Localisation of Theiler's murine encephalomyelitis virus protein 2C to the Golgi apparatus using antibodies generated against a peptide region.
The picornavirus 2C protein is highly conserved and indispensible for virus replication. Polyclonal antibodies against Theiler's murine encephalomyelitis virus (TMEV) 2C protein were generated by immunisation of rabbits with a peptide comprising amino acids 31-210 of the protein. Antibodies were used to investigate the localisation of 2C in infected cells by indirect immunofluorescence and confocal microscopy. Analysis of infected cells revealed that the distribution of 2C changed during infection. Early on, the protein was localised in the perinuclear region with punctate staining in the cytoplasm and at later stages, it was concentrated in one large structure in close proximity to the nucleus and occupying almost 50% of the cell size. Dual-label immunofluorescence using wheat germ agglutinin (WGA) and anti-TMEV 2C antibodies suggested that 2C, and therefore virus replication, is targeted to the Golgi apparatus. At late stages of infection Golgi staining was dispersed, indicating potential reorganisation of membranes. Infection was accompanied by "rounding up" of the cells and a redistribution of actin around the putative replication complex. The results suggest that TMEV behaves similarly to FMDV which also forms replication complexes in the perinuclear region.
Jauka
Tembisa
T
Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown, Eastern Cape, South Africa.
Mutsvunguma
Lorraine
L
Boshoff
Aileen
A
Edkins
Adrienne L
AL
Knox
Caroline
C
D000818:Animals; D000914:Antibodies, Viral; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D006224:Cricetinae; D003593:Cytoplasm / Q000737:chemistry; D019085:Fluorescent Antibody Technique, Direct; D006056:Golgi Apparatus / Q000737:chemistry* / Q000821:virology; D018613:Microscopy, Confocal; D011817:Rabbits; D017945:Theilovirus / Q000502:physiology*; D014764:Viral Proteins / Q000032:analysis* / Q000276:immunology; D014779:Virus Replication
D000914:Antibodies, Viral; D014764:Viral Proteins