J Bacteriol Virol.  2011 Jun;41(2):117-122. 10.4167/jbv.2011.41.2.117.

Investigation of Murine Norovirus Replication in RAW264.7 Cells by Strand-specific RT-PCR

• Ji GY
• Jang SY
• Paik SY
• Ko GP
• Jeong WH
• Lee CH

• KMID: 2168629
• DOI: http://doi.org/10.4167/jbv.2011.41.2.117

Abstract

Murine norovirus (MNV) is a non-enveloped virus with a positive-sense RNA genome and causes lethal infection in mice. MNV has been used as a model virus for human norovirus (NV) whose in vitro cell culture system has not been available to date since MNV and NV are genetically related. In this study, the genome replication of MNV was investigated using strand-specific RT-PCR in RAW264.7 cells. Reverse transcription (RT) using a sense primer followed by PCR showed that negative-sense RNAs were first detected in RAW264.7 cells between 6 and 9 [3 and 6] hours post infection (h.p.i.). However, these negative-sense RNAs were not detected when cells were treated with a translation inhibitor cycloheximide. Then, RT with an antisense primer followed by PCR was performed to detect positive-sense RNAs. RT-PCR results revealed that the amount of positive-sense RNAs began to increase from 9 [6] h.p.i., indicating the accumulation of the newly synthesized (+)RNA genome. Furthermore, cycloheximide abrogated the increase of newly made RNAs during MNV infection. In conclusion, strand-specific RT-PCR using a sense or antisense primer, in combination with cycloheximide treatment, enabled us to detect positive-sense and negative-sense RNAs selectively and provided a useful tool to understand the replication cycle of MNV.

Keyword

Murine norovirus; Strand-specific RT-PCR; RNA synthesis

MeSH Terms

Animals
Cell Culture Techniques
Cycloheximide
Genome
Humans
Mice
Norovirus
Polymerase Chain Reaction
Reverse Transcription
RNA
Viruses
Cycloheximide
RNA

Figure

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  Figure 1. Detection of murine norovirus RNA in RAW264.7 cells by strand-specific RT-PCR. RAW264.7 cells were infected with murine norovirus at MOI of 1. RNAs were extracted at 3 hrs time interval and cDNAs were synthesized using either sense primer or antisense primer. Resulting cDNAs were subjected to PCR using both sense and antisense primers. M, Mock-infected. NC: negative control without primer.

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  Figure 2. Removal of free (–)RNA from murine norovirus stock. Virus stock was pre-treated with RNase at a concentration of 500 μg/ml or 7% polyethylene glycol (PEG) overnight. Virus stock treated with RNase or PEG was used for strand-specific RT-PCR. STO, virus stock. –, negative control.

• 

  Figure 3. Synthesis of murine norovirus (–)RNA and (+)RNA in RAW264.7 cells. Cells were infected with murine norovirus in the presence of absence of CHX (10 μM). Cycloheximide was present in the culture medium prior [before], during and after virus infection. At the indicated times after virus infection, RNAs were extracted and RT reactions were performed using only sense primer for detection of (–)RNAs or antisense primer for detection of (+)RNAs. (A) In the absence of CHX. (B) In the presence of CHX. (C) The intensities of the gel bands of the PCR products were converted to numerical forms using Scion Image program and plotted against time after virus infection. •, (–)RNA in the absence of CHX; ○, (+)RNA in the absence of CHX; ▾, (–)RNA in the presence of CHX; ▽, (+)RNA in the presence of CHX.

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  Figure 4. Semi-quantitative RT-PCR of murine norovirus. RAW264.7 cells were infected with murine norovirus. RNA was extracted at 3, 6, 9 h.p.i. and RT reaction was performed using antisense primer only. The resulting cDNAs were serially diluted 1:10 and subjected to PCR using both sense and antisense primers. Cycloheximide treated sample was included at 9 h.p.i.

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  Figure 5. Detection of murine norovirus in the extracellular media. Cells were infected with murine norovirus and the extracellular media were harvested at the indicated times after virus infection. RNAs were extracted from the extracellular media and subjected to semi-quantitative RT-PCR by 10-fold serial dilution.

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