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. 2010 Dec 27;108(2):804–809. doi: 10.1073/pnas.1013155108

Fig. 3.

Fig. 3.

Copackaging of M and S RNAs into VLPs. (A) Intracellular proteins, intracellular RNAs, and released VLPs were collected from cells expressing all of the viral structural proteins, M RNA, and S RNA (M+S) at 3 d posttransfection. M1+S represent samples in which M1 RNA was used in place of M RNA. Intracellular proteins (IC) and proteins in purified VLPs were subjected to Western blot analysis using anti-RVFV antibody (Left two panels). Intracellular RNAs (IC) and RNAs in purified VLPs were detected by Northern blot analysis using an RNA probe that hybridizes to viral-sense M RNA or M1 RNA (Right Top) or viral-sense S RNA (S) (Right Bottom). Lane 1 and lane 2 represent intracellular samples from mock-infected cells and RVFV-infected cells, respectively. (B) VLPs produced from cells supporting the replication of M and S RNAs (M+S) or from those supporting the replication of M1 and S RNAs (M1+S) were inoculated into cells coexpressing L and N proteins (lanes 1 and 2) or L protein-expressing cells (lanes 3 and 4). Intracellular RNAs were extracted at 24 h postinoculation and subjected to Northern blot analysis to detect viral-sense M RNA (Top, lanes 1 and 3), M1 RNA (Top, lanes 2 and 4), or viral-sense S RNA (Bottom). (C) Cells expressing L protein were inoculated with either M+S VLP sample, obtained from cells supporting the replication of M and S RNAs, or a mixture of M VLP sample and S VLP sample (M VLP+S VLP), obtained from cells supporting M RNA replication and S RNA replication, respectively. Both VLP samples carried the same amount of M RNA. Similarly, the amount of S RNA was also the same in both VLP samples. Intracellular RNAs were extracted at 24 h postinoculation and subjected to Northern blot analysis to detect viral-sense M RNA (Top) or viral-sense S RNA (Bottom). (D) VLPs produced from cells supporting S RNA replication were concentrated ∼10 times (S VLP). Cells coexpressing L and N proteins (lanes 1 and 2) and those expressing L protein (lanes 3 and 4) were coinoculated with VLPs carrying M RNA (M VLP) and S VLP (lanes 1 and 3) or those carrying M1 RNA (M1 VLP) and S VLP (lanes 2 and 4). Intracellular RNAs were collected at 24 h postinoculation and subjected to Northern blot analysis to detect viral-sense M or M1 RNA (Top) or viral-sense S RNA (Bottom).