Analysis of murine hepatitis virus strain A59 temperature-sensitive mutant TS-LA6 suggests that nsp10 plays a critical role in polyprotein processing

Abstract

Coronaviruses are the largest RNA viruses, and their genomes encode replication machinery capable of efficient replication of both positive- and negative-strand viral RNAs as well as enzymes capable of processing large viral polyproteins into putative replication intermediates and mature proteins. A model described recently by Sawicki et al. (S. G. Sawicki, D. L. Sawicki, D. Younker, Y. Meyer, V. Thiel, H. Stokes, and S. G. Siddell, PLoS Pathog. 1:e39, 2005), based upon complementation studies of known temperature-sensitive (TS) mutants of murine hepatitis virus (MHV) strain A59, proposes that an intermediate comprised of nsp4 to nsp10/11 ( approximately 150 kDa) is involved in negative-strand synthesis. Furthermore, the mature forms of nsp4 to nsp10 are thought to serve as cofactors with other replicase proteins to assemble a larger replication complex specifically formed to transcribe positive-strand RNAs. In this study, we introduced a single-amino-acid change (nsp10:Q65E) associated with the TS-LA6 phenotype into nsp10 of the infectious clone of MHV. Growth kinetic studies demonstrated that this mutation was sufficient to generate the TS phenotype at permissive and nonpermissive temperatures. Our results demonstrate that the TS mutant variant of nsp10 inhibits the main protease, 3CLpro, blocking its function completely at the nonpermissive temperature. These results implicate nsp10 as being a critical factor in the activation of 3CLpro function. We discuss how these findings challenge the current hypothesis that nsp4 to nsp10/11 functions as a single cistron in negative-strand RNA synthesis and analyze recent complementation data in light of these new findings.

FIG. 1.

Polyprotein processing of MHV-A59 and assembly of MHV and TS-LA6 infectious clones. (A) pp1a and pp1ab, encoded by ORF1a and ORF1ab, are processed by two virally encoded proteinases into 16 mature proteins. PLP1 and PLP2 cleave the first three nsp's, while 3CLpro cleaves nsp4 to nsp16. The p150 intermediate is comprised of nsp4 to nsp10/11. T1, transmembrane region 1; T2, transmembrane region 2; T3, transmembrane region 3; 3CL, 3CLpro; RdRp, RNA-dependent RNA polymerase; Hel, helicase; ExoN, exoribonuclease; XendoU, endonuclease; MT, methyltransferase. Black boxes indicate structural proteins that comprise roughly one-third of the genome, and the hatched box represents the nsp that contains the mutations of interest. (B) The MHV infectious clone is divided into seven fragments, which are assembled after unique restriction sites are used to digest the cDNA from plasmids, and appropriate sticky ends allow the fragments to ligate together in the proper order and orientation. The MHV-E fragment was mutated to generate the icTS-LA6 mutant.

FIG. 2.

Verification of viral replication and genotype. (A) Transfected cells were harvested in TRIzol reagents, and total RNA was extracted and reverse transcribed. The cDNA was amplified by PCR with primers designed to detect leader-containing cDNAs (N gene and leader). Bands correspond in size to subgenomic mRNAs for N, M, E, and S (lane 2). (B) Mutant viruses were plaque purified, and the nsp10 region was amplified by RT-PCR and cloned into TopoXL for sequencing. The sequence read of the PCR product shows that the mutation was present in the plaque-purified virus.

FIG. 3.

Growth characteristics of TS mutant icTS-LA6. (A) A plaque assay was performed to determine if icTS-LA6 produced plaques at the permissive and nonpermissive temperatures. wt-icMHV (hatched bars) grew at the permissive and nonpermissive temperatures, while icTS-LA6 (solid bars) produced plaques only at the permissive temperature. (B) Virus growth was evaluated at the permissive temperature for wt-icMHV (+) and icTS-LA6 (•). wt-icMHV was delayed in growth at 32°C from 9 to 14 h but recovered by 16 h. (C and D) A temperature shift experiment was performed using double cultures of cells infected at an MOI of 1 PFU/cell with the infection initiated at 32°C. At 6 h (indicated by arrow), half of the cultures were shifted to the nonpermissive temperature, supernatants were harvested at 2, 4, 6, 9, and 12 h p.i. from both cultures, and titers were determined by plaque assay. wt-icMHV grew equally well at both temperatures (C), while icTS-LA6 shows a TS phenotype, with growth declining rapidly after a shift to the nonpermissive temperature (D). ▪, infections initiated and maintained at 32°C; ▴, infections initiated at 32°C and shifted to 39.5°C at 6 h p.i.

FIG. 4.

Northern blot analysis of icTS-LA6. Cultures of cells were infected at an MOI of 1 PFU/cell, and intracellular RNA was isolated at 8 h p.i. The RNA was separated on 1% agarose gels, blotted onto a nitrocellulose membrane, and probed with an MHV N-gene-specific probe. icTS-LA6 and wt-icMHV grown at the permissive temperature of 32°C generate equivalent quantities of subgenomic RNA at 8 h p.i. Numbered bands correspond to mRNAs 1 to 7.

FIG. 5.

Real-time PCR analysis of icTS-LA6. Cells were infected in triplicate with wt-icMHV, icTS-LA6, and mock at an MOI of 1 PFU/cell and maintained at the permissive temperature of 32°C and the standard temperature of 37°C. Cells were harvested in TRIzol reagent at 8 h p.i., total RNA was isolated, and 5 μg of RNA was used for reverse transcription using random hexamer primers to generate cDNA. Viral cDNAs were normalized to the housekeeping gene GAPDH. (A) Dilutions were prepared to normalize the total cDNA of all infections to approximate levels using the housekeeping gene GAPDH. All four normalized samples amplified at similar cycle threshold values indicating equivalent starting template concentrations. (B) Subgenomic mRNAs were detected using primers to the leader and the first 122 nt of the N gene. Cells infected with wt-icMHV and maintained at 37°C generated the most subgenomic mRNAs, while cells infected with wt-icMHV and icTS-LA6 and maintained at the permissive temperature were reduced by ∼2.5 logs. icTS-LA6 infections initiated and maintained at 37°C generated extremely reduced concentrations of subgenomic mRNAs. (C) Genomic mRNAs were detected using primers to 122 nt of ORF1a. Cells infected with wt-icMHV and maintained at 37°C generated the most genomic mRNAs, while cells infected with wt-icMHV and icTS-LA6 and maintained at the permissive temperature were reduced by 2 to 2.5 logs. icTS-LA6 infections initiated and maintained at 37°C generated extremely reduced concentrations of genomic RNAs. (D) Comparisons of the reductions of subgenomic and genomic mRNAs for icTS-LA6 and wt-icMHV suggest that RNA synthesis is equivalent in both viruses at the permissive temperature. Black, subgenomic; white, genomic.

FIG. 6.

Processing of nsp10 and the structural proteins. Immunoprecipitations were conducted to evaluate the processing of nsp10 and the structural proteins S, M, and N. Cells were infected at an MOI of 2 PFU/cell with infections initiated and maintained at the permissive temperature. (A) Anti-nsp10 antibody was used to immunoprecipitate proteins from the cytoplasmic fraction of cells. nsp10 was detected and processed at the permissive temperature, as was the p150 intermediate. (B) Anti-MHV antibody was used to detect structural proteins in wt-icMHV grown at 37°C and compared to icTS-LA6 and TS-LA6 grown at 32°C.

FIG. 7.

Temperature shift immunoprecipitations of icTS-LA6. Double cultures of DBT cells were infected with mock (M) (lane 1), wt-icMHV (lanes 2 and 5), icTS-LA6 (lanes 3 and 6), and TS-LA6 (lanes 4 and 7) at an MOI of 10 PFU/cell at the permissive temperature of 32°C for an hour, medium was then removed and replaced with medium lacking Met-Cys but with actinomycin D, and infections were incubated at the permissive temperature for an additional 3 h. At 4 h p.i., labeled medium was added to the infected cells, and half the cultures were maintained at the permissive temperature (P), while the other half were shifted to the nonpermissive temperature of 39.5°C (NP). Immunoprecipitations were performed on the cytoplasmic lysates using antibodies indicated above each figure. wt-icMHV infections initiated and maintained at 37°C were performed as a control (C). (A) Immunoprecipitation analysis was performed using anti-nsp10 antibody (α-nsp10). At the permissive temperature of 32°C, nsp10 is processed in wt-icMHV, icTS-LA6, and TS-LA6 (lanes 2 to 4). For infections initiated at 32°C and then shifted to the nonpermissive temperature of 39.5°C, processing of nsp10 was ablated in icTS-LA6 and TS-LA6, while p150 appeared to accumulate (lanes 6 and 7). wt-icMHV continued to efficiently process this protein at 37°C and 39.5°C (lanes 5 and 8). (B) Anti-nsp8 antibody was used to pull down viral proteins that were processed at the permissive and nonpermissive temperatures. At 32°C, nsp8 was processed in wt-icMHV, icTS-LA6, and TS-LA6 (lanes 2 to 4). For infections initiated at 32°C and then shifted to 39.5°C, nsp8 was ablated in icTS-LA6 and TS-LA6, and p150 appeared to accumulate (lanes 6 and 7). wt-icMHV continued to process this protein at 37°C and 39.5°C (lanes 5 and 8). (C) Immunoprecipitation analysis conducted with anti-nsp5 antibody. At 32°C, the results show that nsp5 was processed in wt-icMHV, icTS-LA6, and TS-LA6 (lanes 2 to 4). After a shift to 39.5°C, processing of nsp5 was not seen in icTS-LA6 and TS-LA6, (lanes 6 and 7). wt-icMHV processing of nsp5 was observed at 37°C and 39.5°C (lanes 5 and 8). (D) Immunoprecipitation with anti-nsp12 antibody showed that nsp12 was processed in wt-icMHV, icTS-LA6, and TS-LA6 at the permissive temperature (lanes 2 to 4). After a shift to the nonpermissive temperature, processing of nsp12 was ablated in icTS-LA6 and TS-LA6 (lanes 6 and 7), while wt-icMHV continued to efficiently process this protein at 37°C and 39.5°C (lanes 5 and 8). (E) Anti-nsp2 antibody was used to pull down viral proteins that were processed at the permissive and nonpermissive temperatures by PLP1 and PLP2. (C) At 32°C, the results show that nsp2 is processed in wt-icMHV as well as mutants icTS-LA6 and TS-LA6. (D) For infections initiated at 32°C and then shifted to 39.5°C, the results show that the processing of nsp2 continued as observed at 32°C, with wt-icMHV showing a greater increase in processed nsp2.

FIG. 8.

Location of the TS mutation on the nsp10 structure. A homology model of the MHV-A59 nsp10 protein was generated using the coordinates of the X-ray crystal structure of SARS-CoV (PDB accession number 2FYG) using the program Modeler. The mutants icTS-LA6 (nsp10:Q65E) and nsp10-E2 (nsp10:D47A,H48A) were mapped onto the structure. Both mutations occur within a predicted disordered region, proximal to zinc-binding finger 1, and are exposed to the surface.

FIG. 9.

Complementation with TS-LA6 revisited. Sawicki et al. established that crosses between TS mutants within nsp4, nsp5, and nsp10 failed to complement each other as shown by the cis-trans test and biochemical complementation analysis. This was the basis for including them in complementation group I and suggests that nsp4 to nsp10 act as a single cistron. (A) The TS mutation in nsp10 effectively renders the entire pp1ab polyprotein unprocessed, and therefore, it cannot complement any other TS mutant from complementation group 1. (B) Our results suggest that TS-LA6 cannot complement any virus bearing a TS mutation in pp1ab. However, limited proteolysis may occur in trans to process proteins in pp1b. Complementation between TS-LA6 and TS-ALB22 suggests that 3CLpro of TS-ALB22 cleaves nsp12 of TS-LA6 in trans to rescue virus replication. The dotted line represents trans cleavage of nsp12. Hatched boxes show proteins potentially cleaved in trans. ⧫, 3CLpro cleavage site.