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Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) is a small enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. It causes the porcine reproductive and respiratory syndrome in swine. The virus has 7 structural proteins: Of the seven, the N protein is the nucleocapsid that comprises a core of the virus particle. We have expressed the N protein of PRRSV PL97–1/LP1 strain using a heterologous gene expression vector derived from Sindbis virus, called pSinrep5. Immunofluorescence analysis showed that the N proteins were mainly found in the cytoplasm as well as in the nucleus of BHK-21 cells transfected with pSinrep5-N-derived RNA. Moreover, expression of the N protein did not change the incompetence of RNA replication of Mutant/nt14900 that lacks a 3′ cis-acting replication element and the efficiency of RNA replication of Mutant/nt14800 that has a low level of RNA replication. Overall, our findings are consistent with previous results and help to understand a role of the N protein in PRRSV biology.
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Figure 1.
Schematic view of the PRRSV N protein expression vector. (A) Organization of PRRSV genomic RNA. The full-length PRRSV ORF7 was amplified from the genome of PL97–1/LP1 strain, as described in Materials and Methods. (B) Schematic presentation of the PRRSV N protein expression vector. The Sinrep5, derived from Sindbis virus, was used for the expression of the PRRSV N protein. The full-length PRRSV ORF7 (N protein) was inserted immediately downstream of the subgenomic promoter (arrows) of Sindbis virus, generating pSinrep5-N.
Figure 2.
Intracellular localizaton of the PRRSV N protein. BHK-21 cells were transfected with the RNA transcripts derived from one of either pSinrep5, pSinrep5-N, or pBAC/PRRSV/FL (9) that is a full-length infectious PRRSV cDNA. After incubation for 20 h, cells were fixed and staining with a PRRSV N-specific monoclonal antibody and a FITC-conjugated anti-mouse IgG (green). At the end of immunostaining, the cells were stained with PI (red) for the nucleus. Also shown are merged images (yellow).
Figure 3.
Schematic view of two PRRSV replicons, Mutant/nt14800 and Mutant/nt14900. While the Mutant/nt14800 replicon is competent for replication, with a low level of RNA replication being produced, the Mutant/nt14900 is replication-incompetent due to a lack of 3′ cis-acting replication element (14). Dashed lines indicate a deleted region within the genome. Luc, luciferase gene. EMCV IRES, internal ribosome entry site of encephalomyocarditis virus.
Figure 4.
Replicability of two PRRSV replicons in the presence or absence of PRRSV N protein. BHK-21 cells were transfected with the RNA transcripts derived from either of Mutant/nt14800 or Mutant/nt14900 alone or co-transfected together with the RNA transcripts derived from pSinrep5-N (+N). (A) After incubation for 20 h, cells were directly lysed with lysis buffer for luciferase assay, as described in Materials and Methods. Luciferase activities were measured twice and the mean values were presented. RLU, relative light units. (B) After incubation for 20 h, cells were fixed and staining with a PRRSV N-specific monoclonal antibody and a FITC-conjugated anti-mouse IgG.
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