Analysis of the subcellular localization of the proteins Rep, Rep' and Cap of porcine circovirus type 1.
Porcine circovirus type 1 (PCV1) encodes two major ORFs. The cap gene comprises the major structural protein of PCV, the rep gene specifies Rep and Rep', which are both essential for initiating the replication of the viral DNA. Rep corresponds to the full-length protein, whereas Rep' is a truncated splice product that is frame-shifted in its C-terminal sequence. In this study, the cellular localization of PCV1-encoded proteins was investigated by immune fluorescence techniques using antibodies against Rep, Rep' and Cap and by expression of viral proteins fused to green and red fluorescence proteins. Rep and Rep' protein co-localized in the nucleus of infected cells as well as in cells transfected with plasmids expressing Rep and Rep' fused to fluorescence proteins, but no signal was seen in the nucleoli. Rep and Rep' carry three potential nuclear localization signals in their identical N-termini, and the contribution of these motifs to nuclear import was experimentally dissected. In contrast to the rep gene products, the localization of the Cap protein varied. While the Cap protein was restricted to the nucleoli in plasmid-transfected cells and was also localized in the nucleoli at an early stage of PCV1 infection, it was seen in the nucleoplasm and the cytoplasm later in infection, suggesting that a shuttling between distinct cellular compartments occurs.
Finsterbusch
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T
Robert Koch-Institut, viral infections; Nordufer 20, 13353 Berlin, Germany. finsterbuscht@rki.de
Steinfeldt
T
T
Caliskan
R
R
Mankertz
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002466:Cell Nucleolus / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry*; D017925:Circovirus / Q000502:physiology*; D003593:Cytoplasm / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis; D008164:Luminescent Proteins / Q000032:analysis; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000502:physiology; D010449:Peptide Mapping; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013552:Swine; D014764:Viral Proteins / Q000032:analysis*; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D000097573:Red Fluorescent Protein