lunes, 15 de septiembre de 2014

Plasmid-based human norovirus reverse genetics system produces reporter-tagged progeny virus containing infectious genomic RNA

Plasmid-based human norovirus reverse genetics system produces reporter-tagged progeny virus containing infectious genomic RNA



Plasmid-based human norovirus reverse genetics system produces reporter-tagged progeny virus containing infectious genomic RNA

  1. Mary K. Estesa,d,1
  1. Contributed by Mary K. Estes, August 7, 2014 (sent for review April 27, 2014: reviewed by Ian Goodfellow and John Parker)

Significance

Human noroviruses are the predominant cause of acute gastroenteritis worldwide, but they remain noncultivatable. A tractable system is needed to understand the host restriction to cultivation. We established a reverse genetics system driven by a mammalian elongation factor-1α promoter without helper virus. This system supports genome replication, particle formation, and particles containing a GFP-marked genomic RNA. RNA from these particles is infectious. The system also produces infectious murine norovirus, confirming its broad applicability to other noroviruses.

Abstract

Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. HuNoV replication studies have been hampered by the inability to grow the virus in cultured cells. The HuNoV genome is a positive-sense single-stranded RNA (ssRNA) molecule with three open reading frames (ORFs). We established a reverse genetics system driven by a mammalian promoter that functions without helper virus. The complete genome of the HuNoV genogroup II.3 U201 strain was cloned downstream of an elongation factor-1α (EF-1α) mammalian promoter. Cells transfected with plasmid containing the full-length genome (pHuNoVU201F) expressed the ORF1 polyprotein, which was cleaved by the viral protease to produce the mature nonstructural viral proteins, and the capsid proteins. Progeny virus produced from the transfected cells contained the complete NoV genomic RNA (VP1, VP2, and VPg) and exhibited the same density in isopycnic cesium chloride gradients as native infectious NoV particles from a patient’s stool. This system also was applied to drive murine NoV RNA replication and produced infectious progeny virions. A GFP reporter construct containing the GFP gene in ORF1 produced complete virions that contain VPg-linked RNA. RNA from virions containing the encapsidated GFP-genomic RNA was successfully transfected back into cells producing fluorescent puncta, indicating that the encapsidated RNA is replication-competent. The EF-1α mammalian promoter expression system provides the first reverse genetics system, to our knowledge, generalizable for human and animal NoVs that does not require a helper virus. Establishing a complete reverse genetics system expressed from cDNA for HuNoVs now allows the manipulation of the viral genome and production of reporter virions.

Footnotes

  • Author contributions: K.K., K.M., T.M.S., S.G., T.O., R.T.-T., A.N., S.E.C., R.L.A., and M.K.E. designed research; K.K. performed research; K.K., K.M., T.M.S., S.G., T.O., R.T.-T., A.N., S.E.C., R.L.A., and M.K.E. analyzed data; and K.K., K.M., S.E.C., R.L.A., and M.K.E. wrote the paper.
  • Reviewers: I.G., University of Cambridge; and J.P., Cornell University.
  • Conflict of interest statement: R.L.A. and M.K.E. have received research support and served as consultants to Takeda Vaccines, Inc.
  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1415096111/-/DCSupplemental.

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