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. 2007 Oct;81(19):10280-91.
doi: 10.1128/JVI.00017-07. Epub 2007 Jul 18.

Processing of open reading frame 1a replicase proteins nsp7 to nsp10 in murine hepatitis virus strain A59 replication

Damon J Deming  1 , Rachel L Graham, Mark R Denison, Ralph S Baric
Affiliations

Processing of open reading frame 1a replicase proteins nsp7 to nsp10 in murine hepatitis virus strain A59 replication

Damon J Deming et al. J Virol. 2007 Oct.

Abstract

Coronaviruses express open reading frame 1a (ORF1a) and ORF1b polyproteins from which 16 nonstructural proteins (nsp) are derived. The highly conserved region at the carboxy terminus of ORF1a is processed by the nsp5 proteinase (Mpro) into mature products, including nsp7, nsp8, nsp9, and nsp10, proteins with predicted or identified activities involved in RNA synthesis. Although continuous translation and proteolytic processing of ORF1ab by Mpro is required for replication, it is unknown whether specific cleavage events within the polyprotein are dispensable. We determined the requirement for the nsp7 to nsp10 proteins and their processing during murine hepatitis virus (MHV) replication. Through use of an MHV reverse genetics system, in-frame deletions of the coding sequences for nsp7 to nsp10, or ablation of their flanking Mpro cleavage sites, were made and the effects upon replication were determined. Viable viruses were characterized by analysis of Mpro processing, RNA transcription, and growth fitness. Deletion of any of the regions encoding nsp7 to nsp10 was lethal. Disruption of the cleavage sites was lethal with the exception of that of the nsp9-nsp10 site, which resulted in a mutant virus with attenuated replication. Passage of the attenuated nsp9-nsp10 cleavage mutant increased fitness to near-wild-type kinetics without reversion to a virus capable of processing nsp9-nsp10. We also confirmed the presence of a second cleavage site between nsp7 and nsp8. In order to determine whether a distinct function could be attributed to preprocessed forms of the polyprotein, including nsp7 to nsp10, the genes encoding nsp7 and nsp8 were rearranged. The mutant virus was not viable, suggesting that the uncleaved protein may be essential for replication or proteolytic processing.

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Figures

FIG. 1.
FIG. 1.
MHV genome organization, proteolytic processing of the replicase polyproteins, and putative cleavage sites of nsp7 to nsp10. (A) The 5′ two-thirds of the MHV genome encode the ORF1 replicase proteins pp1a and pp1ab. ORF2 to ORF7 encode the major structural proteins S, E, M, and N, along with several accessory proteins. (B) The replicase polyproteins are processed by three proteases to produce 16 mature proteins. PLP1 is responsible for cleaving between nsp1-nsp2 and nsp2-nsp3 (black arrows), while PLP2 cleaves between nsp3-nsp4 (open circle). Mpro processes the remainder of the polyproteins (open triangles). The replicase proteins include a number of functionally conserved domains, including the two PLP proteases, an ADP-ribose-1‴-monophosphate-processing enzyme (X), three hydrophobic transmembrane domains (TM, MP1, and MP2), the nsp5 protease (Mpro), RdRp, a putative zinc-binding domain (Z) and helicase (Hel), an exonuclease (ExoN), an endoribonuclease (EU), and an S-adenosylmethionine-dependent ribose 2′-O-methyltransferase (OMT). (C) The amino acid sequences of the Mpro cleavage sites falling within the nsp7 to nsp10 region of the replicase polyproteins are shown (open arrows denote points of cleavage) along with their P1 Gln amino acid positions. A second putative cleavage site falling between nsp7-nsp8 is identified with a question mark and a vertical dotted line illustrating its proposed cleavage site.
FIG. 2.
FIG. 2.
RT-PCR verification of the replication deficiency of non-syncytium-forming cleavage mutant viruses. RNA was extracted from cells over three passages of electroporated DBT cells and supernatants. PCR was completed with primers specific for leader-containing N gene transcripts and GAPDH as a control for RNA quality and successful RT reactions. RNA from cells infected with wild-type MHV was used as a positive control.
FIG. 3.
FIG. 3.
Replication kinetics of viable cleavage mutants and MHV9/10 revertants. (A) Comparison of growth curves for MHV (black circle and solid line), MHV7/8A (black square and solid line), MHV7/8B (black triangle and dashed line), and MHV9/10 (black circle and dashed line). (B) Replication fitness of revertant MHV9/10 passage 15 viruses. Growth curves compare MHV (black circle and solid line) to the passage 15 isolates MHVp15-1 (black square and solid line) and MHVp15-3 (black triangle and dashed line). Growth curves were performed on DBT cell monolayers infected at an MOI of 0.05 PFU/cell. Supernatants were sampled for replicating virus at 0, 6, 12, 18, and 24 hpi, and titers were determined by plaque assay. Data points represent the average of three replicate experiments, and error bars show the standard deviation. The limit of detection (lod) is represented by a horizontal dashed line at 1.7 log10 PFU/ml.
FIG. 4.
FIG. 4.
Characterization of the nsp9 genetic components of MHVp15-1 and MHVp15-3. Shown is a comparison of MHV (black circle and solid line), the attenuated parent virus MHV9/10 (black square and solid line), MHVQ4319T (black triangle and dotted line), MHVK4298R (black square and dotted line), and MHV9/10K4298R (black circle and dotted line). Growth curves were performed on DBT cell monolayers infected at an MOI of 0.05 PFU/cell. Supernatants were sampled for replicating virus at 0, 6, 12, 18, and 24 hpi, and titers were determined by plaque assay. Data points represent the average of three replicate experiments, and error bars show the standard deviation. The limit of detection (lod) is represented by a horizontal dashed line at 1.7 log10 PFU/ml.
FIG. 5.
FIG. 5.
ORF1a polyprotein processing in recombinant viruses. Cultures of cells were infected with MHV, MHV7/8A, MHV7/8B, MHV9/10, MHVp15-1, or MHVp15-3 for 4.5 h. The cultures were radiolabeled for 3 h, and antisera against nsp8 (22 kDa), nsp9 (12 kDa), or nsp10 (15 kDa) was used for immunoprecipitation. DBT cells were infected at an MOI of 1 PFU/cell and labeled with [35S]Met/Cys-containing medium from 6 to 9 hpi in the presence of actinomycin D. At approximately 9 hpi, cells were lysed and proteins were immunoprecipitated with polyclonal sera against nsp8, nsp9, or nsp10 and then resolved by SDS-PAGE and identified by fluorography. Bands corresponding to nsp8, nsp9, nsp10, and the fused nsp9-nsp10 (nsp9-10) are indicated. The protein corresponding to nsp8 isolated from MHV7/8B-infected cells (+) migrated slower than those of MHV or MHV7/8A.
FIG. 6.
FIG. 6.
RNA synthesis in recombinant and wild-type viruses. Cultures of cells were infected with wild-type MHV or recombinant viruses, and intracellular RNA was harvested from DBT cells at 12 hpi. RNA was separated on a 1% agarose gel, transferred to nylon filters, and hybridized with a biotinylated RNA probe specific for N protein mRNA. The filters were incubated with a chemiluminescent substrate and exposed on film.
FIG. 7.
FIG. 7.
Immunofluorescence of MHV in cells infected with cleavage mutant virus. Cells were infected with either MHV7/8A, MHV7/8B, or MHVp15-3, fixed and permeabilized with MeOH, and then dually stained with antibody specific for nsp8 (MHV7/8A and MHV7/8B) or nsp10 (MHVp15-3) and nucleocapsid or membrane protein. The green fluorescent anti-nsp image (A and D) was overlaid with the corresponding red fluorescent anti-N (B) or anti-M image (E) to determine points of colocalization (yellow) between the nsp and either N, representing localization with the replication complexes (C), or M, which is excluded from sites of replication (F).
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