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.

Multiple regions in dengue virus capsid protein contribute to nuclear localization during virus infection

“…Mutants of JEV and porcine reproductive and respiratory syndrome virus, whose C proteins lack nuclearlocalization ability, did not replicate to high titres, and reversions were associated with enhanced replication (Mori et al, 2005;Lee et al, 2006). In this study, the four dengue virus mutants did not show marked variations in replication in PS cells, despite a lack of C protein nuclear localization in c(K73A,K74A) and c(R85A,K86A).…”

Characterization of the Nuclear Export Signal in the Coronavirus Infectious Bronchitis Virus Nucleocapsid Protein

“…Similar to that proposed for the nuclear/nucleolar trafficking signal of nidovirus N proteins (20,30), and for the export of the influenza A virus NS2 protein (16), mutation of the NES in the IBV N protein and subsequent disruption of cellular trafficking may be an attractive target for the generation of attenuated recombinant vaccines.…”

“…Likewise, several different N proteins of arterivirus (which, together with the coronaviruses, forms part of the nidoviruses) have been shown to localize to the nucleus/nucleolus (32,39). Mutagenesis studies of the arterivirus porcine reproductive and respiratory syndrome virus N protein NLS revealed that nuclear localization of the protein is a fundamental part of the virus life cycle (20).…”

Pathogenicity and genetic characteristics associated with cell adaptation of a virulent porcine reproductive and respiratory syndrome virus nsp2 DEL strain CA-2

“…The culture supernatant was collected at different time points (6, 12, 24, 36, and 48 h postinfection) and stored at À80 C. MARC-145 cells grown in 6-well plates were inoculated with 100 ml of serially 10-fold diluted virus suspensions per well and incubated for 4-5 days at 37 C. The titer of PRRSV was measured by plaque assay using MARC-145 cells in duplicate as described previously (Lee et al, 2006), and then defined as plaque forming units (PFU) per ml. The plaque morphology was assessed by staining with 1% crystal violet in ethanol at 96 h post-infection (hpi).…”