title: Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication.
abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus replicating in the cytoplasm, but the nucleocapsid (N) protein is specifically localized to the nucleus and nucleolus in virus-infected cells. A 'pat7' motif of 41-PGKK(N/S)KK has previously been identified in the N protein as the functional nuclear localization signal (NLS); however, the biological consequences of N protein nuclear localization are unknown. In the present study, the role of N protein nuclear localization during infection was investigated in pigs using an NLS-null mutant virus. When two lysines at 43 and 44 at the NLS locus were substituted to glycines, the modified NLS with 41-PGGGNKK restricted the N protein to the cytoplasm. This NLS-null mutation was introduced into a full-length infectious cDNA clone of PRRSV. Upon transfection of cells, the NLS-null full-length clone induced cytopathic effects and produced infectious progeny. The NLS-null virus grew to a titer 100-fold lower than that of wild-type virus. To examine the response to NLS-null PRRSV in the natural host, three groups of pigs, consisting of seven animals per group, were intranasally inoculated with wild-type, placebo, or NLS-null virus, and the animals were maintained for 4 weeks. The NLS-null-infected pigs had a significantly shorter mean duration of viremia than wild-type-infected pigs but developed significantly higher titers of neutralizing antibodies. Mutations occurred at the NLS locus in one pig during viremia, and four types of mutations were identified: 41-PGRGNKK, 41-PGGRNKK, and 41-PGRRNKK, and 41-PGKKSKK. Both wild-type and NLS-null viruses persisted in the tonsils for at least 4 weeks, and the NLS-null virus persisting in the tonsils was found to be mutated to either 41-PGRGNKK or 41-PGGRNKK in all pigs. No other mutation was found in the N gene. All types of reversions which occurred during viremia and persistence were able to translocate the mutated N proteins to the nucleus, indicating a strong selection pressure for reversion at the NLS locus of the N protein in vivo. Reversions from NLS-null to functional NLS in the tonsils suggest a possible correlation of viral persistence with N protein nuclear localization. These results show that N protein nuclear localization is non-essential for PRRSV multiplication but may play an important role in viral attenuation and in pathogenesis in vivo.
authors.affiliation: Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
authors.forename: Changhee
authors.initials: C
authors.lastname: Lee
authors.forename: Douglas
authors.initials: D
authors.lastname: Hodgins
authors.forename: Jay G
authors.initials: JG
authors.lastname: Calvert
authors.forename: Siao-Kun W
authors.initials: SK
authors.lastname: Welch
authors.forename: Rika
authors.initials: R
authors.lastname: Jolie
authors.forename: Dongwan
authors.initials: D
authors.lastname: Yoo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D008969:Molecular Sequence Data; D009154:Mutation; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics*; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics*; D014066:Palatine Tonsil / Q000821:virology; D019318:Porcine Reproductive and Respiratory Syndrome / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology; D017422:Sequence Analysis, DNA; D013552:Swine / Q000821:virology; D014766:Viremia / Q000821:virology; D014779:Virus Replication / Q000502:physiology*
chemical_list: D019913:Nuclear Localization Signals; D019590:Nucleocapsid Proteins