Figure 8.

Complementary-strand DNA synthesis from cytoplasmic or nuclear capsids. (A) Depiction of the strategy for complementary-strand synthesis: (a) Immunoprecipitation of B19V capsids with the PLA2 antibody; (b) 5’ virus-unrelated sequence; (c) extension reaction by T7 polymerase at 37 °C; and (d) amplification of the primer-extended DNA by PCR. (B) Positions in the B19V genome of the 3’ (yellow) and 5’ (red) regions targeted by the hybridization and extension assay. (C) Viral particles were immunoprecipitated from cytoplasmic or from nuclear fractions with the antibody against the PLA₂ region (PLA2) and used for complementary-strand DNA synthesis. The primer-extended DNA was amplified by PCR and amplicons of the expected size were visualized by agarose gel electrophoresis. As controls, viruses were immunoprecipitated 5 min pi or incubated with the T7 polymerase for only a few seconds.