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. 2013 Jan 20;435(2):210-3.
doi: 10.1016/j.virol.2012.10.001. Epub 2012 Oct 23.

Murine hepatitis virus nsp4 N258T mutants are not temperature-sensitive

Dia C Beachboard  1 , Xiaotao Lu, Susan C Baker, Mark R Denison
Affiliations

Murine hepatitis virus nsp4 N258T mutants are not temperature-sensitive

Dia C Beachboard et al. Virology. .

Abstract

Coronavirus replicase nsp4 is critical for virus-induced membrane modifications. An nsp4 mutant (N258T) of murine hepatitis virus (MHV) has been reported to be temperature-sensitive (ts) and to alter membrane targeting. We engineered and recovered all four possible codon variants of N258T in the cloned MHV-A59 background. All mutant viruses demonstrated impaired replication compared to wildtype MHV, but no nsp4 N258T mutant virus was ts, and all variants colocalized with viral protein markers for replication complexes, but not with markers for mitochondria. This study emphasizes that complete genome sequencing may be necessary, even with directed and confirmed reverse genetic mutants.

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Figures

Fig. 1
Fig. 1
Analysis of nsp4 N258T codon variant mutants of MHV. (A) Proposed topology of MHV nsp4: nsp4 has 4 membrane-spanning regions (TM1–4, black rectangles) and three loop regions (loop 1–3). Previously reported mutations in loop 1 are indicated as the gray double-headed arrows (glycosylation sites) and a gray dot (E226A/E227A) (Gadlage et al., 2010, Sparks et al., 2007). The N258T (AAT to ACX at nt positions 9493–9495) substitution is shown as a black dot. (B) Titers were determined by plaque assay in DBT cells at 30 °C and 40 °C. EOP was calculated as the titer at 40 °C divided by the titer at 30 °C. Titers represent the average titer of two independent experiments. 1 Codon variant previously reported by Clementz et al. (2008), 2 Codon variant previously reported by Sawicki et al. (2005). (C) DBT cells were infected at an MOI of 0.1 PFU/cell with the indicated viruses and incubated at 30 °C for 28 h and titers were determined by plaque assay. Error bars represent the standard error of the mean of two independent plaque assays done in duplicate. (D) DBT cells were infected at an MOI of 0.1 PFU/cell with the indicated viruses and incubated at 30 °C with a temperature shift to 40  °C at 6 h p.i. and titers were determined by plaque assay. Error bars represent the standard error of the mean of two independent plaque assays done in duplicate.
Fig. 2
Fig. 2
(A) Nsp4 N258TACA codon variant localizes to the replication complex. DBT cells were infected at an MOI of 5 PFU/cell for 16 h at 30 °C or 7 h at 40 °C. Cells were fixed in methanol, probed for nsp4 (red) and nsp8 (green) or PDH (green) and imaged on a Zeiss LSM510 confocal microscope. Yellow pixels represent colocalization of overlapping red and green pixels. The scale bar in the bottom right corner of merged images represents 10 μm. (B) Pearson's correlation coefficient was calculated for nsp4-nsp8 or nsp4-PDH for both WT and N258TACA at 30 °C and 40 °C (n=5). Error bars represent standard deviation.
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