The localization of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence

The nucleocapsid (N) protein of infectious bronchitis virus (IBV) localizes to the cytoplasm and nucleolus and contains an eight-amino-acid nucleolar retention motif. In this study, a leucine-rich nuclear export signal (NES) (291-LQLDGLHL-298) present in the C-terminal region of the IBV N protein was analyzed by using alanine substitution and deletion mutagenesis to investigate the relative contributions that leucine residues make to nuclear export and where these residues are located on the structure of the IBV N protein. The analysis indicated that Leu296 and Leu298 are required for efficient nuclear export of the protein. Structural information indicated that both of these amino acids are available for interaction with protein complexes involved in this process. However, export of N protein from the nucleus/nucleolus was not inhibited by leptomycin B treatment, indicating that N protein nuclear export is independent of the CRM1-mediated export pathway.

mentioning

confidence: 99%

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

Reed

Howell

Harrison

et al. 2007

“…However, Rowland and coworkers (24) reported the accumulation of N in the nuclei and nucleoli of virus-infected cells, as well as in cells transfected with constructs that expressed the N protein tagged with the green fluorescent protein. Recently, others have reported the nucleolar localization of the N proteins of equine arteritis virus, which is another arterivirus, and coronaviruses (15,29,39).…”

mentioning

confidence: 99%

“…The presence of N in the nucleolus is a unique observation, since the entire life cycle of PRRSV and other arteriviruses is considered to be restricted to the cytoplasm. Two conserved stretches of basic amino acids have been identified in the N protein at positions 10 to 13 and 41 to 47, and these sequences resemble two classical types of nuclear localization signal (NLS), Јpat4Ј and Јpat7Ј, respectively (24). Subsequently, the Јpat7Ј NLS was shown to be functional and sufficient for translocation of N to the nucleolus.…”

mentioning

confidence: 99%

Colocalization and Interaction of the Porcine Arterivirus Nucleocapsid Protein with the Small Nucleolar RNA-Associated Protein Fibrillarin

Yoo

Wootton

et al. 2003

Porcine reproductive and respiratory syndrome virus (PRRSV) replicates in the cytoplasm of infected cells, but its nucleocapsid (N) protein localizes specifically to the nucleus and nucleolus. The mechanism of nuclear translocation and whether N associates with particular nucleolar components are unknown. In the present study, we show by confocal microscopy that the PRRSV N protein colocalizes with the small nucleolar RNA (snoRNA)-associated protein fibrillarin. Direct and specific interaction of N with fibrillarin was demonstrated in vivo by the mammalian two-hybrid assay in cells cotransfected with the N and fibrillarin genes and in vitro by the glutathione S-transferase pull-down assay using the expressed fibrillarin protein. Using a series of deletion mutants, the interactive domain of N with fibrillarin was mapped to a region of amino acids 30 to 37. For fibrillarin, the first 80 amino acids, which contain the glycine-arginine-rich region (the GAR domain), was determined to be the domain interactive with N. The N protein was able to bind to the full-length genomic RNA of PRRSV, and the RNA binding domain was identified as the region overlapping with the nuclear localization signal situated at positions 41 to 47. These results suggest that the N protein nuclear transport may be controlled by the binding of RNA to N. The PRRSV N protein was also able to bind to both 28S and 18S ribosomal RNAs. The protein-protein interaction between N and fibrillarin was RNA dependent but independent of N protein phosphorylation. Taken together, our studies demonstrate a specific interaction of the PRRSV nucleocapsid protein with the host cell protein fibrillarin in the nucleolus, and they imply a potential linkage of viral strategies for the modulation of host cell functions, possibly through rRNA precursor processing and ribosome biogenesis.

“…8b, f, j and n, which can be explained by the fact that the N-terminally truncated N proteins lack the nucleolar location signal (NoLS) sequences (42,43). The presence of fluorescence in the nucleoli in Fig.…”

Section: Type I N Protein Is Functional In the Type Ii Prrsv Backgroundmentioning

confidence: 91%

“…The N protein is a basic protein and has been shown to be a serinephosphoprotein (58). It has been detected not only in the cytoplasm but also in the nucleus and nucleolus, and a nuclear localization signal has been identified (42,43). The N-terminal half of the N protein is presumed to be mostly disordered and involved in RNA binding (12,62), while the C terminus is believed to be the conformation-determining region of the protein (37,55,57).…”

Section: P Orcine Reproductive and Respiratory Syndrome Virus (Prrsv)mentioning

confidence: 99%

Disulfide Linkages Mediating Nucleocapsid Protein Dimerization Are Not Required for Porcine Arterivirus Infectivity

Zhang

Chen

Sun

et al. 2012

The nucleocapsid (N) proteins of the North American (type II) and European (type I) genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) share only approximately 60% genetic identity, and the functionality of N in both genotypes, especially its role in virion assembly, is still poorly understood. In this study, we demonstrated that the ORF7 3= untranslated region or ORF7 of type I is functional in the type II PRRSV background. Based on these results, we postulated that the cysteine at position 90 (Cys90) of the type II N protein, which corresponds to an alanine in the type I protein, is nonessential for virus infectivity. The replacement of Cys90 with alanine confirmed this hypothesis. We then hypothesized that all of the cysteines in the N protein could be replaced by alanines. Mutational analysis revealed that, in contradiction to previously reported findings, the replacement of all of the cysteines, either singly or in combination, did not impair the growth of either type II or type I PRRSV. Treatment with the alkylating agent N-ethylmaleimide inhibited cysteine-mediated N dimerization in living cells but not in released virions. Additionally, bimolecular fluorescence complementation assays revealed noncovalent interactions in living cells among the N and C termini and between the N-terminal and C-terminal regions of the N proteins of both genotypes of PRRSV. These results demonstrate that the disulfide linkages mediating the N dimerization are not required for PRRSV viability and help to promote our understanding of the mechanism underlying arterivirus particle assembly. P orcine reproductive and respiratory syndrome virus (PRRSV)is an enveloped, single-stranded, positive-sense RNA virus belonging to the family Arteriviridae in the order Nidovirales, which also includes the families Coronaviridae and Roniviridae (7,8,16,47). Other members of the family Arteriviridae include equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV) (47). The PRRSV genome is about 15 kb in length, and the coding region is flanked by untranslated regions (UTRs) at each end (the 5= and 3=UTR, respectively) (36). The genome contains at least 10 open reading frames (ORFs), of which ORF1a and ORF1b encode the two long nonstructural polyproteins pp1a and pp1ab and ORF2 to ORF7 encode at least eight structural proteins, glycoprotein 2 (GP2), small envelope (E), GP3, GP4, GP5, ORF5a, membrane (M), and nucleocapsid (N) (13,25,35,36,47,56,61). The structural proteins are expressed from a set of coterminal subgenomic mRNAs (sgmRNAs), which are synthesized by a unique discontinuous transcription strategy (40). The transcription-regulating sequence (TRS) of the leader (5=UTR) and the downstream body TRS (TRS-B) preceding the ORF coding region of each structural protein in the viral genome are believed to play key roles in mediating this process.Based on genetic and antigenic differences, PRRSV strains are classified into two distinct genotypes, North American (t...