were evaluated. (D) Whole brain, spinal cord, and liver were harvested at 4 dpi and homogenized, and infectious virus titers were measured by plaque assay. (E) Five- to seven-week-old C57L/B6 mice were infected intrahepatically with 10,000 PFU/300 µL of rA59 and rA59 ^I-KO, and whole liver was harvested a 2 dpi and homogenized, and infectious virus was measured by plaque assay. (F) Five- to seven-week-old C57L/B6 mice were infected intrahepatically with 10,000 PFU/300 µL of rA59 and rA59 ^I-KO and weighed daily. (A and C) One representative experiment from three independent experiments is shown: n = 8–10 biological replicates. (B) Two independent experiments were combined n = 9. (D) One representative experiment of three independent experiments: n = 4–5. (E and F) Two experiments combined: n = 10. *P < 0.05.

Fig 4

Immune cell infiltration is delayed in rJHMV^IΔ57-137-infected mice. (A) Five- to seven-week-old C57L/B6 mice were infected intranasally with 60,000 PFU/12 µL of rJHMV or rJHMV^IΔ57-137, and brains were collected at indicated time points and processed for flow cytometry. Representative flow plots to show gating of CD45- (y-axis) vs CD11b- (x-axis) positive cells at 4 and 6 dpi. (B) Frequency and number of CD45+ CD11b− cells. (C) CD45^intCD11b+ microglia. (D) CD45+ CD11b+ cells. (E) CD45+ CD11b+ Ly6G− monocytes and macrophages. (F) CD45+ CD11b+ Ly6G+ neutrophils. (G) CD45+ CD11b− CD3+ T cells. One representative experiment from three independent experiments is shown: n = 4–5. *P < 0.05 and **P < 0.01.

Fig 5

Immune cell infiltration is modestly increased in A59^I-KO-infected mice. Five- to seven-week-old C57L/B6 mice were infected with 30,000 PFU/30 µL of rA59 and rA59^I-KO intracranially. Brains were collected at indicated time points and processed for flow cytometry. Frequency and count of CD45+ CD11b− cells, CD45 intermediate CD11b+ microglia, CD45+ CD11b+ cells, CD45+ CD11b+ Ly6G− monocytes and macrophages, and CD45+ CD11b+ Ly6G+ neutrophils. These data are obtained from two independent experiments: n = 4–5. *P < 0.05.

Fig 6

rJHMV^IΔ57-137 attenuation is partially reversed in IFNAR^−/− mice. (A) IFNAR^−/− BMDM were infected with rJHMV and rJHMV^IΔ57-137 at 0.01 MOI. Cell lysates and supernatants were prepared and analyzed at the indicated time points by plaque assay for infectious virus. (B and C) Five- to seven-week-old IFNAR^−/− mice were infected intranasally with 20,000 PFU/12 µL of rJHMV and rJHMV^IΔ57-137, and weight and survival were monitored. (C) Whole brains and spinal cords were harvested at 4 dpi and homogenized, and infectious virus was measured by plaque assay. (D) IFNAR^−/− BMDM were infected with A59 and A59 ^I-KO at 0.01 MOI. Cell lysates and supernatants were prepared and analyzed at the indicated time points for infectious virus. The data in A and D were obtained from one experiment representative of three independent experiments: n = 3 biological replicates. The data in B are two combined from independent experiments n = 7 and those in C are combined from two independent experiments n = 7–8. *P < 0.05 and **P < 0.01.

Fig 7

Generation and analysis of V5-tagged JHMV I and A59 I viruses. (A) Schematic of JHMV genome with 3′ end amplified to show insertion of the V5-tagged MHV I within ORF4. (B) 17Cl-1s were infected with 0.01 MOI of rJHMV, rJHMV^JHM-I, or rJHMV^A59-I. Cell lysates were collected at 12 hpi for western blot probing V5 tag and actin. (C) 17Cl-1s were infected with 0.01 MOI of rJHMV^JHM-I. V5 and actin were probed at 4, 8, 12, and 16 hpi. (D) 17Cl-1s were infected with 0.01 MOI of rJHMV, rJHMV^JHM-I, or rJHMV^A59-I. Cell lysates and supernatants were plaque assayed at the indicated time points for infectious virus. The data in B–D were obtained from one experiment representative of three independent experiments: n = 3 biological replicates.

Fig 8

MHV I colocalizes to Golgi. 17Cl-1s were infected with rJHMV^JHM-I (upper panels) or rJHMV^A59-I (lower panels) at 0.1 MOI and fixed at 8 hpi as described in Materials and Methods. Slides were stained for DAPI, V5 tag expression, and (A) Golgi, (B) ER, and (C) TOM70 and visualized by confocal microscopy. Each image is representative of 7–9 images, and Pearson coefficients were measured for 6–8 images with an average total of three infected cells per image.

Fig 9

Absence of JHMV I reduces virus assembly and RNA/PFU ratio. 17Cl-1s were infected with (A) rJHMV^JHM-I at 0.01 MOI, and cells and supernatant were harvested at 16 hpi. Virions were concentrated via centrifugation and fractionated. Fractions were examined by western blot for spike and V5 protein expression, as well as viral titer by plaque assay. (B) BMDMs were infected with rJHMV and rJHMV^IΔ57-137 at 0.01 MOI, and cells and supernatant were harvested at 12 hpi. Infectious virus and gRNA were measured by plaque assay and qRT-PCR, respectively. Ratio of RNA to plaque-forming unit (infectious virus) was measured. (C) Analysis of N and I binding. Infected cell lysates were collected at 12 hpi prepared and treated with anti-V5 antibody beads. Bound samples were eluted and analyzed for nucleocapsid and V5 expression. (D) Electron microscopy of BMDMs infected with (D) rJHMV and rJHMV^IΔ57-137 or (E) A59 and A59^I-KO at 0.01 MOI and harvested at 12 hpi. Larger panels at 25k resolution with a scale bar of 500 nm and smaller panels at 50k resolution with a scale bar of 200 nm. Examples of rJHMV virions are marked with magenta arrows. Blebbed Golgi cisternae seen only in rJHMV^IΔ57-137 marked with blue star. Golgi stacks containing virions marked with white asterisk. Collections of vesicles containing virions only found in rA59-infected samples marked with cyan asterisk. Number of virions per field quantified blindly. The data in A were obtained from one experiment representative of three independent experiments: n = 1 biological replicates. The data in B are representative of two independent experiments, n = 12, those in C are representative of one independent experiment, and those in D and E are representative of two biological replicates, and n = 10–12 images quantified.