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. 2008 May 25;375(1):118-29.
doi: 10.1016/j.virol.2008.01.018. Epub 2008 Mar 4.

Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles

Mark A Clementz  1 , Amornrat Kanjanahaluethai, Timothy E O'Brien, Susan C Baker
Affiliations

Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles

Mark A Clementz et al. Virology. .

Abstract

Coronaviruses are positive-strand RNA viruses that replicate in the cytoplasm of infected cells by generating a membrane-associated replicase complex. The replicase complex assembles on double membrane vesicles (DMVs). Here, we studied the role of a putative replicase anchor, nonstructural protein 4 (nsp4), in the assembly of murine coronavirus DMVs. We used reverse genetics to generate infectious clone viruses (icv) with an alanine substitution at nsp4 glycosylation site N176 or N237, or an asparagine to threonine substitution (nsp4-N258T), which is proposed to confer a temperature sensitive phenotype. We found that nsp4-N237A is lethal and nsp4-N258T generated a virus (designated Alb ts6 icv) that is temperature sensitive for viral replication. Analysis of Alb ts6 icv-infected cells revealed that there was a dramatic reduction in DMVs and that both nsp4 and nsp3 partially localized to mitochondria when cells were incubated at the non-permissive temperature. These results reveal a critical role of nsp4 in directing coronavirus DMV assembly.

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Figures

Fig. 1
Fig. 1
MHV replicase highlighting predicted nsp4 topology and analysis of N-linked glycosylation. (A) The first two-thirds of the MHV genome (ORF 1a and ORF 1b) encode the viral replicase proteins. The nonstructural proteins (nsps) are synthesized as polyproteins processed into precursors then 16 mature replicase products. (B) Analysis of p150 and nsp4 after tunicamycin and endo H treatments. HeLa-MHVR cells were infected with MHV-JHM, untreated (U) or treated with 1 µg/ml of tunicamycin (T) for 1 h prior to and during labeling. Endo H treatment (E) was performed after immunoprecipitation with α-nsp4. (C) HeLa-MHVR cells were infected with vTF7.3, and co-transfected with plasmids encoding PLP2 and substrate encoding either WT or mutant nsp4. Proteins were radiolabeled (35S-trans-label) and immunoprecipitated with α-nsp4 antibodies. IP products were untreated or treated with endo H, separated on 5–10% gradient SDS-PAGE gel, and visualized by autoradiography. (D) Predicted topology of nsp4 indicating the location of two glycosylation sites and a ts lesion within the luminal loop.
Fig. 2
Fig. 2
Sequence analysis of mutant infectious clone virus. DBT cells were infected with WT-A59 icv, nsp4-N176A icv, or Alb ts6 icv and at 12 h.p.i. RNA was isolated. RT-PCR was performed on viral RNA using primers listed in Table 1, and PCR products were sequenced across the nsp4 region.
Fig. 3
Fig. 3
Growth characteristics of infectious clone viruses. (A) Titer of WT-A59 icv, Alb ts6 icv, and nsp4-N176A icv following infection at the permissive or nonpermissive temperature for 15 hours, when supernatant was harvested and virus titrated at 33 °C. Plaques were counted 48 h.p.i. Black bars, WT-A59 icv; white bars, Alb ts6 icv; gray bars, nsp4-N176A icv. Viral titers were performed in triplicate; error bars indicate standard deviation from the mean. (B) One-step growth curve of WT-A59 icv, Alb ts6 icv, and nsp4-N176A icv at the permissive temperature of 33 °C. Supernatant was collected at the indicated time points and viral titer was determined by plaque assay.
Fig. 4
Fig. 4
Temperature shift assay on infectious clone virus. Two sets of DBT cells were infected with WT-A59 icv, Alb ts6 icv, or nsp4-N176A icv at an MOI of 0.1 and incubated at 33 °C. At 6 h.p.i., one set of infected cells was shifted to 39.5 °C. Supernatant was harvested at two hour intervals and virus production was measured by plaque assay. Arrow indicates time of temperature shift.
Fig. 5
Fig. 5
Proteolytic processing of infectious clone virus. DBT cells were infected with WT-A59 icv, Alb ts6 icv and nsp4-N176A icv, at 4 h.p.i. radiolabeled with 35S-trans-label for 2 h, and at 6 h.p.i. cell lysates were prepared and subjected to immunoprecipitation with (A) α-nsp4, (B) α-nsp5, (C) α-nsp8, and (D) α-nsp9/10. All replicase antibodies also detect the p150 precursor.
Fig. 6
Fig. 6
TEM analysis of DMV formation by the WT-A59 icv and Alb ts6 icv at the permissive and non-permissive temperatures. Two sets of DBT cells were infected with WT-A59 icv or Alb ts6 icv at an MOI of 1.0 and incubated at 33 °C (A and C). At 3.5 h.p.i., one set of infected cells (B and D) was shifted to 39.5 °C. At 5.5 h.p.i., cells were harvested and processed for TEM analysis. DMVs can be visualized by TEM as darkly ringed vesicles in the cytoplasm of MHV-infected cells indicated by the arrows. Asterisks denote mitochondria. Scale bar equals 1 μm.
Fig. 7
Fig. 7
Localization of MHV replicase proteins and a mitochondrial marker in DBT cells infected with WT-A59 icv and Alb ts6 icv. Two sets of DBT cells were infected with WT-A59 icv or Alb ts6 icv at an MOI of 1.0 and incubated at 33 °C. At 3.5 h.p.i., one set of infected cells was shifted to 39.5 °C. At 5 h.p.i. cells were labeled with MitoTracker Red fluorescent dye. At 5.5 h.p.i., cells were harvested, fixed, and permeabilized for immunofluorescence assays. Permeabilized cells were then incubated with antibodies to either nsp4 (A) or nsp3 (B). Scale bar equals 10 μm.
Fig. 8
Fig. 8
MHV replicase protein localization in icv-infected HeLa-MHVR cells using antibodies to the mitochondrial protein pyruvate dehydrogenase (PDH). Two sets of HELA-MHVR cells were infected with WT-A59 icv or Alb ts6 icv at an MOI of 1.0 and incubated at 33 °C. At 3.5 h.p.i., one set of infected cells was shifted to 39.5 °C. At 5.5 h.p.i., cells were harvested, fixed, and permeabilized for immunofluorescence assays. Permeabilized cells were then incubated with antibodies to PDH and either nsp4 (A) or nsp3 (B). Scale bar equals 10 μm.
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