Journal of Microbiology

Research Support, Non-U.S. Gov'ts

Packaging of Porcine Reproductive and Respiratory Syndrome Virus Replicon RNA by a Stable Cell Line Expressing Its Nucleocapsid Protein: Byung-Hak Song , Jeong-Min Kim , Jin-Kyoung Kim , Han-Saem Jang , Gil-Nam Yun , Eun-Jin Choi , Jae-Young Song , Sang-Im Yun , Young-Min Lee; J. Microbiol. 2011;49(3):516-523. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-1280-1

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Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae family, is one of the most common and economically important swine pathogens. Although both live-attenuated and killed-inactivated vaccines against the virus have been available for a decade, PRRSV is still a major problem in the swine industry worldwide. To explore the possibility of producing single-round infectious PRRSV replicon particles as a potential vaccine strategy, we have now generated two necessary components: 1) a stable cell line (BHK/Sinrep19/PRRSV-N) that constitutively expresses the viral nucleocapsid (N) protein localized to the cytoplasm and the nucleolus and 2) a PRRSV replicon vector (pBAC/PRRSV/Replicon-ΔN) with a 177-nucleotide deletion, removing the 3′-half portion of ORF7 in the viral genome, from which the self-replicating propagation-defective replicon RNAs were synthesized in vitro by SP6 polymerase run-off transcription. Transfection of this replicon RNA into N protein-expressing BHK-21 cells led to the secretion of infectious particles that packaged the replicon RNA, albeit with a low production efficiency of 0.4×102 to 1.1×102 infectious units/ml; the produced particles had only single-round infectivity with no cell-to-cell spread. This trans-complementation system for PRRSV provides a useful platform for studies to define the packaging signals and motifs present within the viral genome and N protein, respectively, and to develop viral replicon-based antiviral vaccines that will stop the infection and spread of this pathogen.

pVC, a Small Cryptic Plasmid from the Environmental Isolate of Vibrio cholerae MP-1: Ruifu Zhang , Yanling Wang , Pak Chow Leung , Ji-Dong Gu; J. Microbiol. 2007;45(3):193-198.; DOI: https://doi.org/2543 [pii]

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Abstract: A marine bacterium was isolated from Mai Po Nature Reserve of Hong Kong and identified as Vibrio cholerae MP-1. It contains a small plasmid designated as pVC of 3.8 kb. Four open reading frames (ORFs) are identified on the plasmid, but none of them shows homology to any known protein. Database search indicated that a 440 bp fragment is 96% identical to a fragment found in a small plasmid of another V. cholerae. Further experiments demonstrated that a 2.3 kb EcoRI fragment containing the complete ORF1, partial ORF4 and their intergenic region could self-replicate. Additional analyses revealed that sequence upstream of ORF1 showed the features characteristic of theta type replicons. Protein encoded by ORF1 has two characteristic motifs existed in most replication initiator proteins (Rep): the leucine zipper (LZ) motif located at the N-terminal region and the alpha helix-turn-alpha helix motif (HTH) located at the C-terminal end. The results suggest that pVC replicates via the theta type mechanism and is likely a novel type of theta replicon.

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